Co-culture of Chlorella vulgaris with nitrogen-fixing Azotobacter chroococcum in nitrogen-deplete medium to increase algal lipid content

Co-culture of Chlorella vulgaris with nitrogen-fixing Azotobacter chroococcum in nitrogen-deplete medium to increase algal lipid content | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Co-culture of Chlorella vulgaris with nitrogen-fixing Azotobacter chroococcum in nitrogen-deplete medium to increase algal lipid content Caleb Arruda, Andreas Kirmaier, Kyle Ferris, Gabrielle Bailey, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6604448/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Reliance on fossil fuels significantly contributes to climate change. Sustainable biofuels are a promising alternative. The high lipid content and environmental benefits of microalgae lend themselves strongly to biofuel production. This study focuses on taking an industrially relevant, lipid-rich microalgae strain and engineering a unique solution to further increase the lipid content. Although lipid yield is greater than terrestrial biofuel feedstocks, it remains a key limiting factor preventing microalgal biofuel from becoming a positive investment. Both microalgal-bacterial co-culture and nitrogen deprivation have individually increased lipid yields in prior art. Nitrogen depletion forces microalgae into a stressed state in which lipids accumulate rapidly, while certain bacteria have been shown to have symbiotic relationships with algae increasing lipids or biomass. Co-culture with a nitrogen-fixer supplies low levels of bioavailable nitrogen to the algae, allowing for higher photosynthetic efficiency while keeping the algae in a stressed state. These effects enhance lipid production. However, the combination of nitrogen-fixing Azotobacter chroococcum and lipid-rich Chlorella vulgaris under nitrogen-deplete conditions has yet to be tested. This study combined co-culture of C. vulgaris with nitrogen-fixing A. chroococcum under nitrogen-deplete conditions and found a significant 8.3-fold increase in lipid content per cell compared to nitrogen-replete monoculture algae. This was paired with a nearly 2-fold increase in algal lipids compared to a nitrogen-deprived control as well as an Escherichia coli co-culture nitrogen-deprived control. Climate change microalgae co-culture biofuel Chlorella vulgaris Azotobacter chroococcum Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction First generation biofuels, typically derived from edible terrestrial crops, have significant sustainability limitations (Elgarahy et al. 2021 ; Asase et al. 2024 ). Several of these can be overcome using microalgae as a biofuel feedstock (Lum et al. 2013 ). Microalgae grow significantly faster than terrestrial plants, fix more carbon from the atmosphere, and use less cultivatable land and fresh water. Many species are also rich in lipids, the precursor macromolecules for biofuel (Chisti 2007 ; Halim et al. 2012 ). These are in the form of fatty acids such as oleic acid (C18:1) or linoleic acid (C18:2), which are often bound to glycerol in a triacylglycerol molecule. Such lipids can be turned into biodiesel through simple chemical synthesis. Despite these advantages, the production of microalgal biodiesel does not yet have a high enough yield to be a viable energy source (Bondioli et al. 2012 ; Khoo et al. 2023 ). Nutrient deprivation is among the most widely explored methods of increasing lipid productivity in microalgae populations (Praveenkumar et al. 2012 ; Gao et al. 2013 ; Procházková et al. 2014 ). This approach forces the microalgae into a stressed state. Under unfavorable environmental conditions, the microalgal metabolism shifts to lipid production to store energy. Deprivation of key nutrients is achieved by reducing the amount of certain organic compounds added to the growth media. Depletion of nitrogen has emerged as a common and effective target for increasing lipids (Bondioli et al. 2012 ). Manipulation of phosphorous, sulfur, and heavy metal levels also shows potential (El-Agawany and Kaamoush 2022 ; Saber et al. 2024 ). However, nutrient deprivation stunts the growth of the algae, as carbon is focused into lipid synthesis pathways rather than cell replication (Yang et al. 2018 ; Chen and Wang 2021 ). This directly reduces efficiency of this method of biofuel production, as the growth phase of the algae does not overlap with the lipid induction phase. Nitrogen is an essential nutrient for algae that promotes lipid, carbohydrate, and protein synthesis (Chen and Wang 2021 ). Nitrogen levels are positively correlated with biomass accumulation and inversely with lipid metabolism (Chen and Wang 2021 ). In microalgal media, nitrogen is often added as ammonium chloride (NH 4 Cl), which becomes bioavailable as nitrate (NO 3 –). Nitrogen starvation decreases the photosynthetic thylakoid membrane content of cells, activates acyl hydrolase, and encourages hydrolysis of phospholipids, all of which work to increase the intracellular content of acetyl-CoA (Yaakob et al. 2021 ). Acetyl-CoA is the foundational molecule for lipid biosynthesis (Chen and Wang 2021 ). When nitrate levels were reduced by Yang et al. 2018 so that a Chlamydomonas reinhardtii population were put into nitrogen starvation, biomass accumulation was reduced by 31.7% while lipid production was enhanced by 93% up to 113.46 ± 1.78 mg L − 1 . Lipid productivity has also been bolstered by co-culture, which can take advantage of numerous biochemical pathways (Ray et al. 2022 ; Tong et al. 2023 ). Co-culture models can take the form of microalgae grown with other microalgae, bacteria, or fungi. Escherichia coli is a notable microalgal co-culture partner which has demonstrated several synergistic interactions (Tong et al. 2024 ). Literature has shown that E. coli provides CO₂ and essential growth factors and concurrently receives oxygen and photosynthetic byproducts in return (Tong et al. 2024 ; Kang et al. 2025 ). This metabolic exchange not only supports mutual growth but also enhances lipid accumulation in algal cells. Yamada et al. 2023 reported that a Chlamydomonas reinhardtii–E. coli co-culture significantly increased lipid yields compared to monocultures. This improvement was attributed to enhanced carbon flux and reduced oxidative stress in the microalgal cells, creating a more favorable environment for lipid biosynthesis (Yamada et al. 2023 ). Co-culture and stress induction can also be implemented synergistically to increase lipid production. A study by Xu et al. ( 2018 ) investigated the impact of co-culturing C. reinhardtii with A. chroococcum bacteria concurrently to nitrogen deprivation. A. chroococcum is nitrogen-fixing: it converts atmospheric nitrogen into ammonia, a usable form of nitrogen for algae (Xu et al. 2018 ). When co-cultured with C. reinhardtii in a nitrogen-deplete media (N- TAP), A. chroococcum released useable nitrogen into the liquid media (Xu et al. 2018 ). Most importantly this led to increased lipid content, but growth rates and therefore biomass accumulation were also positively affected. This co-culture had a maximum lipid productivity 19.4 times greater than the control group of C. reinhardtii in N- TAP media. These results suggest combined co-culture and stress induction could be an affordable method to dramatically increase lipid production while limiting the biomass disadvantages. While the A. chroococcum co-culture model was shown to be successful in C. reinhardtii , its low baseline lipid content makes it an inefficient choice for a scaled-up model. Mata et al. ( 2010 ) suggest Chlorella sp. could be an effective choice for scaled-up production. Chlorella vulgaris is a green microalga with a fully sequenced genome (Cecchin et al. 2019 ). It has been the subject of numerous papers relating to biofuel production, including those pertaining to scaled-up systems (Ahmad et al. 2020 ; Parashar et al. 2023 ; Al-Hammadi and Güngörmüşler 2024 ). It can yield high quantities of lipids, with a dry weight that is ordinarily 14–40% lipids without any modification. Although, even higher numbers have been reported (Gouveia and Oliveira 2009 ; Mata et al. 2010 ). This contrasts with C. reinhardtii , which has a lower dry weight of approximately 18–25% lipids (Scranton et al. 2015 ; Enamala et al. 2018 ). Evidently, C. vulgaris could allow for faster lipid yields and higher maximum lipid content. The purpose of this study was to determine how A. chroococcum co-culture influences lipid accumulation in the naturally lipid rich and industrially relevant C. vulgaris . Experimental design consisted of three replicates of each: C. vulgaris in nitrogen-replete media (N + TAP), C. vulgaris in N- TAP media, co-culture of C. vulgaris and E. coli in N- TAP, and co-culture of C. vulgaris and A. chroococcum in N- TAP. This work and the results described herein improve the understanding of microalgae-bacteria co-culture and represent forward progress in enhancing the viability of C. vulgaris derived biodiesel. Materials and methods Chlorella vulgaris (15-2075) was obtained from Carolina Biological Supply. Azotobacter Chroococcum (43 [NCIB 11694]) was obtained from the American Type Culture Collection. Escherichia coli (MG1655) was obtained from collaborators at Northeastern University. Nitrogen-replete tris-acetate phosphate media (N + TAP) (Gorman and Levine 1965 ; Bono et al. 2013 ) was used for maintaining the microalgal stock in a T50 culture flask at 28 +/-3 ℃ under a constant light intensity of 898 µmol photons m − 2 s − 1. Cultures were agitated on an orbital shaker at all times. Stress induction was achieved with nitrogen-deplete tris-acetate phosphate media (N- TAP) (Bono et al. 2013 ). Lysogeny broth (Chapman 1945 ) and Ashby’s Mannitol Broth (Ashby 1907 ) were used for suspending the E. coli and A. chroococcum cultures respectively. Culture conditions Algae cultures were grown over thirteen days that were broken into two phases. On day zero, C. vulgaris stock was split into twelve T25 flasks at a concentration of 1.0 × 10 7 cells mL − 1 . For the first phase, the cultures were all grown in N + TAP for five days. On day five, all cultures received a media change and the cocultures were inoculated. Except for the N + TAP control group, the media was replaced with N- TAP to begin stress induction. Three flasks were inoculated with E. coli while three others received A. chroococcum . Bacteria were inoculated at a 1:10 ratio to the algae. Analytical methods Growth was tracked using optical density at 750 nm. A standard curve was verified with cell counting on a manual hemocytometer. The samples were prepared with strict aseptic technique, read in a 96-well plate, then discarded. For lipid quantification, lipid standards and a 1 mg mL − 1 Nile Red stock (Sigma-Aldrich, USA) were thawed at 37°C and protected from light. Microalgal culture samples (1.2 mL) were centrifuged at 4500 rpm for 5 min, and pellets were resuspended in 400 µL of N- TAP medium to standardize assay conditions and avoid lipid induction. Samples were then normalized to 1.05 × 10⁹ cells mL − 1 based on the most dilute sample. An opaque 96-well plate was prepared with 80 µL TAP-N for standards, 100 µL for true blanks, and 90 µL for Nile Red blanks. Nile Red was diluted to 10 µg mL − 1 in DMSO, protected from light, and 10 µL was added to each well (excluding blanks) using a P20 pipette to avoid crystallization-related errors. Wells were mixed by pipetting. The plate was incubated in the dark at 37°C for 10 min. Fluorescence was measured using a BioTek-Cytation 5 plate reader with excitation at 530 nm and emission at 590 nm and compared to a triolein standard (ThermoFisher, J62419.14). Fluorescence microscopy was performed on the Olympus BX53 microscope. Nile Red stock solution (1 mg/mL in DMSO; Sigma-Aldrich, USA) was thawed at 37°C and protected from light using aluminum foil. A working solution of 2 µg mL − 1 Nile Red was prepared in DMSO immediately prior to staining. For each sample, 10 µL of culture was combined with 10 µL of the diluted stain in a microcentrifuge tube and mixed by pipetting. Samples were incubated in the dark at 37°C for 10 min before 2.5 µL of the stained culture was mounted on a microscope slide and covered with a glass coverslip. Slides were visualized with a Cy3 filter set. Transmitted light was blocked manually, and the reflected light LED was activated at ~ 10% intensity. Images were acquired using a 20 s exposure time. All optical density measurements were performed in duplicate. Lipid quantification was read in quadruplicate. The data are expressed as mean ± standard deviation (SD). Results Culture Growth Optical density (OD) measurements were correlated with manual cell counts to establish a standard curve for estimating cell concentration. This was recorded daily (Fig. 1 , except on days 4 and 11). Near day 5, cultures transitioned out of the exponential growth phase, at which point a scheduled media replacement was performed switching all groups to N- TAP besides the three N + TAP control cultures (which received fresh N + TAP). This intervention reinitiated growth that continued until approximately day 12. Figure 1 displays the growth curves for each experimental group as well as the raw OD values. Samples taken for OD measurements were diluted 4x after day 8 as they were approaching the functional limit of the plate reader. The N + TAP C. vulgaris monoculture attained the highest cell density, reaching approximately 5 × 10⁸ cells mL − 1 . The remaining groups—N- TAP C. vulgaris monoculture, the E. coli co-culture, and the A. chroococcum co-culture—achieved peak concentrations near 3.5 × 10⁸ cells mL − 1 . Among these, the N– C. vulgaris exhibited the highest density, followed by the E. coli and A. chroococcum co-cultures. Lipid Quantification The Nile Red lipid quantification assay was performed on Day 12. Figure 2 shows the results of the quantification normalized to cell density. All wells were seeded at 1.05 x 10 9 cells mL − 1 for uniformity. Lipid densities were 1.36 µg 10⁻⁷ cells⁻¹ and 5.77 µg 10⁻⁷ cells⁻¹ for the N + TAP and N- TAP groups respectively. This was a 4.2-fold increase, or 324%. The co-cultures had lipid densities of 5.35 µg 10⁻⁷ cells⁻¹ and 11.25 µg 10⁻⁷ cells⁻¹ for E. coli and A. chroococcum respectively. The A. chroococcum co-culture showed an 8.3-fold (730%) increase compared to the N + TAP monoculture, a 1.9-fold (95%) increase compared to the N- TAP monoculture, and a 2.1-fold (110%) increase compared to the E. coli co-culture in lipid density. The E. coli co-culture performed similarly to the N- TAP monoculture, while the A. chroococcum co-culture outperformed it significantly. Samples were compared to a triolein standard. The standard curve used 4 points on a graph of log-concentration versus fluorescent signal. Design Verification As described above, microalgal proliferation was monitored via optical density (OD) measurements at 750 nm. However, bacterial growth proved more challenging to quantify directly due to their relatively smaller size and lower biomass contribution compared to the algae. This rendered OD measurements insufficiently sensitive. As such, a supplementary viability test was used to confirm the presence of live bacterial populations within the co-culture systems at several time points. Bacterial viability in the co-culture groups was assessed using a drop-streak test (Fig. 3 ). A 20 µL drop of culture medium from each experimental flask was applied to the surface of a solid agar plate—Luria-Bertani agar (Cold Harbor 2006) for E. coli and Ashby’s agar for A. chroococcum . The plates were tilted, allowing the drop to streak downwards. After 2–3 days of incubation, the appearance of colonies indicated the presence of viable bacteria. Colonies of E. coli and A. chroococcum are visually distinct with E. coli colonies appearing opaque and cream-colored and A. chroococcum colonies appearing transparent and gel-like. One droplet streak was performed in triplicate (on the same plate) for each co-culture flask on day six post inoculation. The presence of bacteria was verified again on day twelve with brightfield microscopy (40x magnification). Fluorescence microscopy was used to verify lipid quantification results (Fig. 4 ). This revealed a stark contrast between the A. chroococcum co-culture group and the N + TAP control group. After Nile Red staining and a short incubation, the neutral lipid droplets in each sample fluoresced under the Cy3 filter. This allowed for the lipid content to be assessed in a visual, qualitative way. The N + TAP group (Fig. 4 a) showed weak fluorescence with individual lipid droplets being visible while the A. chroococcum co-culture (Fig. 4 b) fluoresced so brightly the contrast between lipid drop and the rest of the cell was indistinguishable. Statistical comparisons between all experimental groups were conducted using one-sample t-tests in MATLAB (2025a). A post hoc power analysis was performed using G*Power (v3.1.9.7) to confirm the adequacy of the sample size and the robustness of the findings. Effect sizes were calculated and reported as Cohen’s d to indicate the magnitude of differences observed (Table 1 ). Results with p < 0.05 were considered statistically significant. Statistical analyses confirmed that the N- TAP C. vulgaris monoculture, the E. coli co-culture group and the A. chroococcum co-culture group both displayed significant increases in lipid content compared to the N + TAP C. Vulgaris monoculture control group. Each with large effect sizes (d > 0.8), indicating strong biological effects in addition to statistical significance. Table 1 Nile Red lipid quantification results comparing descriptive statistics, p-values, and Cohen’s d for each group Discussion Lipid content increased 324% in the N- TAP C. vulgaris monoculture compared to its N + TAP counterpart. Since these groups were identical in all other experimental parameters, the increased lipid accumulation can be attributed to nitrogen deprivation. Stress induction through nitrogen deprivation was the intended result of changing the media to N- TAP on day five. This observation is consistent with previous literature, which identifies nitrogen deprivation as a key stressor that redirects algal metabolic energy from growth and biomass accumulation toward lipid storage (Yaakob et al. 2021 ; Chen and Wang 2021 ). This method for inducing lipid production has been extensively validated as a standard technique for microalgal metabolic manipulation. Our findings further support this well-established approach. Co-culture of C. vulgaris with A. chroococcum under nitrogen-deficient conditions for seven days yielded a 730% increase in lipid content per cell relative to the N + TAP C. vulgaris monoculture. This value represents the difference between a wild-type microalgal culture and the nitrogen-fixing co-culture model. When compared directly to the N- TAP C. vulgaris monoculture, a statistically significant 90% enhancement in lipid content per cell was observed (t-test: t(4) = 4.74, p < 0.001). These results indicate that co-culture with A. chroococcum enables nearly a doubling of lipid yields beyond what is achieved through nitrogen deprivation alone. In contrast, the nitrogen-deprived C. vulgaris–E. coli co-culture exhibited no statistically significant difference in lipid content compared to the N- TAP C. vulgaris monoculture (t-test: t(4) = 0.39, p = 0.72). This finding suggests that the lipid increase observed in the E. coli co-culture relative to the N + TAP control was solely due to nitrogen deprivation. The lipid yields were not enhanced by the presence of E. coli . This result is notable, as it underscores the differing effects of bacterial co-culture partners. Moreover, this supports the idea that co-culture with A. chroococcum is beneficial for reasons not typically associated with E. coli. Previous literature has identified several merits to bacterial co-culture that make it a promising method to increase microalgal lipid content. These include the exchange of hormones, growth factors, vitamins, or stress induction from resource competition (Magdouli et al. 2016 ; Tong et al. 2023 ). In this experiment, lipid content was observed to be 110% greater in the A. chroococcum co-culture compared to the E. coli co-culture. These groups were otherwise identical in cultivation conditions. Given this increase and the lack of statistical difference between the E. coli co-culture and the N- TAP C. vulgaris monoculture, it is evident the A. chroococcum provided some benefit that the E. coli could not. Further work must be done to explain specific pathways and break down the molecular mechanisms involved. However, this result supports the idea that the observed lipid increase was not due to the previously mentioned standard bacterial co-culture benefits, but rather nitrogen fixation, a unique ability of A. chroococcum and the primary reason for its selection in this design. Nitrogen fixation is the process by which atmospheric N 2 , which is not actively usable by algae, is converted into bioavailable ammonia (Walker and Yates 1978 ). Xu et al ( 2018 ) found that when C. vulgaris is co-cultured with nitrogen-fixing A. chroococcum in nitrogen-deplete conditions, this nitrogen supplementation was beneficial to biomass and lipid accumulation. Although it may be intuitive to assume supplemental nitrogen would be deleterious to lipid accumulation, this has not been observed. Instead, findings suggest that A. chroococcum nitrogen fixation is just enough for the algae to be enhanced photosynthetically without compromising the effects of stress induction (Xu et al. 2018 ). The nutrient deprived microalgae may see the provided ammonia as a finite resource, perhaps due to its method or rate of production, and use it to facilitate increased lipid production without exiting its stressed state. Xu et al ( 2018 ) also showed that co-culture with A. chroococcum leads to upregulation of several important genes in triacylglycerol synthesis in C. reinhardtii. These genes include acetyl-CoA carboxylase (ACC), diacylglycerol acyltransferase 1 (DGAT), phospholipid diacylglycerol acyltransferase (PDAT), and the 5 genes that encode the various amino acid chains of DGAT2. They also observed a downregulation of phosphoenolpyruvate carboxylase (PEPC2), promoting generation of acetyl-CoA which can be used in fatty acid and therefore triacylglycerol production (Deng et al. 2011 ). Further experimentation must be performed to elucidate the pathways that allow nitrogen fixation to further increase lipid accumulation when paired with nitrogen-deplete conditions. One limitation of this study is that success of the A. chroococcum co-culture over the E. coli co-culture control is not sufficient to conclude that nitrogen fixation is solely responsible for the observed high lipid content. There are other possible ways by which A. chroococcum may induce lipid production, such as by the secretion of a nutrient or growth factor. As both E. coli and A. chroococcum are members of the class Gammaproteobacteria , they share some potentially beneficial secretions, including indole-3-acetic acid (El-Essawy et al. 1984 ; Heo et al. 2019 ). The E. coli co-culture successfully ruled out any of these as the dominant factor. The observed upregulation of genes involved in lipid synthesis and general increase in lipid content could have been caused by nitrogen fixation or by some unknown secretion unique to A. chroococcum in this study. Ultimately, this work provides a baseline for enhancing lipid yield in nitrogen deprived C. vulgaris through co-culture with the nitrogen-fixing bacteria A. chroococcum . The results are encouraging for the prospect of pushing lipid accumulation past the limits of environmental modifications. By proving that co-culture with A. chroococcum can significantly increase lipid content in C. vulgaris , this experiment represents a key next step in increasing the feasibility of microalgal based biofuel. Further work would be necessary to explore the scalability of the system from the laboratory setting. For example, this model could be tested in a photobioreactor or raceway pond. Dynamics of biomass harvesting must be also studied. The growth rate of both species in the co-culture after medium renewal or harvesting should be explored such that bacterial overgrowth is limited. Expression or metabolomic profiling could offer insights into the exact pathways and molecular mechanisms being targeted by this co-culture pairing. Declarations Funding This study was funded entirely by the Northeastern University Department of Bioengineering. Competing Interests The authors declare they have no financial or non-financial interests or associations relevant to the content of the report. Data Availability The datasets generated during the current study are available upon reasonable request to the authors. Code Availability The MATLAB code generated during this study is available upon reasonable request to the authors. Author’s contributions All authors contributed to the study conception and design. Material preparation was performed by all authors. G.B. and K.F. performed bacterial preparation and culture. Data collection and analysis were performed by A.K., C.A, K.F, G.B. and T.C. Figures were prepared by C.A. The first draft of the manuscript was written by C.A., A.K., G.B., K.F., and T.C. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Acknowledgement The authors would like to thank Professors Samuel Chung and Esin Sozer for their valuable insight throughout the conception and execution of the study. This work was supported by the Northeastern University Department of Bioengineering. We also acknowledge the Northeastern University Wet Lab Makerspace for providing access to equipment, lab space, and common reagents. We extend our gratitude to Helen Kurkjian and team for their help with materials acquisition and experimental support. We additionally thank Professor Timothy Lannin for his thoughtful review of the manuscript. References Ahmad MT, Shariff M, Md. Yusoff F, et al (2020) Applications of microalga Chlorella vulgaris in aquaculture. 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Science of The Total Environment 802:149765. https://doi.org/10.1016/j.scitotenv.2021.149765 Saber H, Galal HR, Abo-Eldahab M, Alwaleed E (2024) Enhancing the biodiesel production in the green alga Chlorella vulgaris by heavy metal stress and prediction of fuel properties from fatty acid profiles. Environ Sci Pollut Res 31:35952–35968. https://doi.org/10.1007/s11356-024-33538-w Scranton MA, Ostrand JT, Fields FJ, Mayfield SP (2015) Chlamydomonas as a model for biofuels and bio‐products production. The Plant Journal 82:523–531. https://doi.org/10.1111/tpj.12780 Tong CY, Honda K, Derek CJC (2023) A review on microalgal-bacterial co-culture: The multifaceted role of beneficial bacteria towards enhancement of microalgal metabolite production. Environmental Research 228:115872. https://doi.org/10.1016/j.envres.2023.115872 Tong CY, Honda K, Derek CJC (2024) Enhanced microalgal growth and metabolites through co-cultivation with Escherichia coli in algal organic matter solution. Algal Research 77:103351. https://doi.org/10.1016/j.algal.2023.103351 Walker CC, Yates MG (1978) The hydrogen cycle in nitrogen-fixing Azotobacter chroococcum. Biochimie 60:225–231. https://doi.org/10.1016/S0300-9084(78)80818-9 Xu L, Cheng X, Wang Q (2018) Enhanced Lipid Production in Chlamydomonas reinhardtii by Co-culturing With Azotobacter chroococcum. Front Plant Sci 9:741. https://doi.org/10.3389/fpls.2018.00741 Yaakob MA, Mohamed RMSR, Al-Gheethi A, et al (2021) Influence of Nitrogen and Phosphorus on Microalgal Growth, Biomass, Lipid, and Fatty Acid Production: An Overview. Cells 10:393. https://doi.org/10.3390/cells10020393 Yamada R, Yokota M, Matsumoto T, et al (2023) Promoting cell growth and characterizing partial symbiotic relationships in the co‐cultivation of green alga Chlamydomonas reinhardtii and Escherichia coli . Biotechnology Journal 18:2200099. https://doi.org/10.1002/biot.202200099 Yang L, Chen J, Qin S, et al (2018) Growth and lipid accumulation by different nutrients in the microalga Chlamydomonas reinhardtii. Biotechnol Biofuels 11:40. https://doi.org/10.1186/s13068-018-1041-z Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6604448","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":452770208,"identity":"f99082b2-8ba8-4d11-8841-6ae85ed3c07f","order_by":0,"name":"Caleb Arruda","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCUlEQVRIiWNgGAWjYLACHhDB3gAiDyAJH8BUiaqFB6QiAUQwE6tFIoFILfyzzz6TeFPDIM8/8+3Bx5U/7sjx958HMnYwyPHdSMCqReJcupnknGMMhjNu5yUbnkl4ZixxI5nZ8OwZBmNJHFoYzrCxSfOwMSQw3M4xk2xIOJzYcIOZTbKxjSFxAw4t8mAt/xgS5G+eAWupn3/+MPtPoJZ6XFoMQFp42xgSDG7wgLUkGBxIZmNsBItg12J4ho3Zcm6fhOHGMznGhg1phw033kg2BjpMwnDmmQdYtcidYWO88eabjbzc8TOGDxtsDsvLnT/48GNjm40833Ec3ocGHBEio2AUjIJRMAqIBwBj5l8cD49FYwAAAABJRU5ErkJggg==","orcid":"","institution":"Northeastern University","correspondingAuthor":true,"prefix":"","firstName":"Caleb","middleName":"","lastName":"Arruda","suffix":""},{"id":452770209,"identity":"547537ab-af14-4316-9c24-9c5c5e4c2e54","order_by":1,"name":"Andreas Kirmaier","email":"","orcid":"","institution":"Northeastern University","correspondingAuthor":false,"prefix":"","firstName":"Andreas","middleName":"","lastName":"Kirmaier","suffix":""},{"id":452770210,"identity":"5ef676ed-aced-4f23-867a-c044667dd074","order_by":2,"name":"Kyle Ferris","email":"","orcid":"","institution":"Northeastern University","correspondingAuthor":false,"prefix":"","firstName":"Kyle","middleName":"","lastName":"Ferris","suffix":""},{"id":452770211,"identity":"f60fafed-3bb9-4add-bd0e-ccfe9453bf87","order_by":3,"name":"Gabrielle Bailey","email":"","orcid":"","institution":"Northeastern University","correspondingAuthor":false,"prefix":"","firstName":"Gabrielle","middleName":"","lastName":"Bailey","suffix":""},{"id":452770212,"identity":"184728fd-fc94-4893-abaf-46554c0c2215","order_by":4,"name":"Trevor Callahan","email":"","orcid":"","institution":"Northeastern University","correspondingAuthor":false,"prefix":"","firstName":"Trevor","middleName":"","lastName":"Callahan","suffix":""},{"id":452770213,"identity":"58309f52-afd8-4410-abfa-6eed24d4370f","order_by":5,"name":"Ajay Sharma","email":"","orcid":"","institution":"Northeastern University","correspondingAuthor":false,"prefix":"","firstName":"Ajay","middleName":"","lastName":"Sharma","suffix":""}],"badges":[],"createdAt":"2025-05-06 15:08:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6604448/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6604448/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82178705,"identity":"2aefd892-9029-410e-a207-50e2f0af2594","added_by":"auto","created_at":"2025-05-07 11:26:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":130094,"visible":true,"origin":"","legend":"\u003cp\u003eAverage growth curves (a) and cell density (b) over thirteen-day trial. Data are expressed as the mean ± standard deviation, n=3\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6604448/v1/ad882458d6ebafe7f9e372e0.png"},{"id":82178706,"identity":"31da2b55-9028-4cea-ae50-4bc8d1179093","added_by":"auto","created_at":"2025-05-07 11:26:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":68817,"visible":true,"origin":"","legend":"\u003cp\u003eLipid quantification assay results for the four experimental groups on day 12. Individual data points are plotted (n=3) and error bars represent one standard deviation p \u0026lt; 0.01 (**), p \u0026lt; 0.001 (***)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6604448/v1/a1beedd002dd89b14d8cffce.png"},{"id":82179247,"identity":"dda66246-5168-47c0-8f38-1edbac08c410","added_by":"auto","created_at":"2025-05-07 11:34:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":555541,"visible":true,"origin":"","legend":"\u003cp\u003eDrop streak test showing bacterial viability one day post co-culture inoculation (a, c). \u003cem\u003eE. coli\u003c/em\u003ecolonies (a) were visually distinct from \u003cem\u003eA. chroococcum\u003c/em\u003e colonies (c) and were grown on different agar medium. Bacterial presence in both co-cultures was confirmed once again on day twelve with brightfield microscopy (b, d). Scale: 100 µm\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6604448/v1/8a807218ed63784ae25da548.png"},{"id":82179254,"identity":"cc71e6fd-b374-441a-ade5-6fd86ec79709","added_by":"auto","created_at":"2025-05-07 11:34:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":521011,"visible":true,"origin":"","legend":"\u003cp\u003eCy3 fluorescence microscopy images of Nile Red stained \u003cem\u003eC. vulgaris\u003c/em\u003emonoculture (a) and \u003cem\u003eC. vulgaris-A. chroococcum\u003c/em\u003e co-culture (b) Scale: 20 µm\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6604448/v1/c37d6b8c647178016ae280bb.png"},{"id":84164382,"identity":"bc6ed8cf-4359-428e-a3a5-9a3c5ee9780f","added_by":"auto","created_at":"2025-06-08 15:31:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1787254,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6604448/v1/2ec12c19-2a62-4d43-ad2a-da527a91f8a6.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eCo-culture of Chlorella vulgaris with nitrogen-fixing Azotobacter chroococcum in nitrogen-deplete medium to increase algal lipid content \u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFirst generation biofuels, typically derived from edible terrestrial crops, have significant sustainability limitations (Elgarahy et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Asase et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Several of these can be overcome using microalgae as a biofuel feedstock (Lum et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Microalgae grow significantly faster than terrestrial plants, fix more carbon from the atmosphere, and use less cultivatable land and fresh water. Many species are also rich in lipids, the precursor macromolecules for biofuel (Chisti \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Halim et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). These are in the form of fatty acids such as oleic acid (C18:1) or linoleic acid (C18:2), which are often bound to glycerol in a triacylglycerol molecule. Such lipids can be turned into biodiesel through simple chemical synthesis. Despite these advantages, the production of microalgal biodiesel does not yet have a high enough yield to be a viable energy source (Bondioli et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Khoo et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNutrient deprivation is among the most widely explored methods of increasing lipid productivity in microalgae populations (Praveenkumar et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Gao et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Proch\u0026aacute;zkov\u0026aacute; et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). This approach forces the microalgae into a stressed state. Under unfavorable environmental conditions, the microalgal metabolism shifts to lipid production to store energy. Deprivation of key nutrients is achieved by reducing the amount of certain organic compounds added to the growth media. Depletion of nitrogen has emerged as a common and effective target for increasing lipids (Bondioli et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Manipulation of phosphorous, sulfur, and heavy metal levels also shows potential (El-Agawany and Kaamoush \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Saber et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, nutrient deprivation stunts the growth of the algae, as carbon is focused into lipid synthesis pathways rather than cell replication (Yang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Chen and Wang \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This directly reduces efficiency of this method of biofuel production, as the growth phase of the algae does not overlap with the lipid induction phase.\u003c/p\u003e \u003cp\u003eNitrogen is an essential nutrient for algae that promotes lipid, carbohydrate, and protein synthesis (Chen and Wang \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Nitrogen levels are positively correlated with biomass accumulation and inversely with lipid metabolism (Chen and Wang \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In microalgal media, nitrogen is often added as ammonium chloride (NH\u003csub\u003e4\u003c/sub\u003eCl), which becomes bioavailable as nitrate (NO\u003csub\u003e3\u003c/sub\u003e\u0026ndash;). Nitrogen starvation decreases the photosynthetic thylakoid membrane content of cells, activates acyl hydrolase, and encourages hydrolysis of phospholipids, all of which work to increase the intracellular content of acetyl-CoA (Yaakob et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Acetyl-CoA is the foundational molecule for lipid biosynthesis (Chen and Wang \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). When nitrate levels were reduced by Yang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2018\u003c/span\u003e so that a \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e population were put into nitrogen starvation, biomass accumulation was reduced by 31.7% while lipid production was enhanced by 93% up to 113.46\u0026thinsp;\u0026plusmn;\u0026thinsp;1.78 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLipid productivity has also been bolstered by co-culture, which can take advantage of numerous biochemical pathways (Ray et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Tong et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Co-culture models can take the form of microalgae grown with other microalgae, bacteria, or fungi. \u003cem\u003eEscherichia coli\u003c/em\u003e is a notable microalgal co-culture partner which has demonstrated several synergistic interactions (Tong et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Literature has shown that \u003cem\u003eE. coli\u003c/em\u003e provides CO₂ and essential growth factors and concurrently receives oxygen and photosynthetic byproducts in return (Tong et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Kang et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This metabolic exchange not only supports mutual growth but also enhances lipid accumulation in algal cells. Yamada et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e reported that a \u003cem\u003eChlamydomonas reinhardtii\u0026ndash;E. coli\u003c/em\u003e co-culture significantly increased lipid yields compared to monocultures. This improvement was attributed to enhanced carbon flux and reduced oxidative stress in the microalgal cells, creating a more favorable environment for lipid biosynthesis (Yamada et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCo-culture and stress induction can also be implemented synergistically to increase lipid production. A study by Xu et al. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) investigated the impact of co-culturing \u003cem\u003eC. reinhardtii\u003c/em\u003e with \u003cem\u003eA. chroococcum\u003c/em\u003e bacteria concurrently to nitrogen deprivation. \u003cem\u003eA. chroococcum\u003c/em\u003e is nitrogen-fixing: it converts atmospheric nitrogen into ammonia, a usable form of nitrogen for algae (Xu et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). When co-cultured with \u003cem\u003eC. reinhardtii\u003c/em\u003e in a nitrogen-deplete media (N- TAP), \u003cem\u003eA. chroococcum\u003c/em\u003e released useable nitrogen into the liquid media (Xu et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Most importantly this led to increased lipid content, but growth rates and therefore biomass accumulation were also positively affected. This co-culture had a maximum lipid productivity \u003cem\u003e19.4 times greater\u003c/em\u003e than the control group of \u003cem\u003eC. reinhardtii\u003c/em\u003e in N- TAP media. These results suggest combined co-culture and stress induction could be an affordable method to dramatically increase lipid production while limiting the biomass disadvantages.\u003c/p\u003e \u003cp\u003eWhile the \u003cem\u003eA. chroococcum\u003c/em\u003e co-culture model was shown to be successful in \u003cem\u003eC. reinhardtii\u003c/em\u003e, its low baseline lipid content makes it an inefficient choice for a scaled-up model. Mata et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) suggest \u003cem\u003eChlorella sp.\u003c/em\u003e could be an effective choice for scaled-up production. \u003cem\u003eChlorella vulgaris\u003c/em\u003e is a green microalga with a fully sequenced genome (Cecchin et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It has been the subject of numerous papers relating to biofuel production, including those pertaining to scaled-up systems (Ahmad et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Parashar et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Al-Hammadi and G\u0026uuml;ng\u0026ouml;rm\u0026uuml;şler \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). It can yield high quantities of lipids, with a dry weight that is ordinarily 14\u0026ndash;40% lipids without any modification. Although, even higher numbers have been reported (Gouveia and Oliveira \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Mata et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). This contrasts with \u003cem\u003eC. reinhardtii\u003c/em\u003e, which has a lower dry weight of approximately 18\u0026ndash;25% lipids (Scranton et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Enamala et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Evidently, \u003cem\u003eC. vulgaris\u003c/em\u003e could allow for faster lipid yields and higher maximum lipid content.\u003c/p\u003e \u003cp\u003eThe purpose of this study was to determine how \u003cem\u003eA. chroococcum\u003c/em\u003e co-culture influences lipid accumulation in the naturally lipid rich and industrially relevant \u003cem\u003eC. vulgaris\u003c/em\u003e. Experimental design consisted of three replicates of each: C. \u003cem\u003evulgaris\u003c/em\u003e in nitrogen-replete media (N\u0026thinsp;+\u0026thinsp;TAP), C. \u003cem\u003evulgaris\u003c/em\u003e in N- TAP media, co-culture of \u003cem\u003eC. vulgaris\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e in N- TAP, and co-culture of \u003cem\u003eC. vulgaris\u003c/em\u003e and \u003cem\u003eA. chroococcum\u003c/em\u003e in N- TAP. This work and the results described herein improve the understanding of microalgae-bacteria co-culture and represent forward progress in enhancing the viability of \u003cem\u003eC. vulgaris\u003c/em\u003e derived biodiesel.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e \u003cem\u003eChlorella vulgaris\u003c/em\u003e (15-2075) was obtained from Carolina Biological Supply. \u003cem\u003eAzotobacter Chroococcum\u003c/em\u003e (43 [NCIB 11694]) was obtained from the American Type Culture Collection. \u003cem\u003eEscherichia coli\u003c/em\u003e (MG1655) was obtained from collaborators at Northeastern University. Nitrogen-replete tris-acetate phosphate media (N\u0026thinsp;+\u0026thinsp;TAP) (Gorman and Levine \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1965\u003c/span\u003e; Bono et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) was used for maintaining the microalgal stock in a T50 culture flask at 28 +/-3 ℃ under a constant light intensity of 898 \u0026micro;mol photons m\u0026thinsp;\u0026minus;\u0026thinsp;2 s\u0026thinsp;\u0026minus;\u0026thinsp;1. Cultures were agitated on an orbital shaker at all times. Stress induction was achieved with nitrogen-deplete tris-acetate phosphate media (N- TAP) (Bono et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Lysogeny broth (Chapman \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1945\u003c/span\u003e) and Ashby\u0026rsquo;s Mannitol Broth (Ashby \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1907\u003c/span\u003e) were used for suspending the \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eA. chroococcum\u003c/em\u003e cultures respectively.\u003c/p\u003e \u003cp\u003eCulture conditions\u003c/p\u003e \u003cp\u003eAlgae cultures were grown over thirteen days that were broken into two phases. On day zero, \u003cem\u003eC. vulgaris\u003c/em\u003e stock was split into twelve T25 flasks at a concentration of 1.0 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. For the first phase, the cultures were all grown in N\u0026thinsp;+\u0026thinsp;TAP for five days. On day five, all cultures received a media change and the cocultures were inoculated. Except for the N\u0026thinsp;+\u0026thinsp;TAP control group, the media was replaced with N- TAP to begin stress induction. Three flasks were inoculated with \u003cem\u003eE. coli\u003c/em\u003e while three others received \u003cem\u003eA. chroococcum\u003c/em\u003e. Bacteria were inoculated at a 1:10 ratio to the algae.\u003c/p\u003e \u003cp\u003eAnalytical methods\u003c/p\u003e \u003cp\u003eGrowth was tracked using optical density at 750 nm. A standard curve was verified with cell counting on a manual hemocytometer. The samples were prepared with strict aseptic technique, read in a 96-well plate, then discarded.\u003c/p\u003e \u003cp\u003eFor lipid quantification, lipid standards and a 1 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Nile Red stock (Sigma-Aldrich, USA) were thawed at 37\u0026deg;C and protected from light. Microalgal culture samples (1.2 mL) were centrifuged at 4500 rpm for 5 min, and pellets were resuspended in 400 \u0026micro;L of N- TAP medium to standardize assay conditions and avoid lipid induction. Samples were then normalized to 1.05 \u0026times; 10⁹ cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e based on the most dilute sample. An opaque 96-well plate was prepared with 80 \u0026micro;L TAP-N for standards, 100 \u0026micro;L for true blanks, and 90 \u0026micro;L for Nile Red blanks. Nile Red was diluted to 10 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in DMSO, protected from light, and 10 \u0026micro;L was added to each well (excluding blanks) using a P20 pipette to avoid crystallization-related errors. Wells were mixed by pipetting. The plate was incubated in the dark at 37\u0026deg;C for 10 min. Fluorescence was measured using a BioTek-Cytation 5 plate reader with excitation at 530 nm and emission at 590 nm and compared to a triolein standard (ThermoFisher, J62419.14).\u003c/p\u003e \u003cp\u003eFluorescence microscopy was performed on the Olympus BX53 microscope. Nile Red stock solution (1 mg/mL in DMSO; Sigma-Aldrich, USA) was thawed at 37\u0026deg;C and protected from light using aluminum foil. A working solution of 2 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Nile Red was prepared in DMSO immediately prior to staining. For each sample, 10 \u0026micro;L of culture was combined with 10 \u0026micro;L of the diluted stain in a microcentrifuge tube and mixed by pipetting. Samples were incubated in the dark at 37\u0026deg;C for 10 min before 2.5 \u0026micro;L of the stained culture was mounted on a microscope slide and covered with a glass coverslip. Slides were visualized with a Cy3 filter set. Transmitted light was blocked manually, and the reflected light LED was activated at ~\u0026thinsp;10% intensity. Images were acquired using a 20 s exposure time.\u003c/p\u003e \u003cp\u003eAll optical density measurements were performed in duplicate. Lipid quantification was read in quadruplicate. The data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eCulture Growth\u003c/p\u003e \u003cp\u003eOptical density (OD) measurements were correlated with manual cell counts to establish a standard curve for estimating cell concentration. This was recorded daily (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, except on days 4 and 11). Near day 5, cultures transitioned out of the exponential growth phase, at which point a scheduled media replacement was performed switching all groups to N- TAP besides the three N\u0026thinsp;+\u0026thinsp;TAP control cultures (which received fresh N\u0026thinsp;+\u0026thinsp;TAP). This intervention reinitiated growth that continued until approximately day 12.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e displays the growth curves for each experimental group as well as the raw OD values. Samples taken for OD measurements were diluted 4x after day 8 as they were approaching the functional limit of the plate reader. The N\u0026thinsp;+\u0026thinsp;TAP \u003cem\u003eC. vulgaris\u003c/em\u003e monoculture attained the highest cell density, reaching approximately 5 \u0026times; 10⁸ cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The remaining groups\u0026mdash;N- TAP \u003cem\u003eC. vulgaris\u003c/em\u003e monoculture, the \u003cem\u003eE. coli\u003c/em\u003e co-culture, and the \u003cem\u003eA. chroococcum\u003c/em\u003e co-culture\u0026mdash;achieved peak concentrations near 3.5 \u0026times; 10⁸ cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Among these, the N\u0026ndash; \u003cem\u003eC. vulgaris\u003c/em\u003e exhibited the highest density, followed by the \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eA. chroococcum\u003c/em\u003e co-cultures.\u003c/p\u003e \u003cp\u003eLipid Quantification\u003c/p\u003e \u003cp\u003eThe Nile Red lipid quantification assay was performed on Day 12. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the results of the quantification normalized to cell density. All wells were seeded at 1.05 x 10\u003csup\u003e9\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for uniformity. Lipid densities were 1.36 \u0026micro;g 10⁻⁷ cells⁻\u0026sup1; and 5.77 \u0026micro;g 10⁻⁷ cells⁻\u0026sup1; for the N\u0026thinsp;+\u0026thinsp;TAP and N- TAP groups respectively. This was a 4.2-fold increase, or 324%. The co-cultures had lipid densities of 5.35 \u0026micro;g 10⁻⁷ cells⁻\u0026sup1; and 11.25 \u0026micro;g 10⁻⁷ cells⁻\u0026sup1; for \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eA. chroococcum\u003c/em\u003e respectively. The \u003cem\u003eA. chroococcum\u003c/em\u003e co-culture showed an 8.3-fold (730%) increase compared to the N\u0026thinsp;+\u0026thinsp;TAP monoculture, a 1.9-fold (95%) increase compared to the N- TAP monoculture, and a 2.1-fold (110%) increase compared to the \u003cem\u003eE. coli\u003c/em\u003e co-culture in lipid density. The \u003cem\u003eE. coli\u003c/em\u003e co-culture performed similarly to the N- TAP monoculture, while the \u003cem\u003eA. chroococcum\u003c/em\u003e co-culture outperformed it significantly. Samples were compared to a triolein standard. The standard curve used 4 points on a graph of log-concentration versus fluorescent signal.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDesign Verification\u003c/p\u003e \u003cp\u003eAs described above, microalgal proliferation was monitored via optical density (OD) measurements at 750 nm. However, bacterial growth proved more challenging to quantify directly due to their relatively smaller size and lower biomass contribution compared to the algae. This rendered OD measurements insufficiently sensitive. As such, a supplementary viability test was used to confirm the presence of live bacterial populations within the co-culture systems at several time points.\u003c/p\u003e \u003cp\u003eBacterial viability in the co-culture groups was assessed using a drop-streak test (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). A 20 \u0026micro;L drop of culture medium from each experimental flask was applied to the surface of a solid agar plate\u0026mdash;Luria-Bertani agar (Cold Harbor 2006) for \u003cem\u003eE. coli\u003c/em\u003e and Ashby\u0026rsquo;s agar for \u003cem\u003eA. chroococcum\u003c/em\u003e. The plates were tilted, allowing the drop to streak downwards. After 2\u0026ndash;3 days of incubation, the appearance of colonies indicated the presence of viable bacteria. Colonies of \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eA. chroococcum\u003c/em\u003e are visually distinct with \u003cem\u003eE. coli\u003c/em\u003e colonies appearing opaque and cream-colored and \u003cem\u003eA. chroococcum\u003c/em\u003e colonies appearing transparent and gel-like. One droplet streak was performed in triplicate (on the same plate) for each co-culture flask on day six post inoculation. The presence of bacteria was verified again on day twelve with brightfield microscopy (40x magnification).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFluorescence microscopy was used to verify lipid quantification results (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This revealed a stark contrast between the \u003cem\u003eA. chroococcum\u003c/em\u003e co-culture group and the N\u0026thinsp;+\u0026thinsp;TAP control group. After Nile Red staining and a short incubation, the neutral lipid droplets in each sample fluoresced under the Cy3 filter. This allowed for the lipid content to be assessed in a visual, qualitative way. The N\u0026thinsp;+\u0026thinsp;TAP group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) showed weak fluorescence with individual lipid droplets being visible while the \u003cem\u003eA. chroococcum\u003c/em\u003e co-culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb) fluoresced so brightly the contrast between lipid drop and the rest of the cell was indistinguishable.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eStatistical comparisons between all experimental groups were conducted using one-sample t-tests in MATLAB (2025a). A post hoc power analysis was performed using G*Power (v3.1.9.7) to confirm the adequacy of the sample size and the robustness of the findings. Effect sizes were calculated and reported as Cohen\u0026rsquo;s d to indicate the magnitude of differences observed (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Results with p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant. Statistical analyses confirmed that the N- TAP \u003cem\u003eC. vulgaris\u003c/em\u003e monoculture, the \u003cem\u003eE. coli\u003c/em\u003e co-culture group and the \u003cem\u003eA. chroococcum\u003c/em\u003e co-culture group both displayed significant increases in lipid content compared to the N\u0026thinsp;+\u0026thinsp;TAP \u003cem\u003eC. Vulgaris\u003c/em\u003e monoculture control group. Each with large effect sizes (d\u0026thinsp;\u0026gt;\u0026thinsp;0.8), indicating strong biological effects in addition to statistical significance.\u003c/p\u003e \n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Nile Red lipid quantification results comparing descriptive statistics, p-values, and Cohen\u0026rsquo;s d for each group\u003c/p\u003e\n\u003cp\u003e\u003cimg 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Plt/uUw96zMUx3YVbNm0z0IyP5ZdarrQxqMJwxOVzcF3dTizo+6xursd1+wuamR/Jg4s/ZpapuV4J5o8vOJ7asCACzdcK8r9dDqch1Z+SwD08A5g9erVGBQUVPwK8K/EoZP/eP/75KSZHNaxKl38mvPe81RlWlYUezVwZ8X6g1hA8uKm6TTRNeb0VUcoLWpV3zi4lGZaupy/SdnYTQg/Qmd9PS7eEUKSPL7mB+ob+vBmdFnftoyLh3WipsSQX/w0m4ePHuLwAS2oo2nAuRsukSS3zB1MLS1dtu8/kvsOHeXS6T/Q3lCTddqMYOq7TuB5cRzUKoBaJg4cM3stDx/Yye5NAqht6MZ9l1+RhU9ZxdGA38zep1Js64yuNLNpzOhCFnc463edSZIMvXqQ9b2sGdTkK548c4HZheSmuf1pXa46nxZ9ZSSZG3mJ5c1NOW3dOZLkvUOraaEJOrjV4JK1G7lq5e98lV7A479NoKGuLtv3Hc59R47ylwUT6GZhwIbdJzLrfzgevnXuQOro2PLAtWiS5PJR7ejk04wxpdr27yPlyRn6WBmx59DlzC7qA764up22+vqctPosSXLJ0BYEdDhkwlrGpSnrsFN/jqGhoRPPPMwhSe5c8g0BsN3A6UzJlFFO8tmNXSxnasS+I1YyLU+588WEHKW3jSH7jVF2ZnLjQtmgvAVrtvmBUYnKbeUlP2SrSg6s3no4lc3SLE7oVYOOFZrxRniS8kNK4/lF3fL0bTCI+SS3zBxAA2Mn/nnwjjKfCu5ZMYz6OtbcfSmWJHlk1UgaGdhwfamYjTP7UNvAg7diZMx+eZF+Nsbs+sMSZha1IyJu7KaDgR7HFXUmF3zbgI4+HZhY6kbFeydX0lLPlgeuKT/b6rHtCGiw//CljEstIJnD75r6ETDg0Cl/KAd3CyPZuIIFmw5YUNzZVZXPmYNbUFPDlF+PXsjDRw7wu14NqK1lwuW7bpMk9ywdRh1NHbbqPYx7Dx7hqrkj6WSuyWpNBzNe+VWWTZbEoZ2DKdGz58jpK3nk0E5+0bIadXScuOPMU5I57BPkxJYDlqoUO7NlEo1NnHn1ZakOZ9OxVJB8dP0UWwTa069Gbx45c5HpBeSVzTNoaeHDm48zSjaS/5y13E34xaQtJMknpzfQSh90cAnkwlV/ctWKtXyZks/Q42toY6jH2m2/5Pa9R7j51/ms5G5O/1r9GJ1Z9jf2IUSH838qhb3rutMzeBBzSgacqMhL55zhfdm8ZWu2bduW7Tt0YO9+P/BRQgHJOLap4kCXgB5MKVVm2/S+1Nb34ql7pTqT0gQObOrDcpU7M1lKRt7aRHNdfW6+ULrDmcnBzcuzbrspJMnchPsMttKiV60+jCt1vtsydyD19D149n5RhSN8Fm4cWkhjTRuevB3PPQu+opNXQ75MVua9uPAr3V1r83FiFud+UV90OD9IPi8d3cTuLWvT2sKM2poSaujos2rdttx46HrxyfAvO5yZD1jFwYDN+y5Sz/oMKTucjXvOI0k+vbyV1tpgl+9/poJkVoxqhzPp0Sl27diGbdq2Y8sm9WlqoMuAoAbs0L4dmzdvxrW7X3e8Pl0Hln5NTRNnXo8gKU/ggBYVaOxYkyFROSQVnPaJdDhn9q1LQIv2Dva0s7Mr9bKlhaUdJyw+rF7krZ5e+oPmOoZcf1T1SuTT+5d58Pgl5mXEsHWgA1t+MV91IY/8dPYKdmaLPspZRTd3TqWenjPPhitXCJr9RQO6BfZmehl1acqj43Qy1uW305WzmpSyOOGLZuzx0yqmx95keQstdv1plUpD+saehTTWMObyHfdKpaq6dfBnGmsZcPXe11daSFnqM3ZrGMzxPx8hC1+ymrMhh8xR7XBum9mDlnZNGKPS4ZxRlJvDATXc2Wbg8uL4TXMH0MalOp+pdDgv08fSnDN/O0+SDD2ylgYAJ649UxwjSwljdRdL9h6uWgc+vvAbbXRMuO7IE5X0jyXjxSX6Weuww+CSzsqx1WNpYe7Hu8+y1aLfbcWI1rR1b8HI0heOZFk8dXgvb4RFkcxi3+pOdPbvwlelDr79K76ltokrLz9VjiBM61ON+jaVeD/qdaNQxkndg+kV3E+lnUjKOK1PPVaooxwQ2bXwS5pYVOG9aNVrs4d//o6mFjX5NF3BmFs76GhoqrJfkGREyDUePH6NaRHXWNHGlN/P2q2Snx//mDWczfjj7KMkY1jP05w9hq1ViclMecE9O3czKUvKX8Z1oI1LE74sdeWb8myeObqP10MjSGaxZ1UnNu0xv1QAuXtRf5raVuWTNJLM5beNvWjh0pBPk16PQCSxe1A5egT1ZsrrAZfMJ6zpYcROP60q2VApcXf30Epfl6MXHypJlKdyePcGHDD2D+amPGSQnR7bfLVYOfOwSOixVTTW0uP8jVdKpap6cPIXWmoZcNHmkvOKIjuS/ZrV4PB5u0jm84tglzcGj89unUxT03K8VqrDGdRkTNEAWSG/b16ejTrNKY6/vHkWra18eetJqZ0r/wXreJiy/2Rlh/Pp2Y00Bji0dL0nT2S36o6s322iyhX75CdnWN7SiJOWlxyT/ylxD+f/lA7MDQyQl5aOPLWp2do6OtDX1YehgTFiHlzFjp17kZAlBSCBQq6Aa8UAmBWvwJ2J0xduwqdGc9T0syvZiJYNunRqh9THYXgZmQdNrb++nZgkFBr6aNeuJ+z0S9JbtG4HI7zE1dDP694mAQABqUwLjdu0gSwuDBfuPQUAHN57HOUCa6O8tRHkso80x/+Tp4vaLXpj25FLiIp+iUunDmHepKGQJN5A33Yt8duBEKDoHs53oUyKArkCEo2/ivycKO9H8azVA/PG98K+lZPw5/Fn0NbWVg2TaEJXVx/6ugbQ1dEBQGhq6UBPzwD6uvrQ0vzrevNTQYUC0LDBrPnzYZ5+E6PGLQYhKd7/NAAUpD3D/KkTMHrMGIwdMwajx03BtdCPs2DKf5UiGpdvPUZwm9GIiH2F2JgYxBS/YpGYEIMZw1qpl3qry4cPQ9umBhrXdFVJ96xYC22a1Ub808u4F56Dbt06qC7koSGBrr4mCnJzAQAh1+/DwjkQPvZmABNw9f4TeFX1g5H683wAPAq5jcxCG3RuVb9UqhFm/nEMW38egpiQa4hNM0Snrs1U6oxqDVvBu5wcV+7cRWbcY8ycPAajx4zBmDFjMHbCFITH5ePR7QuATQU0aehfXE7T3APbTl3FzJ9aAoUFb9xr9tfkUFAB8u+dH0g5dDSsEeDlUpwWGf4I0bGpeBZyGF9//RW+/PJLDBw0CLNXH0BOYTZuXglT2cbHIcVvS+fjQVIhclPCMXniBIyfOBFbj19DZloKXib8jcUdZbE4dug6ajfriHKlV6nSNELjVh1Qzc8ZBUkPcO1pAtr17Af7Uu2x+zdDYGHpC3dXbYBxuHUrAg1a9kNFZ2WjUJESit3nHqJ1t+6wUHlSiyYMdXUhzS+ETJqJQ7tPo1qLrgh00isdBF0DbSgK8yGXy3D9/GFkGnmiYf2S/QIAXAKC0aZZMB7dOI34fAt0bdtIJV9DSwPa2hLI5IWIunMV4bEaaNeqgUqMsYUbOnbpBCv9ZBw5cAU1mnaEa+mFsjQM0bBFe1T3d0FB/APcepGIgFoBpQKIO1dDUc6zJtzMAORH4d7jeDTv2BeeVspFzgrjXuBOZDwatusAi6KqPzEyEjGxMlSu6FtqWyXu37qJQokzOjWvVZKoYY5FW8/g9zlfICHsEh4mK9ChSxvloopF/Bs0RyU3XVy+8QDSnDgsnDYRo0cX1ZNjJ+POo3g8CL2CfAs3NGsYXFxOYlgOfxy9goWjOgMo/IDjDFAQUPzt40wBDRgjsGJJHZYb9RghkUlIiLiBH7/+Gl9++SW+HDgIY2atRUZuNu6EXH3jvtsPJTqc/1NGqF6tAhKjbuHRq5KbtCV6phgxZx327t2Jrds2Y0SfZqBcUerHUd8bpcjIy4OJlTm01BYStDCzhCakKJCW3Ciu3iRVb6NqaGvByslBJc3AyBBaWkROzvvcZi18ahQyKUx8GqGpvwH277kIFCbi5MU7aNquCyRFlZ3w97wMvYQtmw8gs2gRBz0DM1Rv0AojJ87G+Qsn0bi8Ar/+tgUyAJK/WP43LToKSRn5cPUuaaAJJb4YPgdtqplg0qiReBxfCG1NSXEtauXdABu37sCOnVuxYfV8eNqY4esRi7F121bs3b8PX7YPUtvap8/evw0WTP8apzfPxJqdV6Cjp+z9SADkpb/AssULsGD+fMybPx8L5i7GjftvLpTzT5Mb+QKP4pIQUKMqdABoaGpCU+317qOstEI8ehwBhwq+sDRSG8AokhD7ElkaunD1VD0m87Oj8TA8BW5+FQDk48rdB3AL8IWlEZAbHY0XUdmoEli5zNUmc/MzIYcWdHTVn/eo/OR5OfmQaJjA0tBQNdvAECaGeiiUAukR97Bs0XwsmD8f8+fPx7wFi3E/MgeFuTnQ1tSHjtqmJRolAw4ooy6S/M1WI99ov6B4dXOVHEnxfwAAmRlJyJVpQFcDyM/PQ0FBAfLzcqHQMkPvwd+ibiX70qU/ipi7B7Hi90MAgBO7fsXMWbMxZ9Ys/LnvHGSIx9PI9z8O8tNi8SQhA75VK6pnKVeIA/Dq8QMk5Zigds1SHSPG4/rNZ6gQVAd2WkD+q3A8iMtFvea1i0My4mORkaWDCj5OJeUAQJGEO+HhsHVzhpY8E09epcDTr7xqDICQe49h7OQCa0NtvHj4Ag7lqsDZXD1KKTLqJSSmFrB3NFNJT058jsj4Qnh5uyIuMhwyPUt4uJV9fipMf4XHcenwrfL27yLm8UMk5RgjKNCjJK/wJS7efYGKtYKgDSAz6jmepytQrU5Jp/TVi2dIS9NHcGWf4rTIF2HIlJuiku+bfzsA5ORlQaGhDW0d9cHJ18dZNkBjWFqodtShawRLA0MU5hciL+MlVvy8EAsWFNWT8xbh6t1YyPNyAC0tvDHuqSFRPQ7U2+VlPDf4XcrstEokbxy/6jLTs5CXI4OulgT5+fkoKChAQX4e8hU6aNfna7SsW7GMI/jD/L2/SPiPNejYAzaMxKoV29/6SAMtDQmoUk2r7zBGcLezRsyjcOTkqeY8e/YIcn1TWJoqT0CUKZCekV0SUChFSmIWNEr1OhWFUsQ8f14SAyA2MhZ5+abwLGejki58HpQj1EZo06kJ7pzZiRMnDuJhnC3atyk92ij8HU+v7kGfPv1x6EaUehYMbFzh6eKAzLRM5Wji68PzLSeLQ3t3IKXAAe2a1FDPEgBomJTDvAUzIQ0/imETliJLrgGNsr5LUrmKZpln689Lt28moWcDJ0wbPQxnw+Kgo6UBBQBTp1o4c/0uQu/fx/379xF6/zr6tKmgXvwfJ+LpE6SlGyKosqd61gehgoBat/DB0V9RLagFwl7lQ1dHG1QQ0kLV6wG3j+3CvURttGoVDGTG4tHzRPj7KxvMES9CkZClgyoBfiplXrOxcoCOIgvxaWkq6QdWjEKr7pOhaW0NTY0EPIxIUMnPi0tAbHwOrC2N4BzcERdu3i/+/e7fvY621SxhauuIvJxEpKSW/ry5mPN9F3w1fjWkEj1oSSRIS8sqlQ8kvUp/cwy8FAKQlOqVampoIj+vALm5JYWy0nORk5Wv0g5RZ23lACNtLbTtNxwbNmzC5s2bsXnzFqz/ZRW+G9gX9euW0Vn5r8rCslmzkGpUE8cu38PDB2G4f/8+wsIeIfTmfvha6uJ+aIR6ob+kWg0psHRYV3TsvwAA8DjsATTNyhU9LkcpP+oJQl4lo2qdygCAqAcPkSm3QVWfcsUxGhoSQCJFYaHqBYKYe5dx/mYimjRqCR09ApCgoEB11dqCpDDsOnIZ1Rs2gIUuIFfIIVFIVDoH4ZfWo1blRrgdlQs9HS3IZQrI1Ba/PbN7MxK1ndGwvi/khTKQErBU27Ug7iHa16uJFTtuQUu3qPel8l0QK0f2QPu+cwEAYXfvQsvMBf6uJZ3oe2eO4cbzbFQt6ky+fPICUok1/N1LYp6G30ehrhV83Z1L0h7dhdzcGm6eZQ9S2Nk4QFOagYSMDJX0bQu+R7u+s6FtVw46mql4+FB1gEGe+ApPE5NgY2sKE4caOHn1DkJDX9eTN9G/UyCMLOyBzGykp5asMgzIsGxkL/QbsQyENrQ0gYxE1RXnM5Pikaf+Fb1G5exESalOqYamBIVZOcgs1SlgVjrScnNUjkd15jaW0NHXRPWWX2HDhg1Fx9lmbFr/C4Z/9xWa16+pclX3P/H2TyF8FM5V2mHWhC+w/ecf8d341YiKTyu+WpSblYaQ6yewYd8Z6JtYwUDnbT+zLtp16Yi4sH1Y8tshvH6E3Is7h7Fo9Q741WkGN3sNGJjawFpLisunz6IQAKDAiT2rcOBOHHS1lD+9RCKBpqQA2zctx5WHypNWXnok5i9cAkO3GmhU/Z/fsBA+ngZNO0I3Jwxjhs2Gf5v28LYpY96X8F5qteuJYFdi6NeDcODsHaRm5EAqLUR6cjz2rJ6HLaeeom3H1tCF8mQCAHKpFFICUmkhCgvykZoUi72/Tseo2ZvQsv+PaFRNdWaCUKJ8vX6YNqYrzh7ai/isfGiW0bjV1DGAp48frMzURq4/RwZ2mLVoAcwy7uD4lWfQ0tJSdh60jVC+gh/8K1ZExYoV4V/RFxam//zv6+GDm8jSMIFOfjbCnzxEWFiYyut+WBhSMgsAALnpCbgfFobUTOWZ8k06qFytIp7fuYgHL5QNw8zYUIwePxP6LkEo76AHl/LV4GCQi13b9xX3xyJCj+OnMYsQ1G4w2tZwQMzLMEQnyFG1SiUAwNOQa8g3tkMFX7UrU0X8ajREBcd8rFi+Fgnpys5E8otrmL14HWBoAv8aLVC/ohVWzJ6HR6+Uj46Q56di5c+z8TzTCq0b1oREQxc+vhWLf7+Kfr7Q0QSCG7WHRX4kVq76A9lS5Sd+dH4/1m48AkMzJ2gbWMDV0hj3LpxFXLaydxFx7yhW7zkHTa2yr/ICGtDSBFLiXxVPw3Owd4Q0MRJXb9wDAMgL0rHut2WILlTOPHgbR/9gNKhuh0UzZuHa46IOtTwXmxZ9h0rBjXHm3t+YzvoBHp3ejt/2huCbsZPQvFYgKvj6oWLFivDz84F/UCMEedjj6d0w5W9NBWKeP8GTZ1FvfSSTnpkLqnjY4MLRE1DudcCNI2sw89fTqN2iofLfd2/B1MUddg4l82mfPXiAzBwL1KjkDQAICw2Bno0T3J1LbqcydfdDJTdt7N66DzlFHyA74QFG/DgCWh5N0b9nbQD2aFDTFyd3bkdEmnI/lxckYc7YYbib4YifhvQDAPgEBiDyyVncDE8HAOQmP8L4EVMAh6rwL2eAipVrQSPtBQ4euVT8/vdO/YGx87ej89ejEWijBWf3itCWxuPCpZvKAFk2VsybgEvPgfq1A6Fh5ICqnra4eOwE8ooOltvHf8W0tcdRs7lyGu7L2JfQNrOASVHdHHHvBEaOmY18bSdU8lJOCQ1/ch/a5vYoZ19yYeTJo/vQcXCEk0vJXN1Hdx/CziEILm+5flKpbhO4W6Ri2fLfkZKt3HPjHp/H3CV/QtfMAp6BTdAiyAVrFs/E/Ujl96IozMDqn+fiSZIxWjWpCUADXj6+8PcvqScN9bQQXL8trCTRWL72N2QVKv/YZ9cPYMn6A9A3sYcEenAoZ4mHoecR9krZWUyNvIUVq/ZBoj6F8TUJoKUpQXLiK7zuXlrbOwCyWJy6fFuZoMjF5l/WIeyVFNpF7f2y6DoEolvTati4dCqOXntRlCrD4Y1zULlSDWw/+VCtxH9A/aZO4X9Ans3NS8bQ0cKINg4ebNKqDTt0aMOq/l7U09Fh1bpdeeTSQ2WsLIZN/G1Yu/ss1dX0pFlcNLQz9XUMWKVGY7Zr3YS2pgb0rtaWN4sWI6Aih3OGtKIE2qxSqwlbNq7NKtUasmGdSqzfbjxJMjf+Pms5mtDB2YvePv5s3bYdA8s70MTGh5uOqN40Lnz6ru2bRz1Yct8F5Wp8ZCr71/MgYMS1u0seHTG1a3XaundmUhkLXQhv9+TqHtbytiWgQSd3P9aoUYOu9ubUgB57fDeXKUV37edGXGKAgyYNLN0YXKMWg2vUZHC1IDrbmhISLTbs+BMjVFeH+Iy9YksPS9brNls9g9LMZ+xY3Y0AOGfz69UShf0/f0kNE2defv7mGq3bF39HAPSvM+RfvGhQASf3qsKiC25lvzT0uPHcS5Lkxsk9CYked18reTSEurzkB2xXw5POXkHs2bsnA9xtGVCvGx/Gvv6W5Ny1bDgtjK3YpHVn9urWga425qzV+is+KVoB5uT64dQ2duXVyEKSCo7vFECPygP5rsVOr+1byXIW+iznWYXt27Wmq50xPSp1YFi0stSza7tY0cmcFg5ebN2uI2sEuFFX34aTlux/y4qcr0m5eeGPNNXWZoWqddm2VWNaG+mxQZehTChaC+fkn5NpoiGhi3d1tm/bnP5+VdmxU1M6OjVlVCFJ5vObeh6s1XFa8VaXDWtNaOqxWaf+vPk0h/K0x2wf5EJNHSs2bdWOdaoFskmr9vR0cOP0dadJkncPLKMRzLnl5LPi7ZDky1v7GOhuQUNzR7Zo24H1gnypq6XPgaPXsuDdf9x/JjeG3ao70KliO0aUXtSmlLkDa9DKoxFfSUl51gNWt9Kjf72h73xUyu2jK+loacZajdqzW4eWtDU15VcT1ymfBpCbwI6VrNmi72KVMutn9KKpXVU+jCPJfI7uE0zf+m/uM3ePrqWbrRmr1W3NPr26M8DTge5+zXjhfsk+nfjkNIM9HegdUIe9+/Rm3SretLb34Z9HlCsmk2RB6nP2qO1DR/fK7Nm7B6t4O9AnqDMfFu1vZC6XjOxGM1NHtu3cg906tKCtiTE7DprGpOIPlceFIzrTxMyJHbr1YpNagbR28OTmkyWrgd87sZbOVmas0bAdu3dsRTszUw4Yt5b5Rb/rmU0zaK5vyHrNOrFPz85s1aYLB3RpxnKewQxPIcl8jutTje7B3Vn0FBVSkcvBjdxYtenoksV95NFs6W7GVl+VvWDQa+e3LqC9qR7dfKqzfbuWdLI2ZIXgHnwar/xFI+4cYnU3C5rbe7BVuw6sVcWLOnrmHDV3q8pCQm9ScPeKMbTQ06R35Tps16YZbY11WLPNt4xNV/6xISfX0cFEg7aufmzXoS0DKwSwfduO9HLx5MXnyphR7XwZWH9k8YJkv07qQmjosWHb/rwalkrKE/l12yqUaBmwbtPWbFynGoPrtmfd8rYcMHkrSfLJqd9oBH3+elz1MWBpkdfYolI56hhYsmHzdmxWrwr1tHTYqs+k/+rCkJpTp06dqt4JFT4yiQ78azTBoEH94eNsjAIpYWRkBv/gxpg6ZymmjB0MHxfrolgJ5FKgYlBtBFV0Lbm8rqGDms07oU2DQBTmZkPX2AZdvx6FJXMnwMOu6H4OiTZqNWmHGgHlIC2UwtG7FuYumoea3rawdaqAqpU8IM1JwG/L16H+l4sxb3hjREUlIaBOByxbvhSNglQXRxA+BwroG9uhfuP6sDbTBaAPJztTWHtWRq+eHWCip9wDFXI5nHwqoW5Nf2i/ffBMUGPpVAG9v/gCdar6wszYBDZ29qhapxVmLVyGoQPbwaDUhQO5pgE8PT3g5OQIZycnODk7oVqdFpgy+2eMH9oHloZvGf38DMlkElSsXgdVSi2GAAAauhaoUqkCDEys0bBxM7jalV6t4/OlUChgbOOGxvXrwlRf9SpThUqVYKWjj8o16yE4yKfMewv/8RQFSErMgYtvZdSrWxd16tRRedWuXQv16rVFh/YNYW6gjaSEZDiVb4DOnevDWKfsv1jLwBrtOneAmSQP6TkyNOz4DX5eNBHlLF7P+pDAN7gZ2jQOQHpiPBS6Fuj/0zTMm/wdbE2VB3Z6YiJsXeugbasg6GnkIjE+G8Et26KGX9n3ugGAk0819OraEgaSAsg19dG002AsXTQF7rYGAAALJ1/07tMVtsYaKJAB3pUbY/7Py9GnXfWyp+MV04B/rRbo0KoW5Pm50DK0Rq/vJmHOxCEwL9on3APro1XjKpAopDAwc8bQSXPxRbtq0Ne3Re26VaCnIYFcroBHQHUEBbgBACpWrQkbY11ASx+BlYPh5OyMNp06wMVWD1K5JoKb9cX82SNga6iHilWqw83RAiSha2yD+o3rwdai5OqemYMPevXtDg8bQxRICUePKpgwZymGDmwJ7Xf/cf+R9PgovEiQof+PI1HDp+zLYoaGBjCwckDNajWhK0tHglQH7bp0Q2VvR/XQYvae1dC+WTDSk+KgaWyPHycvwYiBLaEFQCrNRlo60bRjJ5R3tSoqoUBqcjLcKtVH88YB0FbIkJ6Siap12iG4YsmUWgCw86yKbh2boDA9AXkKHTTr+h1W/DwdPk4lV/oMLd3QrWcnGCED6dkyBNTtiBWrl6F+pZJtaeqbo3WnjrDWyUVqtgx12nyJZUumwsX69awGbdRo2gH1qzojMT4ReublMGzqYkz8sTsMi+8H1kKtJm1R2d0cCYmpKOdbH4tXrkDjKiXvY+dRFe1b1ERm0itIDO3w/cRFGP11m+Lf1S2gBqr7OiA1LQPOPvUwdeZkVHDSh41bLTRtUBFalCM9NQuB1VujdmV35b6uyENyUj7qtGqPwKLfQVGQjeQ8CZq364TyLpbF76/OpWIt9OzUDDqKPFDLCK17fI8l8ybA2VJ5fJvZl0fP3j3haKqFfCnhGVAXcxatxJdd6vzFVFEJKlRvgs6t6wOFudDUM0O3r8Zj/vSfYGWoLGnrXgWd2jSEDmXQ0rfCFz9NwdD+raCna4jqtWvBTF8CuVwGV79qqF7FExIAvpVrwMFMBwoNfQRUCYKznS1atO8E33KWKJTKUaF6ayxYOAPeNoYo51sFfh72IAgtfUvUb9QADpZGxZ9Qz9QJXXr3QYC7FQoKZbB08MSPE+ZiysgvYPy2yQwfQMLXc7eEz1JOwn00qFgTlb76Db/O7q6eLQiCIAiCIAiC8MHe3TEXPnkEQYUCCrkYdxAEQRAEQRAE4b9LdDg/c5qa+ijn7gk7axP1LEEQBEEQBEEQhP/IO6fU5uXlQS7/bz3yU/hnIuRyBSQSjXcuUS4IgiAIgiAIgvBXDAwMVJ4n+s4OZ4cOHXDixAno6f3zl0AXPtzrB8O+Y1cQBEEQBEEQBEF4J6lUiqtXr6JixZLn5b6zw/nixQtkZmaq9FAFQRAEQRAEQRAEQR1JeHt7q1ywfGeHUxAEQRAEQRAEQRA+lLh0KQiCIAiCIAiCIHwUH9DhJOQyGRQKhXoGAIBUQCaTifsBBeFfR7mA1IccuXK5HApxzL8nQi6XQSaT40O+MioUUCg+oKDwr/L6XCp/y7n2bRQKOWQy2Tv3Eb5XjPL9Ze9YOLD4M8rf/zO+fu+y3lkuk0H+js/0z8e//M7K8l7f4/vEFLXPlG0w9bzXXtc/svevs0nIpDKU9dMo5PK/+EzCv8/r/ejv7cd/ax+Vy8usA157/3MkoVC87+dUHp9l1XvKevPN7ZAKyKTvOp6E9/W3O5w5iY/RMcgTwU0HICpdpp6Nl3f3INg3CMfupqpnCYLwD3b70Eq06vgFXmWr57zby5t7UMndH+sPh6hnCWryU2Mwb/QAuDk5wMHJDd2/moDHUZnqYW+nyMKUr7pg6uIj6jnCJyT85iF0aVINdrY28K4YjAW/HEL+u9pwAAAFLu1dg7pVfWBja4fKtVtjy9FbUC927/Q2NK9dCba2tvCt1ACrt5+FTCVIgeuHN6BNgyA4OtjBycUbvQZPwoOojNJBeHbnGPq2rQMHOxu4ewdg3Jw/kZ7/ZptA3dMru9C15bdIUc+QpWPiN+2xcs8D9Zx/hYxX9zGif1uUc7SDs4snBo/+GfEZBephb3h55xR6tqoJezs7uPtUxYwVO5EjVY2Jf3IdX3VrCkd7O5Rz88PIWb8hLUf1l016eQc/9WsDN2cHODi6oE3Pn3D3WaJKTOKz2xg5qBPcyznCzt4RNRt1we7ToSoxZSnIfIy+9TrgSsQbvxr2r/geX03erJ4s/EtlJz/DxMGd4epkB6dyHvjih1mITMlVD3tDTNhF9GvfAA52dnD1CMD4hRuRmacak5P4GONfb9vZHV/+NBcxatsuyIrF/FFfwKucA+wdy6HLgLF4HJuuElPa3aO/oFWrfniVr55Thvw0jOzTGBvPxKjn4MbhZejZbQYK1dIzYm+iW92uCEv96+9A+Av8m7Je3WMNW10C4KCJv6tn8/Hl32mtY8GdlxPUswRB+IdKCL/AYFdDOvg3Z3SWeu7bKbIj2LeRBwFdLt15XT1bKE2WwdHdg2lg6s75v+zgrg2LGeBsRu+afRibrlCPLtPuFcMoAfjD1L3qWcInIjbkIL1t9Fm5cT/uPXKc04f2pKGWLkcvfPdvfnn3Yppr6bDDgPE8evQAB3euQy0DG/5x+HFxzONzG+hopMkGHb7joeNHOHpgK+poGXPe+kultrOIFto6rNf6a+4+eIx/rppJH0cTegV358tkGUnyVchR+trrsmKtzty+7zhXzRlGSz1N9vh+GaXFW1KV8vIyhw2dxvWr5rJJrZ7cvn01x09ZwjyS2xaN5/K1f7B/57ocu3ArZ44Zy6NXX6pv4h9LkfmS3eq409ItmL9sOcQ/lk9lOTNtNu46gTnqwaUkPr3AyvZGrFizC3cdPMaF4wZQX1uH383YURyTExfKxhUs6eLbhBv3HuGauUNppafNzt8tp/LXIAuTw9m2qgOtXCtz2e97uHfbL6zhY0HH8o0ZFp9Pksx+dZ/NKlrT1jWIi3/dysMHtrJr0yrUMXDixmMPit9PRf4rTh81lr9v3shWlRvy102bOH7kcD5LIe8d/pVTZi7j3DHd2WHwHP62ZDaXrDupvgXh36TgFQc296eRfSWu/HMvN/8yhx42OqzR4kemvt7ZypARdZO1PUzpWbkNt+4/ymXTh9BMR5t9R/1WEpT7igOaVaCpQyCX/7mfm9fOoaeVLmu2+olpxZVGOsf2rksdUw/OXbWVuzetYKCbMStU68GoMtolyc8vs6abKe3KN2Z0nnpuaXn8Zfporlm3iX1aVuWM1Vs4ZfRoXg5LY3zoYQ4dP5drF45imxbfccfvizhzwWbmZUdz4vBx/HPjb2xaqRk3bN/IsSPHMTJVfdvC+/qADmcIa9gbUkdbl/omTtx+7oVK/uMrf9BWz5K7rySqpJNkftwdDuozmBcfvFLPEgTh/0n4zf1s4OdMDYCulVv+jQ6ngr9P+YKm+kYEjLls5w31AKGUx2fW0EjTkEt33CpOi76zm/a6upyx/qJKrDp5YTb/nPcjLUz0CGhy6Ix96iHCJ6GAk7+oQwfvZnyR8roVJueqUR1p5VSbD+MK1OKL5MSzczVHVms1mjmvxy5kKfy6pR8r1O3PDAVJZvK7tn70DOrNxNzXBfM59Yt6dK7QirE5JAuT2Ku2Ays2+I6Z8uKt8+XlTTTT0OTMDZdJknO/a0lL55p8nFBYHLN93kAam/nyZnh2ScFSsuMfc8IPfehub0ttLX16VajCyUt3U07y7I7VbFTDhwb6ejSxcGTTdl/z5rMk9U38Y534YzwN9W154FpMcdrN/Ytprm/BzadU20ilLf2xJU3t66l8jxtn9qO+uS9vRys7itvn96eekQ8vP0kvjjm+bjQNdB14PCSZJHnz0CIawJibTj4vjom+u5v2OnqcvlY5mLDr56+oqe/Is2EpxTHMiWWHIEcGtfmR2aV+72IFGfx98RhW9vagrpYO7R3c2O/H2UzIJl/eOcW+7evQ0tyIuoYWrFyjNbccu6O+BeFf5OquuTTRseDmU8+K0x6eXkdLPTOu2ROqElva+indaGhRiXciSoZX9i79noaGbrz4JJMkeXnXHBrpmHHrmZJ9NOzUL7TWM+aafcoBj8fn19NKx4BLtt4sjom5e4DljA05ZdXZ4jSSfHrrABsHuFEDYDm/Fox5Z4ezgAfXL2T9yl400tOiuY0DW3cbzscxuUyPuMOf+rejo4UFdXQMWLFSTS7bdJbS3DQun/kj/dxcqKOpS6dy5Tl43DKmlF29Ce/hb0+pBQipTAs9Bo1HE19i3IixiE5/v8nNsqxXOLD3ICKS/sYUMkEQPpp7x1ajWfsfUbvPDIz5si7yCtTmcr3Do3MbMPePO5g2axac9fEvv/fq47t47BT0bIPQvr5/cZpTQDM0rWKHk/vOqsSqysWcb7pi5sZHWP7bH6hsaQCpXHzXnyJZagzOX7qP2s07ws1CqyhVA+06dUB+wh1cuf9crYRSfOQtXLmXhI5du8JA+VhlQNMCnTu0Qdz9mwiPKEBBylNcvPgCrTt0g7X+65K6aNexCzKfhyL0wStAWx+dv5qERVO/g3Gp1oFrBR/YWWkgMSkLANCg09f4ddU8eNpoF8fY2TuDebnIyil76pmhrTcmTp6IWlW94efjjrqtBmHs952gAaBB128wrH9n+Hp6wtOpAsZMm4ogDyv1TfxDEZdOnIS1V0PUDXYsTg2q0xyetlKcOHdeJbqYPBaHjt5A7VZd4F3qe2zUvhus86Jx8fJTAPk4ceAC/Gu3R83ypsUxwU26wd0wE2dP3gIAGBiaQE8byM4pmcKbl5sHmYLQ19cBAHhWaYXVy1ci2NeiOAYG1qjk64b0xHQUljX7V8cEA36ahO4tq8LHxxsVqjTD1InDYWMIuFZujKkThiHQzQ3lnVzR/4dx6Nm8svoWhH8NBc6fOQp992poUNujOLVCrcbwd9XEyfMXVKJLpOLEocsIatAJlV0MilPrNO8Oe40knD93DwBw5eRxGDnXQaP67sUxftWboKKbNk6cOQUAuHnhKPLMvdGiWVBxjGNgTdSqZI0z504UT3e9f3wNmrf/FlV7TMWUQfVQ8JftFh206T8Ug3u0gpebD1xcgzBp5nh4O+rD1KUyJk8Zj6BAD/h6uaJlr6H4rncDaOmb4ftRk9G2kT8qVPBCQM32mDr2W1gYqm9beF8f0OEEFHIZzBwqY8H8mUi5txNTF2xSDymTlrYWtDW1oauhqZ4lCML/A11zd/y2/wxmju0LY22UeTN9WWQZzzF62Aw0HDQNXRr5QFr41/dufd5y8ejFS1i4usLEVNkABABo6sPNwwpREQ+QVjq8FIVUhiqtvsG5S0fQvrY3FH9zQRLh3yMjPQ7xiVL4evmopJs4ucBSV4qn4Qkq6a+lRT1FEnTh6e2kku7o4g5FRgKiE5ORmRyJmGwZPHzLq8Q4lLOHNlIQHh0HwBCdvvgGzepXUIm5ee4sIpJ04e/tAAAIbtQBHdvUxeszeWTYBcxd8TvcazZCQAVrlbKlXTuxAwVWjbFv50rE39yL2+HKe7OkGRHYdegWpqzdh4GtbLFr+7/oHmVm4tmTOLiV94dZ6XQLKzjZmOLlsyiUVTsqEmPwPC0bXr4lDXAAMDV3gbmRHM/CXwBIxuOYJLhV9MHrcQQAMDUzh421FsJfPgUA+NbpipFDmmLeiD6YOGsxls6fjn6DxqNql1H4oouy8R5YtwO+Gtge+qU2VJD8GMfP34erlzeMigchVMU9PofzYcSWg/vhqx2Og2de3/Mpw97NO1Bv4AL8MqcPjmzZguSyOq3CvwPliAyPhZOjP6xLHp0I6FnCy8YSL59EoawzjyIrDo9jU+FeUbXOMre0gKUFEP7yBQDg2eNYuHj5w7p0r8PYHM72Foh8HgUFgIgnkbCx8YNDqTERSEzg5WCP2GcxyCu6J1TH1B2/7zuDeeP6wVhX8l7tltzEx9h7OhyLthxC12ra2LW7pAN9as8WWFb5Ctt/n4E7x7bgcbyya/vszjGExFljz+FdsEq/jhNXlceb8GE+qMMpAVFQKIV33f6Y/H1r/L5gLHafj1APAwBkJkfg6L59OHD4KA4cv4pcaQ5uXDqNo0cPY9/+g3jyMlm9iCAI/yMVgpujUZA7AMXfWIVNgVWzR+GFQQ3MGNMRmvLCd642JwBQ5CMlPRX6JobQLrmYAUADZpZWkMlzIS+rVQpAQ9sELTu3g4Op1t9esVT4d8nLS0JmLmFuYaKSrmtmAWs9XeS/ZSQ/PTkJEhjAzFRl54K5uRG0tYBChRx5yUnIAmFqaqQSY2JqBm09DRQUqi+XoZT09CpGjp4B32YD0LlJydV5AChMfIIuDXzgXbkBzj3Tw9SpE2GpqxKiom6X4fht5Ti4+NTB1oN7UKu8soumbeqC5Ru3o1UNDwyatBaLp/RVL/rPpUhHYkoOTK3M1TIMYWdiAmmBtMz6MTszAwX5hLmZ6iUTfX0dmJjooUAmBXIzkJKXC1MzY5UY6OnDyMQIhYW5ym1rGcDHyx1pUaHYtmkD/ti4AXdepMGvSgDMSvcwS5NmY+HE4biVboORQwdBdc8pYefdFNv2rEcFVzfM37QfgztWLcrRwpCZv2LsoMao3uZ77No6H1bv+O2Ffzh5FpKTcmBqbqm2LxjC1soM8sKCMtsI8ow0pBTkw9RMtc7SNDCEobEhCgoLAGQiITkLxpbmKgMngD7sTM0gK5RCDiApMQvGZpYo3d8FdGBjZQnKC8Ci0593jWaoH+QFKBdQfi8GNj74dfsONAh0wbB5f2L6sDbFeR2/mYVls/qjfFBb7Nu/CT62ykFhj6Au2LZ9JdzLeWPVrkPo2di71BaFv+uDOpyAcvljAPh20jw08ijE6BGjkVAAaGmq7k7RD89iSO/e6NenN74evQjp+SlYNXs0evfqgz59v8SeU49U4gVB+Gd7ePIX/LwxDAuWzYW1NoqW1pdAInlLw0YoUvb3wzKbo8J/QpqXhcdhoQgJCUVoaChCQh4gIzcP8ZHPEXIvpCgtBM8jyr5i+P+naB9RO5bId+8nEsnrkqoxxf8iIJFIICljH2Txf94U8/ACurbqgGdaNfDr6lkwV+tQKLRN8cWPE7Fs6WwEWmRgxPeDcePp21eo19QxhLGBNgBNmJgYQaO4BSKBsZkpNCSAjoEh9PVKzQL4x3v95ZfVnHrXr/bam7+ZMkVZr7693oCy3gVwcetM9B6xCkN/3of7ofdwNzQM+38Zh42T+mDqsqPqRSHPS8esH7ph2ro7mLfyDzQPslEPKSbR1IGJsSEkAPQNjKGrU/J3GhgZQ1dHCxra2jAyFnMN//XeUkcovX1PLrtEyZ78ej9Vr9dQRv2jrKfUkO/dsXw7TZiaGUEiAfQNDaGvW9Kt1jYwgqGuNqChDSMjI2i8roa1dGFSdOnf0MgY2lpvfDLhbyirhvxb9Cx9sWjJDKTd2YnJc3ZApqmrsrOUr94Vlx89RtjDx7h+aBWsDewwd/1BPH7yGA8f3MWQntVLRQuC8E8mTXmCocOnoVqX71HdwxhJKWmIT06FHER6ahIyc3L/CyeGT5CGPizNzZGXlQupykUqBdJTUqGjYwzt17fsCf+xhLBzaF4zEJUqBSIwMBCVKlXD0ZAHWPRTH1SqXKkorRL6DFlR5jSx/y/6+lYwNZAgLUV1nQNpRiqS8wthaFRyj1RpZpbWIHKRmal6mTw9NQsyakJfRwd6llYwApCRmaMSk5WRhsICwMBA9brCvbMb0LRuS8RZ1sGRwztQxU3tKhsAPXM7tO3UB19/OxaHjm2HXvRpzFu6Qz3s06ZpChtLQ2Qmq0+Kz0F8Rib0DQ3KbGgZmZpBT0+C9AzVe17zcwuQnVUAA30DwMAElvr6yMhQfVYV8/OQnZkNfX3lb7Jj3Q64VumFUd+2hr42AIkeWvT7AX1aV8C2jZuRVuotspOe4JvOTTBzYwiWbT6CYb1rlWQKny9NY1hZGyAjLUVtCngO4pPSoWNgWFZ/EZpm5rDQ00NGhvL+7tfkubnIycxT7scwhq2VMbKS09S6rblISE+HrqE+tABY2xojOy0Vqk84KURCciq09PQh7sb7dyurHvzbKrUYiInft8KGJaOw82QotHVKjRzoGcGxnDOc7G3g7GgNLQ1t2Ng6wMbGFuWcnWBmJOZgCMK/ReT9Wwh9Ho8Dv0+CRzlneLi5oH6nIUiR52HGd23RqONoZIv7eMqgD18Pd6RFRiIzs1SPU56NZ48TUM7FDyVLggj/KVNHH/wwejzGjhuPCRPGY+y4cajo5Ihm3b/EmNFjMWHCBIwZMwaD+jT875wE/0vMzO1hZ6uDJ09VZ/6kRjxHcr42PN+ykI6ZS3nYSArwLDxWJT0yIhwwtoaTlQVMrV1RzkQbLx4+U4mJfhENGSzg6WBbnHZx9yI0bjEQNvW+xKkj2xDoXrJ3UpqL/ZtXY8+RO8VpAGDpURmV3ZwR8fjFP6oT//GZwMvHARFPQ1H6SaWKpERExafD1d2x+F7X0jSsHeFpaYxnj16qpKemPkdilgY8PF0BWMO3nC0iHoSrNNTTU5IQHy+Fl1t5AHlISc+BqZUdVIcjDGBtZoXcjEzIpMrSKRHX0LVZM+y+l4ctR8/jm+7BKiWEz5hEC67lnREb+wBJpc/huYkIT0iEq5dz2fuxkQMqOFrhZVi4SnpKUgKSUojyrsp7lD0rOCH62X0kl74rJCMFkbHJcHZzhgSAm7crEhMfIq702I08HeExMXBwd4Ke6piY8C/zXzrXauPb8fNQz6UAU6YtQpJCUuaGqVBArpBDpvi8TkeC8KkoV601zt8Ow83rl3H5yhVcv3kb+9fPh4WmHobP3Yhdf0yGoRhDKlPdZo2R/+omDpwNKU6LvH0cp0IS0axDA5VY4T9j7OCFkZNmYc7sWZg5cxbmzJ6Eis72aNrzK8ydNwczZ87E3LlzMbB3ozenb/0/0jR3Qr1a/rhwfC+eJ7++ziDH/j17YGAfhFoV3dRKKNmVC0KNQGvs3rYDua8bdLJk7Nx7GI4BwfBy1YGOuSdq13LDoT1bkVT8QPY87NuzByZelRBQQbkg0P3T69Cl7wQ0GTgXh3cuh7OF6h1dEg0pDm1ehB/HzsCrUhc1Xj24iIvhUQioGVhmw/TTJUGdJk2QGH4a566VdPhvXjyM50m6aNagvkp0MU1HtG5eDRcO78ST+JIW/sm9O5Bm4IK6NT0B6KJpmzq4f3EPrhUtsAQAV4/vQES+ORo2qQxAHzXrBeDOpQO48aykyytNf4ITV+7BKzAAZqYSyNJf4OuuPfFQ6oPj58+jYz3P4lhBACSo17A5cl5ex+mLJathh106gbBIoGn9eirRJczRtHVN3DqzE3cjS2ZPXDi8AwkSW9RrEAAAqNWkGbKiLuLkOeUiQgAQeu04wqLkaN6oMQAgqG5L6KU9xKHjt4tjou5dxOXQFDRu0Az/pon2QhnUn5PyVzJj77CSmR6/Gr9TPYv3Dq+ghRYIfTvuuZygnk15fjrv3r7H5Mx3PjBHEIT/uULO+qoOrXwaM1r52Kxil3bMYcWgBrwQVvIcuNJibu6jjYYeF2+/rp4llCbL4OgeNaln5MjJC3/nlnVz6etgTL+6/RmnfFAiSSl/nTGAwc2/YETy67QSGdG36G+iyyGT96hnCZ+IVyGH6WurT5+aHblp115O/LYL9TX0OeHng8UxcXf3MqhKINfsDSlOu7JnEc01tdm060/cs28HB7SrQW0je248/rQ45tH5TXQ21mSN5l9w5549HNavKXW0Tblo41WSpDTjJdtVsaOGkSsnzlrGVcuX8+clP/PnJT9z0YKFPH45nCR5/+TvLGciYaW63bhp9wGuWzqF5R1MWK5SKz6I+fweVKfIjGTPuh40dQjg4l+38JdF4+lgrM3mPaaw+JGn6U/ZrUV1Dp6ysbhc0tPzrOxgTM9KrfnHjj2cPaIPdbV1+NPckuM7Oy6MTfys6OBZi2s27OKyGd/SXFebPX5aTVlRTMqLK6zpbkYblyqct3IDd25Zy8ZV3WhiH8DjN5XPPV8/uRcBsNMXY7h2zeri33XJooX8ZeMh5uS/Wd8In5n8VxzUMoB6Vt6ct2oD1y+fRhdLbdZpM5zpxc9pTeeYni3YofdMvm7Jp0fdYh1PM5bzbch1m/dw0aSBNNHW5oCxf5RsOzeOg5r70dDam3NWbeTvy6fR1VyXdduOZMbrHVmRznF961HTyIkT5v/CLb8tpp+zISvW7MvYt1Qri4bUpaV7A0aLbsU/3t/ucGYnPGS7qhU5cfFR9SySBfx5VA+6lK/Kk3eUDyQWBOHfQMqVE3qwWrO+jCt5djNJ8uyGCbRzDeCpe6mqGUXi759isJcffz90Tz1LUJOXEsPF47+iu5M97Rzc2GvIVIbHlO7hS7lsTCd6VmvP54lvNgCz4sPYurIfpyw9oZ4lfELCbx1lj+bBtLOxondATf68/hgLiht8ZOzNbXQt58jFW2+XKiXn5X2/sn71CrS2tmXVuu2548Q9qu9FIWd3snXdyrS2tmHFqo356+6LlBUFRd/dQ39PV7q5u9PZyYmOjo7FLysrSw6bv794O4+uHmSv1nXpYGtDZzdvDho+h0/UR6s+IxmxYRwzqCPL2dvQycWL309YwcSMgpKAtEdsFuzOniN+KV2ML+6cZp92tWlra0t3n2qcs2Yvc6QqIYx/coPf9mxOB1sblnP357gFfzItR/WXffXkJkcMaEc3RzvaOTizVfdvefl+bFFuAoe0rUsnZxe6upRT+V3tbK0Y1OQrJhcPegmfs6zkZ5z6bXe6OtrQwdmDA4fPZ0xq8bAJyTR+2zKY9duMLRlMIRnz4DIHdm5Ee1sbunpW4pSl26h+bSk74Qknf9edro62dHR251cjFzFWZdtkflYMF48dxPIudrRzKMceX03i01cZKjGl/TalJ6s17MP4fPUc4Z9GQv79JT5eF3nbqpQk35onCMI/lXIluDeP3bellxDH/N+jrEMlZS7C8Fff91/Vv8Kn4+3H1etVG8veh95erkSZMVRA8Y51Kst6Pyp31neU+by86/h813H/rrzX3icGVK6Mq/r+hEJR5iKhxcr6vMLn66/343flvXsffVf5Eu8+D5Z43zjh/9sHdTgFQRAEQRAEQRAE4a+UtbaPIAiCIAiCIAiCIPzHRIdTEARBEARBEARB+ChEh1MQBEEQBEEQBEH4KESHUxAEQRAEQRAEQfgoRIdTEARBEARBEARB+ChEh1MQBEEQBEEQBEH4KESHUxAEQRAEQRAEQfgo/vZzOAsyX2HlvAXItwvC0G96w0BbNT8x4gZWrDyALt+NRYCrkWqmIAj/KMkRd7F08W9IksqLH9wukxYioH4PfN+36dsf5q7Ix4Uj27H72GXkSHVQo0E79OrSFAbaby0hqAk7twsXo/XwVd820FLPfCsZTmxdj2yTaujUupJ65mcqF1uW/IyLT6KKn2yvUMigb+6CoSPGwtX67d+uNDsZe7b8gTM3HkHH2Abte3yJJsFe6mGC8FZZic+wft3vuPcsEa4VaqL/gJ4oZ2WgHlYs9NR2rN5xEtAs3XiSQFtbE5ratvhm6I/wdjYplQfkZ8bit9Wb0WzAD/Cy0VfJK8iKw66NG3DudjhMbb3Qp/8AVCpvqxIjy0/DoR1/4NjlMEDHAs3b9UDbplX/Rr0jfK5iHl3Fut83Izpdiiq1W6N3t9YwM9BUD1OjQOiF/fh9xzHkKvTRqHUPdGpZAzpql7ie3j6FdRt2IyVPEzUbd0TPTo3eaENEhF3E+j93ISYtH4E1W6Jvj7YwN1R9//T4p/jz118RGpEKF59gDOjfC87Whioxb5Lh0K9rUFCuFjo3r6KSk/biOtbuvYOBXw2GtYm4Lvdfw78pM/Yuq1tpELDk6l231bP58OI6Wmqac8elBPUsQRD+YS5sn04tgJVq1GW9uvVYt0491qhencPnbKVCPbhYLn8e051a0GWNRq3ZsW0jGmlpskWvCUwtUI8VypKbdJ9NvM1Zp9sE5qlnvkPErb10szTg4Am71LM+XxkPWN3VnLZuPqxfrwHr1qnH2jVrsmn7/nwYU6geXSwr/iG71/akkYUz23XqxjqV3Kmja8WFGy6rhwpCmVKeXmR9P1taOPqxS9eOdLc2oGtAU956nqUeWuzitkWsW7MG69RR1rd169Rng3q1aWUAGthU4Y2nmWolZPx92gBq69rzXHi6Sk523H12quNBTV0zNm/fmfWredPQ3IO/7blVEpQbw+8616CWthmbtuvM5g2rUluiyyGT/6C09MYEQc39M7+xnIk+PQPqsnP75jQ30GaddiOYnC1XD1VxYNVomunpslr9VmzbtAZ1oMWB49awdPPg8s75tDLUZcXqTdixdQMaa0nYbtBMZpaqsi/tWkhrAwkdvSqzS5f2dLEzZmC9L/kyqeSsmfziEut52dDa2Y9dunWiq40hy/m05v0o9eOoRHjIDT54Ec3ZfTtwzPx9fPn4Ch+8TKYsJ5kXL17jyxvbGdy4Kx88i+OFczeZ/+4/V3hPf7vDmfUqhDXt9QmA9uWb8uEr1ebS4yt/0FbPkruvJKqkC4Lwz/Pr2HZ08OjAV/kKKqRSSguVL5ns7d3NqNt76aCnzR9nbuPrc8PZrfNpomnA5dvvqEUL6rKSHrFf8wACYJO+U5ivHvAW0aEnWaO8JQFw2Ix96tmfrcR7+2hvZM1fD4WSZPE+LJXKqHjrbqzggm9a0MShGi+ExJIk5fmpHNMzmGYujfgyQz1eENTlcGLvYNq61+PNZ6kkyYSnF1jb04It+s17a2dOLpeV7KOFyqhbB36mhb4xZ68/qxZdwN3LR9FYAmobevDis9I7Zj7nfNOQ2oZu3HbqIUlSnp/GOd+3ooVddd6LULbNLm2eTn1NU67acU1ZTJHL36Z+SX19V56+Jy4MCGWTZUeyTSU7Vm7yPeOzlWn3Tv9BG11djl9+Wj28WOqTU/Q002XvkWuK2geF3L9iFI31bbn9/HOSZH7SXdZ01mezPtOZISVJOS/tmkdTXVMu3aG8kJUVeZEB9tqs3uJHRqcpu6pxT86zmosxOwxaXvRuBZzYqxYt3erx7ktlBzPh6SUGuRqx7ZdLimLetHP5CHp5+bOCuzt9yvvRw8OLK3bdY15CKNsF+7JiYAU6uXrQp7wP67f6gYnve5IW3ukDrhUTMoUO6jfpBJ3kixg/dQWk6iGCIPwLFODe7afwCa4Fe10JJFpa0NJWvjQ13z41NjUxGjn5RqhfswZeTwqrVK0hbPVkePoiRi1aKC3swjY0qdcGaXqVEORjgUKpXD2kDMSx9bPQuPUguNTuDA9TI8gUf+tOiE/a08ePQENbVPb2BIDifVhLS/P1DNs3FCTewu87r2HolPmoG+AAANDQNcfYFdtxcMssWOqolxAEVVnRD3HoeCi6DxqKIA9zAICNZ118P6gTbhzfi2ex+epFAAAaGpol+6i2FvLSHmLkqFmo2HIohvVrUBwnzYzCqH6tMXbNNfTo3hTaEjlKH/X5qRE4fOAaWvX6Ed0bV1BuW9cMPw4bCcPce9h54goA4FVUBLQMXFAvqKKyoEQftWrXg2ZeCiLik0ttsRQqEPXsIaJeZYDyHDy+fxc3b95BVHyqeiQKc1Jx/94d3Lh5E09exEAuqqZPwoubR3E6LAc/jRsK26LZqYGN+mBgWz/s2rgRWeoFipw7tBdJ9MLIIb2K2gfaaDf4O9R0lmH77tMAgLsnD+FBkiVGjBgIEy0A0EDtzt+gfTVL7NpyWBlz7hSexZtg/NThcDJTVsh25eth9LedcHLfWjxMAeSJt7H56E30/nYMKrkaAwBsPGtj1ODOOHvwDzxOKXtn7PL9Amxc/C1yM+KQWmiEeb/sw7edA6Fn448tRw+igZsZ4qKiUaXFl9i5fQmsddW3IHyID+hwAjKpDFXq98XiyQNx8JfJ+HXvPfUQQRD+6fIicfd5CixNszFrwk/o1KkjhgyfjrvhceqRKioEt0C9IEssnD8X98KjER/7FEvnT0OOqT86t6utHi4Uy8f163fQ9oelOLB1PpzNdSCTK9SD3qAoTMGtu7GY/Ms+/DrnJ5hoEKK/WeLho1BoGZvi8tG16NO9E7r3Hog/915EwTv68i/v3UYa7dA4yBUntq9Ez84d0ffLH3DruRR1atWAsZ56CUFQFRcThpepEgQEBqqkB/j7IT/xOR5GJ6qkv83GhdNwOdYa06cPhV6pFtmrhzeRY1Ydh0+fRt9mfpDKZCjd4ywoSEJSqgJe3n4liQAMbO1gZayNB/eeAQDqte8Bd5NETJ+/HC9fJSLiyU3Mnf8znKs2R8Nq3ipliykKMH1we3w3ag6GD+qKho2aon7dGqhcpTZW77hcHBb38By6taqPOvUaolmTxqgeVA1fjViM9LL72sK/yKM796Gl4wp/d5tSqZqoUMUX8dF3EVPm7l2Au2H3YOTqDns7ZQcQAKDlgsoVnPHwym0AQMj9EGhZucLDwb4kBiaoWskNT+7dQAaA5KQ4aJk4wd2m9PsDtq5uyE+LR2RkFuKehSM1Qx9VKrurxHhVrgyN9Fg8CU9SSS+RgR2bdqDNd0swpLEpNm05hMKiY+vFrTO4G2+J7ZtW4NGxHQh5/pZBGeFv+6AOpwREnlSCTj9NQ9c6Npg2ZiQevipUDxME4R8s5cULxKUlYt+GVbgWngEbCxOc3bkYNarVx7YzT9TDi+mYe2Hm3HGIvLIGQQE+8PTyw9Rfz+HbGQtRz9dCPVwopocBI+diwpDWgIYCMtlfdzYBQEPHEuN/XoneLSqBsnwo/t46b5825uPZo0eIeXYZv28+DCNLR2TH3Eb/Tg3Rf+jPyHvLV5XwKhEKjTwsHd0fP83cBBM7W0TcOYKWdavh561X1cMF4Q1ZSfHI19aCrZ2VSrqppQU0tXORkZ2rkl6W9BeXsWjVfnQcOBT1/VTrznLBHbFy2Ux42WqjoPDN0RMDA2eUc9TF40cPVK58Jr54iZi0HOTn5AAAbCs0xczJ/XDwl3Hw8fSAb0AwNl5Nx7RFc+Fm8bbFXyTQ0dbAoa2rkG5cF+fuPMCTexfR2EuK8aPG4WkqAORi7rifEJbni/N3niAy4ilWT+6JjUvGYvUOcQz92yUkJkLfyho2RqqLVFla2UCjoBC5OWW0+RVSpCWnwcLSHoZq62ZZ2ZsiOyMN+QCSkxNhaGkOI2PVKSgWNlYoKExClhRwcfUEc18hPFa1ZxseFga5XAZZfi7SUtIBbTPYm5upxJiYWMNAQ4HcrGyV9BKG6PXjZIz78UsMn7oIIwe3h27RR3HyrYd5S2ajc88BWLV2HgJclbMXhP/cB3U4AYAKKQBLzJ4/F1oxZzBu2moAgMZbpjAJgvDPItcxR/tuX2HT7uPYv/MPrFn3J27euILmXtkYN3wi4t/SXop/dBpffj0O5Wr0w6GTF3D1yil8360mVkz4GtvPPlUPF0rRkHxIlSuBhqhYy6YgyldtgrHT1+LU6aNYs2o5Dp+9jj/nfIldKydj+7GH6iUAABqaWkhLjsQzqSeOnT+PtSvX4PzVyxjSwh1TR43Bo8Q3G/iCUJo0JxuFfLPNo6dvAA0NTZWrkW9zZPs6PC+0x3dfdVPPgkSiAcnb1wmHtqkTOnZrgiMb52LR+pPIVwCJEXcxZtx0JOUCWhrKuubO0TX4bsI6NO87BuevXseZ47vRsYoJxgwZiBvPM9Q3W0xWWABHv/qYPW8cvJ1t4ewTjB+/7wNp/EtERaYDkCLpVSJMzSxhZmwAU3Nb9Bo6FT9P/Qm2Jm/ryAr/DkRObi4U0Che+fs1AwMjaOMtu7dCjrzcHGhoaLzRuTAy1INEIgGgjKGGRH3TMDBUzt2VSYFKDZoj0LUQY4ePxo0n8VBQiov7VmLOmuOAri4ABfJysyFXSKCpdhDq6BhDV08Db38Ihzaq1moAR0ttmDhVRO1SV/rNHcujdg1/AFqo0aARbEzF/RX/Ler7xN/mVrM75k74Agd+mYzfDz2Cjq7qc1J+m/IlvH18EBAQgAre3ug5eAbe0o4VBOF/yMYrGEvW/IJuzasUVwTG9r4Y9m0fRD46hTtP0tVKKG1ZNRfhuR5Yv3EdWtStCv9K9bD8z41o4FaA2bOXIkumXkIQPhJNfXw5egHmTPoaVoZFD3nQ0EWfAd/Cxy4Px89eVy8BAFBQAQWAL4d9DxcLZYNCQ98O3wz7DpL4+7h1+4V6kU+TNAeTB7aEdwU/BAT4w9urAsb/vB03Tv2Jat7eqBgQAP+KFRBctw/C48SZuzR9c0vogSiUqVZ42ZkZkMkU0NT6i+aVLAZbNh9GcJM+qONnqp77HjQxaOxijOzqh/GDW8PNxQ21m/WBjndztKvkAk19PQC5WLZgPvTLd8D6X+aiRqAvajToiA3b/oB5+nUsWLZdfaPFFAoJbK28YFnqApemph4kCjnkskIApvhqxFDEX1kDv/Lu8Ausge9Gr0RQxyHo17566U0J/zoSWJiZQaKQQa5220d6eioKJRrQ1CxjUEFLCyamZpBJ86E+ZJeamgkNDU1oQBMmpuaAVAa5WlBGWiokEi2AgIZlAH5dtwZmqWdQt4oPPL188cOsPeg7eAhcjLQg0dGHiak5tDTkkMpUN5Sbm4LcHAU0yvqMwv+bv6gR34cEvYdNR/c6VpgwagTuRWRBR6vkRy7Iy0F6egYyMjKQnpGB7Ny8skdGBEH4n0pPikHYw3Dkq3UQjSxMAYUcubkFqhkAgDw8CX0Jz/K1Ud621OCSri3qVa2MmAePkZotepzC/4a8IBuPwu4jLkW1M6RhpA89fT3k5OSppL9mbmkGXRjA3FR1NQgjAysYSGRIz3jbkhifnrycrOJzdEZGBvIKCiGTFiKj1Hk7MzMbcnHjsApTOycYK+R4FZmgkp4YmwTQAo6Wqs/SVBd7+zpuPElH226tPrghpm3ignnrD+LqucNYsnA+/th2AGtn9kRiSircyrsAeal48SQJVSvVQ9G6KwAAA3s/1PR2xeM7YShjYmQxqrXWiNedD2V6wx5jcPnGDSyY+D2cTAqwe/1c1AyqiDFzdqiUE/59nJyckJsYi1eZqnVh4qsYaJmYwNy8rM6cLmztbJCaEIfMzNLphYiNSoNVORfoAHB0cEROUgoy01WXHI2LSoSZiQssihYp8qnXA2ev3MCebb9h/oKfcezkYbSsYogsuTbsHc1gaW0BDXkaIhNSVLaTmhSLPG0dWNmKW3z+ST60nlOhYeCM2fNmQyfmJEZN/hnZEq3iiSDfzt+OhPg4REZGIi4+Hgc3zsZfPY5VEISP78LORahcsR5O34lVSb9+/joMTL1RoXxZlbU+yrnZIDbmHmJUTig5CI+OgpmjPYz1xOPEhf+N/LSH6FSrCn6aulElPfrhfURGS1GlknL1TnWelYPgbF6Is6duqaQ/vn8Z6dpG8K2gugjFJ0vbEAu2XUJCXAwiI6MQn/gKS8b0Ra2WXyE8IR7RkZGIjn6FRyH7UMFRnLlLc3QOhI+LNi5cuFAqVYYL587B1NMH3s52pdLfdOvWBeTquqJ+1aLVY/8uRT52rJ2DFZsuoEqtpujRvStqV/FA6KmjeJpmhia1AwBdA9g7GSH8xV0o7+gskh+Hp/FJsHN1wodMGJRINAFpApbNnoDHWbb4dtRUHL94F3Exz/F9S09s/uM3xL2rJyv841WoUR16iMKlG8rFpwAA8hRcOncH5Ss2hlOpNYFKaKF2jTrIiXyIB8/ii1PzE0JwLuQFqtWvAgAICq4BRXo4bjwoNZMk5wXOXH6Eyo1qwwRAVmwoJo2eiMdZhmjdtjO6dGwNOzNg/+YDcPBvAV97wNonEJ42xPmzysWIXrt6+hz0bQLh6/nuQR/hf0z9OSl/JevVPVaxMODgiTvVs/jHtC8IgBJDe+4Tz+EUhH+09IhrrGKvQ796PXg9LJo5Wcncvmw0zfR1+NWkbcVxEaFnuWT5L3wZr3yu26Oz62mpo83WfSbxUWQis9Lj+evs72mircXJq4+XegfhrfJfsW01G9bqOl7tOZxy3ji9kyt/28G0HJUMkmRGzG0Gmhnwu6l71bM+U1LOHdyU2gY2XLLhJLNy8/jw2iE2r+pIB792fJ6ofGK3PDuO639ZxiMXHhSXXDWqM/X0Lbl4wwlm5uTy7pktrGily/qdx1P16dKCUBYF103qRW19Zy7ddJo5udk8unE2HfU1+P2MXcVRma8ecPmypbwWFleqrJxzv6lHG+/GjC7jOFd3cs0P1NZz4oWnpZ/DKeeSYe2oZ1aOGw7fYn5BPm+e3MyqLoZs2G0Sc4uiDq8ZRU0Y8tuJa/kqJYtJsU84ZXAb6utZceuZ8FLbK0WWx4H1XVm5wVAqn4CodHn3LBpp2fPEzSRSEc8eQQ509OvA83eeMa8gn89DzrFpgDWrNPmp+P2Ffyl5On9sW5kW9tV46MoT5mQmctWUAdTRs+DvR5TPfSXlvHlmN1es287Uomd1FibfZ4MKFvSr+wXvPk9ketxjDutWl2aWfrz2ev/Ni2H3Wm509G3Di6FRzEqN5Jzv29LY0IkHr8WQJAuSQljVSZ/BbYfwSXQ6s9JecfXk/jTUN+cv++8XvT/524Qe1NO34vJt55XH4KZZNNHV49C5+4tjhH+Gv93hzIy9w0ATHQ4ct0M9i4qMKHav6UJoW3H3ZfFAYUH4p7t3cjNrVnCkgZEJrawsaGRsxf7DFjO1VGvhyPJBhKYl91xOKkqR8dAfs+jpaE4jUzNaWprR1MqRw2f+zhxZSTnhHfJj2DbIijW6TFDr3BRwxsBgatnX5INYhUoOSaZH32RFIx0OnrRbPeuzlZfyksP7NKeJoT4trW1obGBI/xrtePaOsuFCktKo83SzBNt8/1txmiwngdOGdKalqREtraxpqG/E2q0H8nFcVnGMILyLNCuWEwa2opG+AS0tLWlkaMru385kUm7JsRt5YQ21JODYVZdLCsrzObihC31rfftegxsnVg2hlq4dzz1NV0nPS3nGH7o0oKmxEa1sbGhuas7m3YbyWUKpYSxZOlfNGEJbc0OaWljS3NyENs4+XPznSb5ZwxSR5fLLOk6sWO8HlQGxizumUx+2PHotniT57MZ+1q/kRn19I9ra2dHc1IxV6nbj5YfigsOnIDXiJrs2DKSBnj4tLU1pYunM8Qt3sbA4opBzBteipm113o9WDu6R5JPLu1i7ggP1jExpYWZMG6eK/G3/9eJ8kowNO83m1T2pp2dMS3NTWti4cf561QHruyf+ZJCnLY3NLGhtaUFbBw/OWH2YpZsZsuxYjunXksZ6OrS0sqSBoRm7D57LNDHi8Y8j4duXcSqTvDAbIbfuwdjBG16u1urZSIl6hPsRqfCvVB2WJqoLCAmC8M+Tk/oKIQ+eIDtfATtnL/j5lEPpuzNSXz1F6LMkBFSuBgvjkmM6Nf4F7j96gUK5Bpw9KsDHrfQztYR3UhQi7N4tSPXsEejrVureBiIqPASRqURQlcrQV5vvJi/Ixr1b92Dq5ANPF9XHMXzOKMvHkwehiErIgL6RBfwDA2H2ehEhACzMxI1bd2Fs6w1fj1JTHSnDs0cheBGTAkNTO1SqUhGG2v+VO02Ez4U8Hw9D7yEmKQuWdq7w9/eCTqlFMwsy43H93hO4eFWGi/3rKX4KhIfcRI7EBpUC3N6xFq1SWtwzhD1LRkC1ajDVU713TlGYiwf37yEuJRtm1s4IDPSBrvrSuSBiXz7A4+evAE09ePkGoJyt6qMkVFCBJ6E3kQMrVAr0KK6fMpOjEPowFn6Vq8LcWFk55WUkICT0EbLypdA3toR/QABMDcRtFZ8KaV467t25h7RcGZzd/eDjYV9qfyWin4YiIlmOqlWrwKDU+SonJQa37z9BoVwbXhUD4FLG/laQlYjbd8KQI5XAzbsiPJ3f7FNkJkbh3sNwFMi14OFdEe5Ob573FLICPAq5jdiUHFjYuSDQrzy0y7rFVPh/9bc7nIIgCIIgCIIgCILwPsRQriAIgiAIgiAIgvBRiA6nIAiCIAiCIAiC8FGIDqcgCIIgCIIgCILwUYgOpyAIgiAIgiAIgvBRiA6nIAiCIAiCIAiC8FGIDqcgCIIgCIIgCILwUYgOpyAIgiAIgiAIgvBRfPBzOOX5GXj45DkKpIRE/RnDAAgJXL0qwspE7cnlHxUR8+wRMhVGqFC+HCSyXDx8+ARGNq4oZ2euHvyPJs1JQdiTaLiUrwALI131bEH4ryjMScfjR0+RryipBqhQwMzWBd5u9iqxbyXNxoOHz2Hl7AlbC0P1XOEtclNjEJshgYeb498a+ZNmJ+FBeALcfb1hoqetnv3ZS0+IQVRSDrx9vKH7F8+fp6wQL54+RGJ6HsysHFHeoxw0/86P8S+Ukx6Px+ERkEEXbt7esDE1UA8pAxEX+RSRr5KhZ2SB8uW9YaBbxolfEIRPiiw/B08eP0RmvgJ2Tp5wdbLE+xz5+ZkJePAkAlJqwdWzPOwsjNVDUJibhkePniJXKoGTmxecbc3UQyAryMSTR0+RmSeDrbM73Jysi98/LyMJj8JfQEqJymeSSCSQSLTg7FYetpZva5MoEB3+BAojW7g4WKjkFGQl4Wl0Gjy9vKCn/T5/rfBe+IHCz2ykjR4IlP3S0LXkwbsJ6sU+Lnk821d3YJP+00mSifeP0lFXj9N/vage+Y/36vofNNQx5qbzz5QJ8nzePHmEj6JS1UMF4YPdObqchgA1dfSpp6d8aQBsO3gJ5erBb3F+0zRqQpeLt19TzxLeRpHDsd2rs163ccxTz3snKVeN7kzAiScfxqtnCtIkftvKj+buDfksUT1TlSI3iTO/70YLQz2amBjT0MSaA0euYGaBeuSn48nV3axXyY0GBkY0MjCgh3897rvwWD1MlSKHvy/4kc5WpjQ2NqGxiQkbtB3Ch1FZ6pGCIHxCMuPCOLBtLRrpG9DE2IAW9p6cueowZeqBaqLuHWOL6p7UMzCksaEBnbyqc+uJeyoxyS+uskujQOrrGdLEyJC2zr5cuf2CWsw19mxelXpaOjQ1NaG1gyfHL9jJAoUy/9GJ9bQ30aS2jl5x+0XfwIi6WiBgyuXb7qps77X83FyShZzWtTVHzt1FBaXML5CScilz82VMur2FVeq057N4GfNycln0dsJ/6IPHch+E3UR6gSkG/zgBs2fNwPTp04tf06ZNwexpC1G9vOqowceWExuLF5E5qFalOgBAy9QVE+YuRPdm/uqh/3wSDUgkmtAounwceX0bWnQYgCdJeeqRgvDBnt6/BU2r6jh65SZu37iBG9dv4O7dECyd2Oe9rrqlvbiCMRPnQQ4NaH/ql4b+a+TYvGAk5m6/AW0dnfcaLX4t7NQfmL5sNwBdaJY1teQzd2DtLKw68gA6utrQ+IuvZ9vS0Zi17jgmrD6CqOgIrJ87EFuXjcLctSfUQz8Jecn38VW/r5FkWA9X7j/H45AzqG2dgG/6foWHcfnq4cXOb1mEb0atQ4fvF+NZRAzO71uL1NANGDxsFnI/aH6UIAj/fLmYM2wQdl3Lwp9HbyPyxSOM7VUZM0f2x/Yzz9SDi8kzn2PIgAF4isq4eCccT8MuoWV54ts+X+JORE5RUBJGfzUA5yKscOjyfbwIv41Bje0xdlA/nLiboIzJi8YPA/th7w0Zft1/AS9fhmPpuA5YNuVLzPnlHADAtWY7nLp0B7du3sSN6zdw8+Yd3DizG8Fe5vBv2BGdWgaW+mQldiwfg+YdBuNhRj5e3jmOzi3qYvWeEOTGh6JXs4aY8ttxQJaBRRO/Rruuo5BUoL4F4YOo90DfTyGnf1mT5h71+TJNPe/DpbwK59YNv3L5ilU8cvpW8SjGa9LcJJ7Yv5UrV6zghu2HmZSlOhT9+OIG2ulbcsfZaOW/b5/mwSNXWKgS9e/w6sYGGumaccuF5yTJF2dXUE/Xmofu/Y+vGguftMndq7NK45HvfTVThTyDo7o3pJtbRRpqGHPpjhvqEYKavLRozvyxG90dXWlposmGvSYxXz3oLQpSHrNNUEV6ly9PPS03nn4k6oLSXoUeZiUPe3p6OtHeuwmfJ6lHlFBkP2ZNZyN2+X6VSvrcrxvR3rMt4z/Bq5ynfx1OPQNXngwp+WJSnp2ip6kOJ6w8rRJbIps/dQ1iYINBzCh1It27aAiNTfx551lm6WBBED4R8WH7aaOrw+nrzpckSpPYs0Y51u007a1thqtbZtNYx4H7rijb4SSZG3udvpa6/H7eXpLks7PraKprwjX77xfHKLLC2cDTjN1+WkuSDDuykobQ4qLNqjOnFn7bhLYerRn7lqlBW2b3p7m5J0+Gvn2KS1L0Qy4YP5jm+qCBuRtHTl/NFwk5pDyP5w78zmbVPQiANZv14f4zIZSqb0D4IB92SaIgFfeuP0OFCjXh/OaU6w+gwMkt81AnuDFmLlqDDetXoXe7Rhg8ejVeDyy8enACjWsHY8D3k/Dnxg2YMvxL1K/fDXcj0ou3Eh56DdkG9vCt6AhAjjXjv8fomZuhKI5Q9SL0HL7v1w7enm7w8Q/GuLm/IzFbXpyflRiFNXOGIcjfBx6e5dGmx7c4e+sF3mdQ92XIOfzQpw3Ke7ijgn91jJ3zO1JypQCAG3uWon6DXngaX3K1UlEYif5NamP27xdKbQXQ1tFF5J2daNljIvILU9G/eVV8PWI9cjJeolejOliz806paAU2zP8erXv8hNRCAMjGjB964oexyzBv8kC4l3PBkCm/Qw6gMCsJfywer/zbPMqjdddvcO7W20ethE8QX+F2WAwqVvNBeOgtHDt2DJdvhiFf+rYjRtXhdTOwO0wbKxdPhIUOIf+w28E/I1L8vmgcDj7Qx74jB1Df3xYF0pL65t3kWDJ5GJJsmmL+qN7QlMvwgbfff5oKEzD2h5HwazMSQzrXRGGBsq59m7gHd/A4Xor6zWqppNdpUh+Zr27hwdMslfR/PynOnbkOW88aqOphVZxq4VwdNX1tcfH4xbLPa9TH2EXbcXDzbJiUul04Iz0NGrq60NER9xALwqfo0bWryJI5ol4935JELSs0aFgZD+8eQ1SZVaQcF69cgH45X1T2cSpO1XcIRIMAV1w9fR0AcOPqFcDIE3UreRbHSIy80KSmL26euohCAE8f3YfE0AdNqvsVxwBAtbo1kRZ7Gw/CM1XSAeDV3UMYPWsTegybjib+1urZxaycysPWRBNBjXqhaSVbGJiVg5uNAaChB78KXjDUd8SXfXtAp5Co4OeNv1gKQHhPH9ThzEp8iPvRGXhyZz/atmmNli1bFr+aN2uKrr2+x8v092u0AsDTixvwxTez0HzIQty8cws3bt3Hn4uHYPvP43D0ZgqQ/hQDevVFlkUjnLl6G9evXce9q4dgmX4BI8Yug6xoOyFX78HBvQbcrCRA3nNcexCN6g1roawld55f2oZmTZrhyL1sfD9uGr7sUAXrpg9C3yEzkQdAmv4Mgzo0wMh5+9Gw8zeYPO4HKKLOoGWTOvjzUJj65lSEX92F5o2a4EhYIX4YNxX9O1bDbzMGos838yADkJMSi5CQx8gtLGlskvkID72HqATVg0gul8HeoyYGf9EaWpqG6PnNcPTpVheKvAw8Cr2HuKTsUtFEXMwzPHj8ElICgBwJsU+wYv54HL4nx9DhP6CKjwsgTcKwHq3w/cwtaNTtW0ydOByGGdfQplEz7DgvOp2fi7yoF3iWmoYjG2agXadu6N+vNxrUqo5WXX7Cs8SiqS9vkfLkJCbM2ozhsxaiqocpCqXyvzU19LNECdoNmo5TR/+Av48VpIWva66/FnJ4BVbvf4n5P0+HjQFE517NlsWTcTHBEwtm/gBdFpTdeSolPi4JCokVPOxLOl8AYGnjCL3CAiTFp6qk//tlISI+DnZu5aBfeo0gHW3YOpojIfklymw/SjRg5+wOZwdrQF6AiGf38duiMZj06yn0HPwdKjjrqZcQBOETEB0VA0M7J7iYmKik2zg4QZqRgdSUss5fBYiPi4OFgzPMVC5G6cLBxQJJEREoABATGwUjO1uYWajWHw7lrJGW9hLJCsDUxBwazEeOtFAlJis1FYX5BchKLrnYBABgHpbNmYk867oY8U1X1Tx1zEZagTEmzFmEGeO/hkZBOl4PUabExyOgRX+sWLMUTYLskJZeZs0ofIAP6nBGh91HXF4hstMjce7MWZw/f774dfbsGTx+ngNjo/fddC5WzVkI2+pfYta4btAvKtak61D8unoBfO00sXv9ItyMs8K6dctQ3lG585s4B+Gnb9rj1skTiMoGgHhcvf0MfjWqwQBAytNHeJEsQdUgH5V3U8rCisVzUWDZGCfPncQPA/th9IzV2LZqMiLunkdEghSnNizBrptSbD5yGgumDsUXA3/AkdOn0bWSIaZNnYXEt93ywjysWTQT2datcercEfwwqB/GTF+JzasmIeLuaTxJALS1taCpqVF8f6aSBBpaJfdsvqaQy6Fj6oROrWtDW0sPLTv1Rr1gT0BBaGhqQkPlRiUJNCUa0JCgqPEvAVkIHYtArFm9Bj8OHYmvejbGxS3LsPVKMnacuI75k35E3wHfYMeJK/imqSmmjF+AzL9qrQmfhOTkNOjqmqJZh0E4cOYmXr58gcMbZ+Lh2TUY/ONCvG0XR2EKxg0dCYf6P2BIe3/ICt99NUkoItGCk4sbjLSLllZ7T/lxIfhhxHx0+Wke6nkZoVBa1on+8xV5dy+mLj+GSUvmw95IG1L5X3+5WWmpkFMbutqqV+gMDK1gZKAJyt9/wPRfoSAf6dlZ0NLVVltVXhMmZmZQKGR/uUtmvLyNbi3rYtDI+UiVWaFxw5of1oAQBOEfjkhNSwM0daGlpamSY2puAV0QirIGPWVSZGRkQFdHD5pqI9AWliYgCECOzPR0QFsLapuGqbkFCBmkhUClmrVhxAisXLezuDOYl/QYa37fC2hoglSto2PuHcXve2+g35Dh8LD+i2uSElP8OHku6vvbwb/JAEwZ2QuvzwTedbpg6tgB0DewwcQFC1G9vOqgpPDhPuh8EXLnHqQKTxy89BCpKUmIj48vfiWnpOLa2TWw+ovf+7X8mDvYf/0FWnRug9IDr/rmdug96CuUd9TAgd1HUbl+D1Rx1S8VAZiYGUMmz0SeDJBFP8WjuHxUrlIBAPDofgikWrYI9HRVKQMA+UlxuHnjORq36gEPi5I9vnG/Cbhz4wgq2BbgyImL8KreAo2D3EoK6juid49OSH54F5Exybhzei8WzF+AxYsXY8GC+di29wpys6Jx/dIztGjfHW5mJV9C076TcPv6YfjZAoWyv9+YkckVIAm5/H2n4CnJZTK4+nnBweb1SJIUF69eQQ4lOLFjFSaMH4dx48Zh0qS5eJlagKjnZ/D0VRkVifDJcQxsgSv3n+HPpePhU84S+oamaNZjOBaO6I7zB3/DnedlL1B14JcZOBdph58XjIIGAIlEuQy5luSDqhPhnQqxZMpoyN27YvKP7QAAmhoSSCCBllg0CMiLxYSfxiO4x0QMaOEDgNDW1IREolHm47pe09E3gKbmmx1/aWEOCgsVr0fsPh1aWtDT0YXkjUaiAgX5ee/8rl7Tt6+IPWdDEHbnLHpUM8AXHVvgwLVo9TDhf+D+xYNYsGA+Fi9ejIULFuDPrUeQnBaD9UsXY+GixVi8aCEWLlqFsOcp6kUF4T1IoK+nDw2NN+vI/Ly84lmFb9DQgJ6u3hudQQDIzVXOPCE0oKuvhzI2jbw8ZZtDLgPM/VpizoR+2Ll4COo1645x40aiRavuyDH2hZ2ZLqih2t44tHkTcg0D0a9nQ5V04Z/jA1qIObh9PwTm3n7w93CBnr4hTExMSr1MYWhQ1iTWssVHxiCv0BRVfD3UswAA8sJ4PH+eBp+gqupZuH/7CUzN3eFoBjx9+ADZMhNUqVAeABASegu6Di5wdXlzpdzCwmxkZ8thZKx2A6qGNgwN9AAUIiUrGxb2NtBWe4yotbUttCCFlHk48OdyjB4zGiNGjMDo0WMwe/Ee5CpykJEtg6GJ6jOHJJraMDAo3aVWPcNLJBpQfZLQX1AeuZCotRQkagfha8VhigKkJydDIc9FWOgt3L59B3fu3MH1G9dRYOSFru1aw1Dy1upE+IRoaGnD0MgQWlqq+4xvVX8oCjIQHZuhkg4A6U/PY+TkNfCqVgkPrh7Ejl17sPfYBeQpZLh+8QhOXLgL2d8bExHeIeTQWsz5/RwqBXng9KFd2LV7L05eDYMMOTh+cBeu3nmuXuSzsnX5NOy+kwVfNx3s2bUTe3Ztw40H0SjIjse+ndvw8EW8ehEAgKWVOVCYicQs1VsYMrKSkKvQhJGpkUr6v56mIWzMzJGWlAaVCQkKGdKS02Gkb42/mhyrY2gCJycX+FVugOV/rIOvbjzW/3FYPUz4H7i8ezVGjx6DESNGYNTo0Zi99BdExz3B9OEjMGrkCIwYOQqjRk7Ehdsx6kUF4b3Y2NggPyUFqbmqA8+pyYlQ6OnByKiM+7c1dGBuZY70tETkqk2RSo7PgLG5FfQggaWlDfLSs5CjdudOalIy9HSsYKgPABroN2ENTu1bCweDDNwJicCXk1Zj3tiOgFwCc+tSbfvsSOw9cR5VW7aHr/Pbnrsp/H8ru3fyDsxKx5OwF/D09YO56tTuD0KFAqQE2qU/iiIRY/u1w8RlhyHR0gEVCsjVrgrKUh5hx9GrqNmmOcwA3L9/HTCzR3kvWwA5CL39AC4e1WH/5rNmoadvDnMLbSQlxqqkZz0/j46teuDio1R4OtriVfgL5OWqhCAi4ilk2oYw1DbFN9NW4sL587h48SIunD+PTWuHwljTFNaWOkhKUG3opIZfQtf23XDhYRp0dbWRn5mB7MKSueny7HSkFxS8faSZACCB5PVVJIkEVMiRnlK6UyBFZkYK5GVspHhgW0MXZlbmsHCojD+2Hcax48dx/PhxnDh+HNs2rsb4McPgbltGRSJ8ck5snoXa9TrjQYTqTh7zPBIaOkawt3+z0Z2emgl7d3c8v7Efo0aOwvhxY7Bg9SZkK6Q4tHk5Fq3ciQIxXvFfk5aaDTdvN5zZuQKjRo/FuLGjsW73WciQiV8WzcSfuy6rF/mMFCItB3B1NMIfy2dh9NhxGDNuEo5cfoS8tBeYN3UCjl99ol4IAODk7Q0T42zcDYlSSX965xbkprbwcP/UplEZonqlCkgIf4CkrJI1/qXZrxD6IAF+laqWudaBPDseo7/sgGnLD6mk61tYwdTQANlZ777XW/g4OgxdhPOl2h5b186Dt1sQdl28iAsXL+LixQu4cPEoOjfxUi8qCO/Fp7IfCvNeIiyy9FVyOR7cvg9rh4pwKHNNHl0E+gUiK/IlEhNKtStkkbj1IArlg5SPKAzwD4A0MQJR8YklMcjE7VvP4eFfFbaagDQnFSF3n6Byk/7Yve8Yjh/ZhS/a1ULoudOghS98vUraJ9HPQnD/QQ5aNGsGtWtEwj+J+rK1fyXpxWm66Wix14+rGZecwOioKEaVekVGRjIhOUMZLC/kq9hoJiQV/bsMOREX6G5uyJ/mK5dLJhU8+vt4WhjZcteFSJL5/LGNH8vX6MukomWQ5YWZnP9dGxoYePL0vWSSCk7oVokVaw1hPsnC9FBWszHkNzMOlH6rUgo4c1ADWjgG88rDoqWTFblcMaYrTSz8GRpTyOtbp1MLhpz3x5nih74mPrvE+l7m9G/0HdPf+uTbQs7+qgHNnarx2uNkZZI8l8tHd6OptS9DYslHxxZTR6LDxcWPkSjgtnnfEwB/nH+ELOOxKM/OraK2lj43nI5QvkvqCzb2MmK9zuOZWfRZIu4cYgUz0L1GZybkk2QGB7fxZvl6XzCt1JL2t/Ytph60OHLBHhYW/XG5iY/Zsao9y1f/gilv/duET8njc3/SBOCX49axsGiN8/jwS6ztZsZabcYztyguLyuVkdExzCuQk1RQLpcXv0gy6sY+2mjqc9G2qyUbF94t/xXbVrNhra7j1R6LomBGagKjY+MplZNUlHzXr7/vc+snUhdOPPHglUrJz5FCXnp/VJCUcsnwNrRwqcenCaWeqyUr4KuYKCanZRcl5HFYu0p08GvFh7E5JMm0yBus7WnCFn3nf5IP+o64voV2ulocNm9X0SMNZNy77Cfq6jhw79XXjzBQMC05njGvEpUPd1ek89vWfrRwqc0b4SlFMfncu3IUNaHLhVuvv968IAifkMKMp2ziY8la7UYzuagx8PjyNjoaaHPU4mPFca/PV4VF7cbE+4foZKjLryb9qaxDKOfx3yfRWM+Km06FkyRzY6+xsq0eOwxewJyiyvbWoeU00zHmgi3Kx6BkPD9NJ2N9/jB3e9E7kU+v7aGniSG/m7m7OI0kL2wZRy1dSx4LeXtfQ/j/97c7nDf3zSmazPn21/+1d9bhUR3f/3/Hd+PuboQECwkQEtzdtRR3KW3RQrG2QKm3UBwKFNcixYp7cAiQQNzdfZPdff/+2JDsLglS+X0/n0/v63nyPDBz7uy9d+bOnDNz5szADzeQJHOfnaKdAThs7q/qxaiw47NxNNG34YhJMzlxRA+aGVly4ffHqvPj7x2mr5Upm7Xtx9lzZrFnmyY0MXfmmn03SZKyshi2dzbge7N3kiTjbm6jWEePm868qC5DnezoG2zf2JFGFm58b8wE9unYjHo6Zvxi/VmSpLwil8vGd6aOjiHbdx/CCWOH093GmHaewbxw//WKXnb0bXZubE8jS0++N3oCe3dsSh1dcy7f8AdJsiI/iv1auFBb35qDRozngO6tGRDYnU09zTllxQmSZGroNurrGnDnlSiSZE7sJXob69GtQTt+vfooSXLrZ2OoAbB5u94cP2YoG/s2ZaegADYK7se0MpLM5+SeXvRsPZq5KoeRFnDVjL7UhBabte3DSeNHsqG7FfVNPbn/ouL3BP4NlHPDp6OoowE2b9eH48cMo4u5Lh18OvNO1Evlkjy9diqhbcnjt6smUNRIuv0bLaHDb/YLBudbU57M3oGWDBr0KVWPE6vg8okhFDm05PO02s2ey1sXUBu2PBuerp4lQBm//bgnjZ3bMErpGDZp8g26WWhwwEe/VKelPTvDJnaGtHVrzHHjxrCekwmt3TrwfuzfeLj0fxQV3Lj4fWpr6rF9j6Ec2qcNxVqaHLdwi9JZ1cWcM7QxTby6MqGqC0h9cp4h9SxpYuPDkeMmsW+XIOpBh8M++I7F/4PnlQoICCi4e3w1LXW16BPQgWNHDaK1sS6bdprKtIKXqxKVXDWlLXXtmjM8peZkzj1fTaFIS5vBXQdxxMBOFGtocNiHP6iMdWe3LaGhjhabturF0cN700wP7PzeQuZVz8BWcv3CERRp67FTnxEc+/5A2htpMbj3TGYUqJ4CunV+L+qbN+WL6vsS+E9Ea9myZcvUVz1fR1pCHCo0DdGkaQCaNGnyyp+fbwP0GzgQDTztkJuZioxcKfoNHw4fl7pdlBq37oFALyM8fvgYFXp2WPLNBkwZ1qp6R6OpfX0MHNgNhWkv8DwmBS5NumLdpvXo1aoeUHWm5PPkQvQYMhz1nM2RlZKAUpkDhg3vCUuj2hyFAH1zJwweMgj2xhWIjk2Cka0flv+wFmMHtIQGAA0tMdp2H4T2gS5IiY9BXinQZeg0rF3zFZp4vLovVBl9cwcMHDIY9gYSxMQryl7x/XqMHdACGgC0RObo1a8vrPWkSExOg6NvB3y/ZiVcDaWw8wlCY29byCvLkF2sjQ5dusLRwgBiMyc0qm+HjPQUaIqs0bpDIJqHdEJwI1ekJSehXNMCc774HsPa14dczxytW7eASIvIy8qBtXsTdAhpCp1qr2U9hHQZiC4hPshKS0BOkQSNWw/Auo0/o0NTJ9WHEfgfRhuB7Xqjc7AvMlLikVtMdB48A+tXL0d9J5NqqbLiPBRrmqF7t66wNXvVYUVWWY68YjladeoKDwcz9WyBWpEjKzsX7g1aonVgfZW9DQW5WRBbeaFb5zYwrKX7qigtgoSm6NyzE6zq6N/+zZTk50Bs7oYunVrB4GVzlVcgNasI/i07oXkDZwCAoZUnBgzoBa2SNCSkF6B5l5FYv3YV/Jz/lsOl/wPRQkCbHmjn74LYmGhIde0x6/OfMG9yX+gqNcC87EyY2PuiW8dgiHUAI2t3DB06CJa6pYiJT4KBtTc+/epnzJ82EOK3DA4oICDw34d9vRbo3zMY+alxyCwiBo6dj+9XzIKNSc22q8K8TIgsPNC9S1sYiRRae8OWXdGphQfiY6Ih0bTA9E+/xeKPhkOsdKqCR5O26N2uCVITolFQIcbImV9g5aIpMK0qA9BEQLteaNXECfGxUSiWivH+B1/g689mwsJAdTdgbkYGDG2aoHfPltB7542CAv+/0KBweriAgICAgICAgICAgIDAP4AwFyAgICAgICAgICAgICDwjyAYnAICAgICAgICAgICAgL/CILBKSAgICAgICAgICAgIPCPIBicAgICAgICAgICAgICAv8IgsEpICAgICAgICAgICAg8I8gGJwCAgICAgICAgICAgIC/wiCwSkgICAgICAgICAgICDwj/DfZ3AKp4YKCAgICAgICAgICAj8V/C3GJxRV/egaX13BHcbi4QsqXr230N5Csb2a4O53+1XzxH4tyJNw/g+rbBw9YnqJJmkCNk5hSpiArUTffsQBg0Zhfh89RwF6WFnMKD/OMSklqtnvUJpbhy+XzwFjX294ermjg69RuDk5afqYgJKHF03DxPn/4QK9Qwl5KW5mDmwJZxd3eHl5VX9V9/XDz71vPHexB/wD/W4/1U8uXYMg7u1hpuLK/z8W2LV+mMok7zD7KSsAIsm9Mbg0ateWx//7WQnRWDpByPg7eEKb99ALPx6BzKLZOpirxD/5CImD+0Md1dnNG7eDqu3n0GZ9B3er8CfpqI0B9u+/QT+Dbzh6l4P701egmeJBepir1CaFYUVc0bDt5473Dx8MHzSIjxLrKOzFxAQEPiH+esGZ0UuvvpiFeLTMhF25z7i03PVJf4W8pLicfPqIxib2KlnCfxbkUsQ9fwZEtOqBlFZNj4Z1w/f7bqmLimgRmlWBGbP+AiX7kdDKlfPBSrz4zD/o5k4fTMclW9SLEuSMHloT3y69g90HjYDy5fNg0nRIwzs1h27zoarSwsAiLp1EDPnfYPIpJzXOm1oaOuhTY8hGDdmHEaPGoPRo8Zg/PhJaNPIBi8io2Dh6gxt9Yv+ZTy/uhe9ug9CVIkdFq38EoPbuuHz6UMwfck2ddE6Ob/za6zc+jtiE9NRy+fwP0F5TjjG9e2Gn49GYOLszzFjRGtsXzERo2asQOlrZi0S7h1D96498fvjcny4cDmGd/LC59MHYuqCTahUFxb4mynBVzNHYNKnv6DtgOn4bMEkxF5eh559RyAy/TVTI6UpmDKyL5b/chMDx8/Dkvlj8fyPn9Gz93uISBdqTUBA4P8A/kVCD31FE5E5f/zhJzqZW3P970/URd4auayCeTnZzMjIZGFxmUrew9NraC625onbWSQlzMzMYEFhiYpMZUUpc7JzKZWRZUX5zEjPYpmkkiQpq5QwLyeLGRmZLCqtULmuBhkL83OZkZHBvPwi9cxqSgvzmZmRwezcfErl6rmkpKyIWZkZzMrOZUWlTD2bJCmtkDAnK5MZmZksKavlfmQVzMutehdFqu+ioryEWdnZrFD6cbm0knnZ2SytKquirJjZ2TmUyklJaSEzMzJZXKZ4FyRZXJDHjIxMlkpq+e3/FioS2M7bjO8v+FXx//JIBjoZcsY3v6tLCiiRFX+bQ9r4EQBtfVozOkc1Pzc5nOO6+xMAjR1b8UWiavtT59KviynWMuPei7E1iZI0jmjpxAbtPmBxTbMTIPno0i76O5sRADuNXMpydYE3IC+O4cAWLmzRazbz/4s/37+HSn42oRUtPDoxPq8mdeOCgTSyaciwVKmycK1kPT/Hpk6W1NQAW3T4+J3r47+F33+cSm2RG888SKtOu7htIXW0bHjsTrKKbA0F/KBPA5o7h/BRYml16rXdS2kgtudvNxNUpAX+XpJu76elnphLN16qTst4cpKOBiLO++m0iqwy4Re30FxDzA1HHlanJT04QTtdEZesvaAiKyAgIPD/g7+0wsniRHz5xbfwajcWY8Z1hY1uCSIiktXF3orshIeYO2EoGvrVg5u7G5q26Ir9559U5z9/fBe0cIOo/AkmDOgMTzcX+AW0xcFLEdUy1w+uREibkTh6bCd6BDeCd6MueBRfisyYUEwd1Qd+Pl5w9/BAi9a9ceCPx9XXAUBlYTJ+WDQDgY3rw8PdHX6NWmDZDwdRruxtJC3BoQ1foG2QPzw83OHl3RAfLtmEwoqXaxSVuHR4A3q0C4Knhyc8PHwwaOw8PE9SdfGMD7uMKe/1QH1vL7i7uaNV1/dw5WFCdX5u0mPMnzQcjarehX+Lzth7tuZ+j6+ZiUaBg5BYolGdlvz0JJrX88O+i3EKmZ8/QGCrUbh87SSGdw+Bi5MzOvefitjkPBzfthJtmjeGs5MLer03DykFr5/Tj38WitXfrsSSpZ9j95GLKFWbWC3IjMHOjT9g6dKl+GHDr4jLKFYVqBM5Xjy4hO+/+gJLP1uBw6dDUfHyVkoz8Mu61bj6MFblirArR7Fh6zGUyQFUPb6GhiaAMuz4aTOS8soReuZXfP/jHhTKiGc3j2Pt5gMoVqrH8twUbPl5De5HZAAAoh5dwJZfjiA+5hG+X7kUP206hNwSxZS/pDgTv+1ah6VLluDb1ZvxPDGvpqD/QmJvH0S7tgMhbjQSiyZ3hkSiWpkp4efRq203FDn1wIqPh0JLInntChwAmDg1xMIVX6BjC7eaRF1bBAc2QU5S0mtXT/5tnNi8ED3f+wxDZ65Ct+a2KJe868uR45evP8WpFyKsWrkAJjrq+f82NKCtrQVtLUBLaalXW1cbcrkUlNf0kbVSmYsls+fDod0HeL+dNyoq37U+/nuwa9gB3//4LVr72VaneXq6QVsjG2k5tffZpZmpuHs3Bl0HjkZjJ3F1ess+A1HPKA8n/7ijIi/w96Jt4YpPV3yLMX1aVqdZ2zrCyhxIzUxXkVVGU0sLGlo60NSs6SC0tbSgrcH/2RV8AQGB/3DULdB34eTajynSMuG+S7EkSziiuTO7j/pOXeyNFKc+Yof6tmzQ8j1euhPOhJgwfvheCEXm/rwTp5hVXTiwOY3M6nPs+Gncd+ImE2LDOaGzDz0DRjO3agVl07x+1NSxYMdOQ/n7pdu8deM6Y8NvM7ieFb0CB/HczSdMiH7K+WM6UmzizbMP0xUXVuRx7tBgmth4ce2e80xMSuDen+bSRFuPi346U3WXEv48fzANTZ351ZYTTEhK5LGtK2imp8F5354iSV7atZymOrp8/+Ov+Cw6kY9vHGOIhzmbdJjOnKpFopSwE/S1NWLbgR/xztMoRj0J5aBWrnTy7cOUYlKS9ZSdG9jTt/lQXrj9jAmxTzhrZBvqmjbizehiklLO7dWIfq1mUHnd6erOT6ijb88r0cUkycWDgmhk5sMRIyfwwJl7fHx5Lz2tTOjTsAm7D5rIi7ef8/rxDbTRFXPlL7eUSlJGyv0/zaaFSIeuPk3ZOrg5TcQ6bD9oNjMUP8NHZ7awnoMpre092KZda7ram9PCwZd7/ghXL0wVeSk3LBtLfV091msYyJCgJtTX0WP/8ctZICeZeZc+Nvqcs/q4ymUbP+5JG7d+zCwnKU1kO28zjvx0NynL5Mz+7SnWAW1d6rFP/1nMlsq5fXF/Wri3YbLSy8oOv0IPEyN+s+MuSfK3DR/T2Mic/s2asnGTxmzZ4T3GZZOZERfYtrETza1d2bptW/p5OdHMyoObjt2vKey/jIibx7nv1B2S5Pp5PWns3ExlhfPF/fPcf/AKSXL38jE0MQ9gxBtWOGulLIkDA2zYvOc8SmrxAvhXIi/jmcM7eeVRClmZzj7NrRk8eOE7rahlhp+mm5Eux3yyWz3rX0vyk3Ns29CNXQdP5+59B/nj8ll0c3Dgwu+PqIu+wtEfPqSzVye+SCnijC6ebNzqg3eqj/9mirJiOHNQEK2c2vJZau3feFHqYza10Oe0Tw+ppMslLxhsZ8ROw75SSRf4Z5FJirjty6k01LPhnvNR6tk1VOZwyYTedPcO5vfrd3DPrxvZvYUPgzpNYHzum1f9BQQEBP5u/rTBWZ72iEFO+mw3ZBFfenV9Nqo5vZqOZaGa7JtYP6sPLZ06MjJDUp2WGXODfdq2429Xk0l5PNt7WdDGoxcj02rcejbN6U0T87aMLyPJEk5q40MdYx9ej8itkqjgovdb0alBf8Zk1vieledGsrWHOUd8uI0keWX7Yop1rHnomnIHXs6l7wfTq/lg5snI5Af76Gioz692KxlnsiKunDmIE+ZtYH76UwY6mPC9jzZSWb9+fPIn6uuY8PCdDJIl/LhPI/q1HcOsmsdg7PVfGdKyBx8mF3L7wsG0sG/LiNQatSc7LpR927bjwUuJpDyWrd1NOHz+jpoCSK77sBcdvPszU0JSnsLejexpaNOc92MLFAL5zxlSz4iOjYcwPldxhwVxD1jfWMSZK46plPWSxAfHaKuvxcEzvmFOicKqv3PiZ1qL9Ll003XKCp6yhYuYzbpNY0yaotbz0l5wfPcGNHNtzfCMun0pn1xYRzMNLU5ZvJ2FEjlJGc9s+5zG2iKuP/6CLAyjj60J5685qXLd5rn96FRvELMkJCvVXGrLXrCFkyGnf3WiWn7r4oG09uhIpdfJnBfX6W1hxh93KwzHk1vmEACnLdvJCpKlJaWUl2VwZFsP+rUawfCEfJJkZWkOv57Zm2a2zfggvsri/i/m57k9aOyianAqs/OL0TS2COTzdzY4Jdz82Wjqaltwz7k3TDz8WylPZe9m72pwyvj1lC40tGjGB0lKHci/nIriTM4e3o6AmO6e3rQ0EtHSuRkvv5xQrIPUZ6dY38mZ644+ISnn5A4e/xKDM4uzh3WnjZkBoWPLNQeuqwvUUJbBoSEObNh2gor7dsSlLTTQADsMWK4sLfCPIeOvqz6ih50lAW2OmbeuWu+qHTnP71lJSzFoae9Kd1dbQlOfc787SMHcFBAQ+L/gT7vU7lq7CqFJOhg6qDty01ORnpoEHRNzZGY9xbvEDWJhOLbuv4BeYybAy1q3Ot3KPRjHLl9Cv9YOyI94jsjUQoya+QG8bF+69RTj4aNo2Hn7wFoEID8Wt2MT0WfkhwjxMQMAFMbewf5TjzF++jS4W9W4luiZWMLTwwqJaXEAKrDtl90I7D0J/Vp5VssAemjg0wBFGVkoLwH+2LcXlVZtMKJf8xoRTUMs+OkgNn81GbeOb8PzYkvM/HD0S09PAICjnx9sdSqRnFaC0rh7+P1KHEaMHg/LGu8kuIWMxPWbJ9HEJBXrd51B99ET4GOnV51v4doCRy9fwqB2TsgNf474DDkCmvjUFIB8hN6LgFeAPyx1gfKkOISnZGLI5A/R1M0YAJCZnIyk+BKMGDsKLmaKO0xLDUNGiQ4aNvBWKquGq6cPoMjQF0sWzoK5vsJfrVnPcThy+igGd/HGnTMn8CzFEIuWzYO7rREAwNTWG59+tgTayQ9w+twjABVIToxHQkJC1V8y5AAu/nYEOs4dsHjhaBjpagDQROfhH+H3M4fRppEtUPnmyImvIAfkAOTyd3Maksuk0BE5YXD/3tABINYXIzn8Ji6FpqJfv4HQk2bgSVgYohJzEdKjF7SzH+H3C8/UixEAAHkptq+ahimf7cOM5RsxvFN9dQmBP0lRwn1sP3QFPUdNgr+jUgfyr6YcK6YOwNaLOThy+S6iol7gefh9DG2hgwE9B+JRXO2uopBm4NMPZsO7xweY0rcBwAqFi77GG1xw/xeoJAK69MfcubPQ3FsTXy74BJce1eGeKbLG1GnTEHd1K4aNmoWbj57izIG1mDhjNXT0taCp+S94X/8RSOHo1wIfzJmPwV0b4eDGVdi456a6UDUPTv2MweOWY/DcrYh48QJRUZE49PMsbF8yCsvWnVcXFxD4P0cqKUFyYjziq3XFJJRVVCI3IxXx8QodMj4+HpnZwikE/638KYMz6/k5/LDxNwAFmDokBLZ2DrBzcMbCNWdQXlKEjNS3bxDpkc+Rkq+HNsGN1LOqiYx4jFK5PbqG+FenMS8Rd6Li4RvUGGIAabGxyCoQoXuPVtUycVGPkS8xQlBgzXUAwJIyZKblwcLUFiyOw+OYPDRv1QJaKlIyJGUkQcfIFHrahbh86wl8W7aGrX7tr+zpvXuwdW0FX9caQxEACtKSkCWVw9pKH1Ev7iGr0ggtmqnez0syol4gOVcbbYIbq2dVE/k8DMWwQBOfGuNYmh6Hh7FZaNy0ETQAJMcnoKjEFF1a1xjHCbGPkS+zRKtmAdVpsU8fokhkAh9f5+o0ZdIS42FoYgtLC6Vn1hAjpF1n+LlYITk5HjqmTvCwV40cbGvvDlszDWTmFSDmxmH4e7nB1dVV8ecZiAsRSUiNz4GFjSfMRTXXaeoZoXXHHvB1MQZkCqNRQ00BfL0+SJB89bBWDY3q/Z7KSdUQ0NbRga6opu7S02KQIynDD5+NgX+TQIS0aoUWzZqi+5A5KNDSQ0lutlIBAgAgK8nCihlDMPbTg5j79W58NX+guojAX+D+1d8QlW+A9wf3Vs/611KQcBNbD93E6Fmr0L+tHzQBWDj6YsWKlTAquoPtRy6pXwIA2P/DUpxPMMfi+RMgrShHeXEpKqVyyGUVKJHLIa/qf/4n0bHC8LETMXvB5zh3+jhc+AiLlv4EibpcFW3fm4t9v6xA0u096BDUAh+tPIQxS1ahX1N3iE0VE40C/zS6aN9rGD6cNQcHfr+ACR3NsWT+QsTk1TYxK8eR7dtg6N4Dny0aB0tDXWhqG2Hg1IUY09UPuzZvQ3ap+jUCAv+3JIQeR2A9N7i91BVd/XHx+XMseq8r3NwUOqSbmxtGz9ykfqnAfwm1W0+vpRwbv16JeKkN3h87GZMmTsCECRMwcfIUDO/bCcjPQnTC2wcOKi0ph1xTDAtD5YGrApeP7cXJy2EAgIjwx9C2cYKbp1W1RGpcDNLSgKDAJgCA6IjHkIls0dinZrWuXFICauhCbGRQnQYAaXF3cD+yAEGtgqFRXgKJhDA2UJVhSSYuX3sE9/pNYSYG8opLYWJuomKUZry4hW1bj6BYCpSVlEHf2Azq6w63/rgI6teHfz0LFBYUQK6jAyMTpd+SFOD3fTtw83EayiukkGuIYGGoWJVUUIErx/fhxMVHAIAnj+5Cy8oB7u7m1RLPn9xGUo4WAhv6AQBiY8IgM7CCl1dNEJeopw8hNbOGq2dNwIjox89gZtEYHi761WnKiET6kMokaouNMtz54xgu338KXT1DSMtKUV6qek5jeWkFysvl0NPWhpmjN94fNxbjxk/A+PHjMHH8SLhYmEBXpI3K8jKoFM0yXDr5Gy7djQW0tKGhUYkyiWoId2mFTN2cfAVlI1UDQKW0HBVKxcilMsjUjvogVA1VHW09aMMIP/56BUlJ8YiOjkZ0dDRi4+KRlpqAT6d2ULn+3055QTymD+uGpTufYs2Ok/hy9sB//XEdfzcXj52HjWdbtGhmo571r6UiPxv5cjnsXVQnzUwsLWBiqoms7FrOHZTk49SJk0iKvYOuzT1hY2sPexcP7L6ZgGehW+Dh4Ivfr7/9GPbfQQVCzx/FqYv3Vc5tNXZqgC4h9RAb9wC5dRohOug5ZgFuP3qBhIRY3L15HmO7O+BRZCrqNardO0bg7yE+/CYOHDyBAuXZAG1T9OveGvnZYYhLq+2MZAmyMwphYeECc5WgYmK42NmjKDMTpf/DwbEE/jsxtvfEiHFjMW78eEyYMA7jJ46Gi7kFWvcejNGjx2DChAkYPXoMenSse0FG4D+bdzY44+/8hrU7L2PUx99h5y8bsHHTZmzevBmbNqzHLz8ugZNJAZ5Eq0YWfR1GJgbQlBchOSunOi0z4g+8P2YaQp8rIoI+ffAEjq7N4WBSc11U5D0UaFqgqZ+vQib8AXSsPOHsUuOWa2FuC21pHmJjle5HXoKNX3+NSqsQ9OvZGNA3gpmpBp5FRNbIADh7cB1OPyrF2HEjAOjA2sgQWclpNYM1i/DlrAn4YedJaGkDts42SIoPQ3JRTRmFCTewassRtB/8PuqZa8PQ0AzaklLkKPkcP7q4C+PHTkNsrhwWZgbQYgmSsmpWz7IjL2LUmCm4+UzxfiKfPIeLWxCcq2xSWVEKvl31HUoMLeDt7QIAiI54Bj0rNzg61JjHLx4/h719AFytX6bk4/7DGHj4+8OmjkiXDZsFozw5HPcfJlWnyfNjMHPaaGw6dBtNgkKgVRGD4xdvq1x348IRJJTpoZGvB8xdAvDD+l+wdctmbNmyFZs2fANva2M0CmmCxJhLePS85gDrwuQwfDhuJA6fiQCMDaCrSyTHZ9UUXJGKyzcfgmpr0dUo9iRDqnSwpK6RAUrys5GTWZP29ME5xOWXQ1ur7uVSV6+msDcrx/lLt2BsYg5ra2tYW1sj59kZ9O7SA6fvp6pf8u+lIgsLxg7B/gcy/Hb2Mma8X+NlIPA3IYnBpYeR8G/XGVaCJV+NsXN9NLLUx4XfT0A53nJU2D0kpWuhiZ/yNokqdAyxaN1RXL92CUd/O4rjJ07g5LHD6NbIFm5+fXD4xD6ENK7uKP9H0MCpX77GyLFzEK10fmNZZgyu3omFu1sALGqddyzD17OGYdDEFdAwNIGNjQ2M9LVwdvd2xFU4oHfHGo8Zgb+fqNsnMHLoGJy5Fa+UWoo/Lt2FqUVjuDsouQhVI0bj5vXx4ulpPFR2KS9Lw8Xb9+HSqD4sDIVOROA/CyuvZvhu7S/YumULNm/eii2bvkMDR3sM/2gJtm/fhs2bN2P79m34YHxn9UsF/ltQ39T5WmR5nNrdl5auHfgiq5Yt65I4tvE2ZbcJP5EkK9Ifc3jfDlz8U00Ql1eQ53Jyjya0r9+DZ24+4vXTO9nM24ohfWYzr4KUl0WyjYMRxy46qHLZ6o+60MK1A5PKSLKcU7t4sf3gVSoyrMzglB4NaVuvHX87f4dPHt7gB0PaUWRgw3WH7lWL7Vs1iXoia3654SjDw59w06qZNBHrcsQHa1heFQHo0vbFNNA15Wc/H2J42D3OG9mZppb1+HtoIkky5clpepkbsvvIT3nvcTiv/P4rg30s6ezbk08SFAFmKnMi2bWhLf3bj+XNh894aveP9LQ05ODpq6sCABRwep8A2tbrylPXH/LG2d0M8rFhUI8PmVNOkjLOHdyENl5defneU17/4yDH9+/OBp42dGswmFlykizjlI5eDOq+WCl4USr71Ldiz0lrq1PkOY/Z0FHE8V/UHe1Smh/D/kGutHDx54/bjvDK+WMc3rExTW2b8OrTXJJlXDKmPXV0LTnrs7W8dusqf1w2g2a6OuwyYjFLXhOdtDDlIdvUM6dTvTbcsu8kL57ez+5B7rT36sDHSWUkK7hwWAghduCKn/fy6sWTnDKwPV1sTejaYER10KA2XmYc8TJoEFPZ29ea9UNG8urtZ6wg+eLqTpprg236TeW5Kzf4y/efsIWPE/XEtlyzW9EGjm/4kHoGbrz+XDkQi4ybl46mFnQ5cOx8Xrhxg3u2rGJ9S0O6NezLmKyaAFf/rfw8pzuNnJszqo6gQb9+PpJGZq9Gqb28bxXb9xjGx4poXTy5YTYBsPeoT3jo4CH+umMnf92xkzu27+C+/edYWPGahvBvpTyFvZtZseXgT9WC1FRy1/cfscewD5hUFdyLJAtenKOToRY/Wf+HirQAeXztbOpo6bLniDk8e+ka92xcQW87Ef07TGR6VWwvWfYzjujfiZ+tq+PsQlk5p3RwZ6OQD/jf/2XXTuztg/QwE7N+q/48dOoqTx/Zxm6BzjQ08+GJqnGMLOPGLyaz7+j5zKg6ivrM5k+pCzFHfbSK12/e4I9Lp9BcpMNJi7arBMgT+PuR5Eayb6ArTZwacfX247x68SSnD21LXS0Dfr7xfLXc7WM/sV3XAQx9rggSmB19nYEuBnSq15qb9p7gpT+OcGjnAOoZOfPApRdKvyAgICDw/4d3MjjDL2+jo4UlV2yu6ehUKeP8Aa3Yuud8SknG3dhHB0NdLlxfl7yC/LRn/HBYZ1qaGtHSxpHjZ3/H1HyFMpsXeYa+Tk7cfEo52mUpP+rdjL1Gfk2SlJXFsa+/Bz9Z8+rvFGc856dTBtPR2pzGppZs02Mkz9x4riIjlxRzz+qFbOThRGMjY/o0bss1O06zVKYkJC3j/rWL6eNsSyMjUzZvP5Rn7ygddE/yyZXf2L9DAM1MjGll686Js79mbHrVqF1F4pMrHNqtBU2NjWjj6MNPvv6V+UoaZ2F6BD9+ryutzIxpYe3AsR99zZS8GoU/4fE5dmnmTQNDYwa26ctDZ0O5YlJX9p/4I0lSLolnvyYenLGy5kiAivS7bOHtxC+2Xa1Oy3x2gY3dPLn1RFh1Wm3kJj3m/IkDaGdtTiNjM7bsNIx/3Impzq8ozOKGLz9mfTdrGhsb09GjCRd89Qtzi94cCy854hanjehCG0sTGpuYs2PfCQwNT63Oz08J48cjutHS1JDmVvacvOBnntq+jIGtJjBHQlKazH5BXpy2/ED1NX9sX04XayPaODXlrbhSkhL+vm0V/T1taWhkxEbBvXn69GkO7NyG6/crDsU+u2MRndwCeSda1bCSSYp4cONyNvNzpYmJMc0tnThk4iJGxL2MgvzfzdZlw+nu34Vxeeo5Cg58N4Me3p0ZlaxqEh3+YSL1rX14PaJU8c0PCaahsRktLS1oampa/WdkpE93rz6MLa47WvG/Fkk6R3Spz57jV6oZOBX8+oMutKrXkVEZNalJ907Qx8aNO04/VRYWIEmW8vdfv2dQAw8aGxnTytaFYz5YybjMmkjS0tTbbOBizPc/2alyZTUyCecMaMZ2vRb8T0epjQg9xeE9gmhmakITM2t27j+Zl+/HKUmU8NPRwXRo0o9JL/sFeRmPbV5Bfx9nGhoZ090nkCt+/o1VgcsF/mHyk59x/sT+tLc2p5GxKRs278YdR25Q+fWf3fwxRWYuPHO/ZmyKD7vMcYPa0crChMamZmzeYSCPXxb6DwEBgf8bNKiIsvJWVErKUF4ph76+AbTqcMatlJSjUgaI9UWQSytRXl4BPbEY2nVdUAXllSgrqwA0NCEWi6r34VFWiZLySojFYmhVR8STo6y0DJraetDT1QYoQ2lpOXREYujU8jukHOVl5ZAR0BOJapUBgIryMlRIZdDR1YOebm1+poSkrAyVMkJXJIau9qvlyKSVKJdIoKGhXfUc6hJKMpo6EIv11OPZgHIpysokr7yLl1RKyiGRyqCnJ4KOthYkZaWAth70dLQAylFaWgZtvZr7o1yK0jIJdJXej1wmVdSNSAyt17iWAgApq3p/GlW/qf7cREV5OSqkcmhp60Isqu3d1YFcirJyCeTUgJ5YBG1N1bJV34UYlFWivFJe9d6I8tIyaGjrKtqB4gKUl5dDKidEIv0qt1miQlKOiko5dPX0oKujhfKycmjp6EFHWxOyygqUV0ohFulD7ecBANKKckgqpYCGNsQivf+ZyIyVFeWokBJisRi1PZK0QgKJVP5KvrRSAkmFDCKxPrQ0FXUgq6Mb0dDQhFhf/EobFyDKy8pATW2I9Wq2AaCqn62QQeW9K76DCuiKRG/sS/+tVFZIUFFZCQ0NrVf7TcpQWlYOrZdjRi1IykshhzbEItX6+F9DLlOMy4QGRCIRtFTak6KvlMo1IBapjl+KPlQGbR1diPTeoY8X+MvUjMGArq4edHVU27BMKkG5RAaRip5U1W+US0ACunriWsZuAQEBgf8/vJPBKSAgICAgICAgICAgICDwtgjTXQICAgICAgICAgICAgL/CILBKSAgICAgICAgICAgIPCP8FqX2tWrV+Pp06fQ0RH2awgICAgICAgICAgICAjUjUwmw+LFi+Hg4FCd9lqDc+XKlXj06BF0df+3gygICAgICAgICAgICAgI/DVkMhm++uorODs7V6e91uAUEBAQEBAQEBAQEBAQEPizCHs4BQQEBAQEBAQEBAQEBP4RBINTQEBAQEBAQEBAQEBA4B9Ba9myZcvUE19HRXEmdm9Yh6N/XIaOmQecbYzVRVBRnI5f1/6Mkxcfw6m+L8wM/vf3gD66dAAHz7xAo2b18ddCLBGA0mnb/yDhN45i99FHaNCsAXT/9p+U4uLRnbj4MBV+Dbyg/Zbls7IMR3asR2SWGD4eNurZfxIZrh7dhfMPM9DQzwNab3kvAAASL08/z88Ix+Z1u2Do1ADWJnrqkgL/KASpoXIQ/Z8hMewqftlzAe6+fjAUaalnQ15ehH1b1yO5xBRerpbq2f9xhJ46gPBMwMPZWj0Lz0LPYtPm7bhw/T50jO3hbGemLqJCSXYkfvr2J5w6dwFXrlzF9Vt3YWJdD7aW+uqifwuyggSsW7cVcmMXOFobI+7BJfx66Aq86jeAvt5fmwtNfHoNx888Rf3G3ni1lv/zKcmOxc5fNuPg0dNIzKqEu4c7RDpvbvzP757Dxs3bcOHqPWga2MLV3lwlvzgtHOs2bsaZPy7g8uXLuHz5Ms6fu4CkLKBRA7fXjzwsxtFf96BUZA87SwMAQPKLGzhw5BHqN62Hl7cXfe8MTt1ORcP6Lq8v7z8EuSQfJw5sx469R/DoeQocXD1h+hY6S2luAnb/sgkHjp1CXHoZPDw9INKpo91Ks7Bv628wcHCHmeGbywaIO2cO40GsFPWqxsGyglhs33Qctt4+MBZrq0jfOrUTD5I04eP2csyU4ty+PUipMIaLnamK7H8qpbmJ2PvLBuw/dgaxacVw9/SEuK73qURSxE1s3LQVp89fQwlM4OFqC021hleam4g9v2zEgWOnEJtWCk9Pz9fUVTb2/XIE+nbuMDN6y7o6exgPYitRz8MWAFBeEIttm4/Dxku9rspw/JdfkaNtCScbE6V0IC3iKvadfQzf+vWgU9Vp5SU8wJ7jN+Ht4wtd1Sr/16Okmv0pwq4exv7fn6Fhc7+/qLOr8uDifhw+G4VGgfWhqDI57p8/jE3bduL2wzRYW2hg785DMHX0gcX/hR75V1/cX4HvSGHKAza31CYA9hn3g3o2SfLpH2soAgg48lRYsnr2/yQbZnemlWtvZqlnvAPxT6/yqy++ZUa5es4/w+5l/Wls055Jleo5fwel/GhIIP3aT2D+O5QvK85kn4YW7D9ps3rWX0DCuf0CWK/NVBZK1PPqJjXyJj9btprphRUkyci7O2muYcRt55PURQX+QYqyovjD0qV8mFCgnvXOXNoyn4aGDXg3pvayJNlxbOtqwPdn7VXP+o8jM/Iivc30OXXlHrUcKQ+t/ojGYjH9AkLYrKE7xfpm/GLTH2pyquQn3+WoAb3Zq3dfhjTzpsi6Ma88zVUX+9sojz1Pe2MRV+66RpI8tfpjmln681l8qbroW1NRnMat3yygg7Eu6zUbyyJ1gf8C0iMusYOfLc3tPdmubTDN9PUY3HMSkwpk6qJKyHhs/Vya6ovp6x/M5o08KRabcMnak5QrSd07uIoGWhp086jPhg0bsUGDhvT29OG4D36mVEmuNu4c/Zb+TXowMktGaVEaH4Yn8P75dWzbbAqzczP45NFzkmT6s1MM9G7Ck3fS1Iv4j6OyMInT+wZS38iSrdq2o6u1MR28Q3jj+etH8uy4G2zn50AzGw+2axtCC0NdBnaaxKRsxVihzqnNc6gncufZe2/3TrKjzjOofmMeuZ5IspQPHjxjWsoNtvHqwoeRKYx48IilcjIj9hkTUnK4ZX53Tly+j5lpcXwRl02SPLN5AQOChzK1RL30/zxy426zc0Mnmlu7sl27VrQw0qN/m/FMyH79gB169Ce6mIno5hvIVs0bUk9bxEmLNlK5FnLibrCdnyPNrN2r6yqg08S662rLXOqJ3Hnmbqp6Vq1kR11gUP3GPHxNua5uKurqRQojHj5imUzG6KdPmJ6bxRnt2/GnfTeYEvOQKTnllBSk82lELCPOfscWvWYwOS2Xjx5FkSQr8l+wd4Afv94Zqv6z/2JkvPX7Nv605TeVen5Xts7vRjPHzkxT7iD/BtbObEdrj95UfIXkvdPraaWnRRfvxhw69nNePbqZliIr7rsQq3blP42ct/7YzW/X/KYyJvz/5J0NzqLUxwyyN6JYbEhnn3aMzFC/9UquGN+Dutqa1NRw4ZmwFLX8/002zutBe/f+1Y3sz7B2fm/a+/Vg1ptG/r+JPV8MoaVdF6a8g0H49pRx7rDmbNRhAgve4XnkJdns52/LodO3qmf9BeRMjXvBiKhEytSb62vYs2wobVx7Mb1YoehJSnP55OFj5hb9lW5O4F15cHQ5dfWdeCOuWD3rnbmybRHNzZvwQVyhehZJUi6tYFR4GBNS89Sz/qPIiQ9lzwAnAuDH3xxSyUt+fIQuBtoc88lWlpFkRS5/mt2fBqa+vPr8bQzIfC4e049fbb2unvG3Uh5/kQ4mRvxqz02S5Nl182htF8iIhDJ10bcmO+YKP5gym4tmv8+gwHGsvZb/bynKS2dOQR2zirICzuzTkA6+XfggNock+eTSLnqa6HHy0ronQdKenaCHsQ5HzNrAUjnJynyu+2QI9Y28eeFJjfH0y6LhdPTuxMjUckrKy1hSUsqSklKWlb+hTyuOYe/GXvxi61WSZMq9Ywxq4stmAYGs79mQQQ092XvkYhZV9fW/fDqcLbt9wKq5uv8zJKV5TM/KV0+uZu+XY6lv6Mj9FyNIkjkJ99izoQ2b95jFuqc9yjhvaAtauLTn3WhF2c9vHaabmZhjFu5UF+blA9/SWh+ErjcvPcpQz66FUi4Z0Zb9J35HkpRmR3JYp6Zs0qwlfVy92SqgIQND+jOugDy1cQEbevmxWWMvNggIoLeHD5f9eEZRjCSTw9o25swvf1Mt/v+AsqJsZubU9TWWc+GwIFo4h/B2pKJ/ehF6hB7mIo6e96u6cDXl6Y/YwtmAHd5bxNwyOclyHlk9m8YiC+469+KlFOcPa0Fz53a8E6WoqxehR+huLuaYBa/W1ZUD39Gmqq4uvmVdLX2/HftN+JZyktKcKA7rHFBTV4GKuorNKuHq+cPp0yCIDb3rsVlAM9bz8eO+y4nMeHqWrRp4sXlQE7r7NGITXz/2GL6EL+cJbh34kr4NOjEy/R0Uqf9psjmohRN7TPqBr5uCexM5qTF88iSaf7f6m5UczSfPYqsn8H76sCPNXTswvqr5lxfl8snjJ8wrev1kyt9OZTHHtnVmt/dqXyj8/0EdPgWvg5DKdNCjx2BoFkbhws0nKrmS9AhceBiPQX2HwkhXDvUYuBXlRYiPjUZUVAxy8ktUM6uolBQjMS4WUVFRiE9MQXmlvDpPXlmKpPgElFbKUFFSgNiYKMTGJaKkXKZSRl1ISgoQHxuDqKhoJCSlo1JWc4PyylIkxCeiTCKDpCQfcVVll0peLVtWWYrE+BhEx8RBUknoar/Fq6QM2enJiIqKQlxCMsoqap6rvCAN6ZkFkFWUISY6GUUlkuq8suI8xMVEK+45OQNSpXumtAyJ8YkoKZeisqwQ8THRiIlLQHG5tFrmJTJpGZISYhEVHYuyCtR+z5QhOyMZ0VFRiI6OQXZesUp2fmYq0rKKICkuQEx0NNKy8iCvup2KsmLEx0YjOjYRgBS62m/nzFZamIOY6CjEJ2WgopKo7bZkleVISYhFZFQU0rLylHKI7NQkJKZkVd/HS4rzsxGfkAypTAOGJmawMDNW8SSQVZQiJSEOUVFRiI1PQqmk5p1JSguQnFkEykoRExONgmIJtHXEsLKxgkhX+QaJorxMxERHISY2HkVllUp5QGlhNhIT00EA+VmpiI6KRHJaJpSq8DUQ+TlpiI6ORlR0DDKyC1Ryi3IzkJKeh4qyEsTFRCMlPRuy6iYlR06Goq3FJ6ZCIn39D8okRUiIT0alpBzpyfFq74MoyElHdFTt30NJQTaSktMhJ5GbloSoqChkZOeryLxEWl6MxPhYREVFIz27AMp3JZOUIikxGaXllchMSUBsfBIKCwuRmJQBUIak2Gjk5Cq1R0qRkZqIqMgoJKVkQFrzOdUgq0BacjyioqJRUApoadbSuJTQ0NCEmYUVjA1FAIDy4lwkJKRCRqAwNx3RUZFITEmHUpdUK5WlBYiLS0BZhdK7kkuRlhiPjKxCZVFIygoRFxOFmLgklFfIUFyQheSUTNT1E9eP/Iz2nYdB07EFHEz0VPoDALh4YC8KjZpjwZz3IQIAHTNMmL8IXrqJOHT4JoBKvHgahsdhYQgLC8Pjx4/xLCISL5vu1d0/4lKcOSaODlEptzZKC3MQGx2F6Jg4FJRUqGdDWl5U1d/GICuvSD37jUhKCxAfE4OoaNXri3NT8fjxY4RVP8MjRMemwdy9DVav/xadA+tDQ/pqv/2u5GYkIz2rCGAlkhPjEBUdh/yiV5/zzciRnRyJdStno1VQcxy6lqwuAADIjLqH389FYtIHC+HvpnCHbdBuKKaOaIeTh3cjVbXpVHP50D7k6DXBwnmjINYAoG2CsXMXwc8oDQcOXq2SKkFYxAs4ejeGs50edPVE0NcXQ19fDJHe653Kzuz8GS8qHfF+/9YAAPuAPjhycBucRHmIiIlFwIBP8MuaRTCs6vL7j5uAgnsHcehCtGpBr0MuQ3pyAvIKJZBVliAuNhoxcUlvPbYrU5KfjhM7f0Kn4CZYvumceraCygzs3XECbft/hCHtfQAA5s4BWLBwCl5cPYjQZ7W31+L4UOw5E4YJ8xYi0EPhFlkvaADmjemE33f9iuQyhRwlefh2zgiM+WQbWnfsAiMteZ3ftDIJ945j3/lojJ88CgCgZeGFLft+Q88GhngeHwk9j87Ys28bXI2B7hOWYeuaj5GXloS42GLM/WYrPpneVVGQrhWmjO6Jw1t/QmRWjT7xJiirQGpiAgpLpagsV+hYcQnJKK98/RjyKkRBZgr2bvgCbQJbYOORUHUBAEBx8i3sPHkPY2ctQ3Mvhdu/d4v+mDehK07s3YzE2tVE3P7jGCIyLbBo7gcwE2kA0EP/6R+jnbc2dh84CQAoSaipq2aeirrybtEf88Z0xondvyJJqa6+mzMCoz/5Ba06dYGRlgxvc4BD4r3j2HsuChOmjIIGAC1zT2zZ+xt6NqyqK7fO2L1vG9ws9TH988348oOOSI5/gfQyM/z4y28Y1NYJ1n5dsP/or7BnCWIjo9By4Exs37gILzcyBPUZDmfZc2zc9Yfar7+enIxkZGQXAZQiLUkxBmblvtqmZZVlSK7Sg1LSs9+gm8iQmZyA6OhoxMTEqPxFR0cjMSnzFT3sJSX5mUhKygRB5GWmvPb3ZBWlVfcUjbTMPBU9IS85EQWlFSgtyEBMfCYkr+nnSwpzEBsdjeiYWOQVVVV2FSIDU1hamakEspFJy5EUr9CRC4olqCgvQGJCMhSqukLXzMwtAViJ1KR4REXFIFvNjtE3MoWVpSk0QOSlJyAxLQ8mBhYoyk9BXn4ptPXEsLKygkhXVT8uys1ETJWOVZv+XlyQhZhohR2Qkp6j8p5lkiIkxCWhQipHWVEuYqNffrMvexw5slISkVtUibKSLMSnpaNcyf74/4a6BfomilIfsam5ET9Yuo2Tu9Znt9HfquTfPvYVfer14p5NK2gIO55+XLPC+fTqYfZp25iG+vrU1zegm08QV+84p+LKE3bpIPu2D6C5iRENDPQpFhuz68CZfJasmB7IiT5Dd3MHrli9jkM7N6OJkT51dUVs3XMinyTW7ib3khsntrBj8wY0MTKigYGYIgNLDhy/mIlZihn1ohe/08ncg1+uWcNBHQNpaqRPXV09tu01mc8Sa2ZJs2PvcFyfVjQUi6hvaMIW7QayX3tfutYbXPcKp7yIGz+bQVc7c+rrG9DAyIQdek/io9g8knL+OH8oNQBCQ4O6+vZcv+8BSfLykXVs3cyHRkaGNNAXUWxsyeFTP2NqrmJeRpJ4mR427lz2/WqO6NGS5sYG1NHRZcsuo/kguuZu8pMecergDjTSF1HfwIhNQ/pycNcmtHPqUb3CKSlI4hdzx9DN0ZIGhobU09Oho3cA1+25XF3OyrFt2azDCE7uG0xtTS227vshi0kmhP3BAR2a0EAsor6hGTv2Hsy2/q4M6DqZha+ZmLt1YitbNXKlnkhEY3MH9hs8mY1dTDjig23VMmlRoZw4qCMtjPWpb6BPaydvzlmxlQVVs+e7lr5HM5sgPktVXhkp59JRwazXfBRLSC4dHswmnWawuOpZ4x78wWHdW9HazJQGBvrU1TNkSLdRvPVU4fK0d+UoAqCGhhZ1RAb8avcDpj47TAexOfdcrmrTslIeWLuMjb0cqK9vQH2xAf1D+vLYZcVsOUmeWj+d9i6duWHjSgZ4OVJfpENDU1t+sHhz9WpAbcjLc/jz8pn08bCngaEhRSJdWjnX5xc/H2FF1bTe9k+H0qdZL84a0Y1iHS36tRzKlBKSZRlcvXgK3ewtKBbr09jUir2Gfsxnr/k+Uu/soLWpDz+cN4UuVkY0tHDnwatJJMu596dP6edmS32xPg0NTdiqy0heV/JcOPjjdHr5duDaH5ezvoMF9XR1ae3oy2+2nFaZgXxx9yQHdmxGE2MDisX6tHGqz0+/2s2XC8ap90/R18ONs6ZNYz0HUxqZuXLyzAm00NMmoEEtbRHHfPQLSbIsO4bLpg+lg5UpxWJ9mlrY8/2pXzAhp2bWsCw3josnDaCFiQFFYgPWa9KFI/t1pLlVAB/WscJZkZvEzj5WnPzpQZLkzb2f0tImhBu3fstgPzcaiHWpb2TJcR//wJyyuudXw898T7HYnn88rVldqiiKZ2c3cw6ctKE6Leb+afZt25giPT0aGlmw9/CZnDm2J/1DRrK2dRl5URo/nTqCP+66wtLke2xgo8dpKw8oSRRychdfNmg/gyXKU7eSPI5q5cTWfZcyvyiT21Z/x2++/Y7fffcdv/3ma/68cTszikgyn+Nb1+enGy8pXVwbcl48sIYtG7jSQF+fYrE+/Zr15OlbNW5CkXdOcUCHABoaKPp7J09/frP5ZLUb1JtWOKPunGDP1g1pZGBAfX19Onr685stp0iSiU8u86uvvuZ33yme4euvv+KeQ1erx5JLez5jUJPRf3mF85NBAew6dAG/njeaVmZG1NHVo7d/Dx69onAffSOySkY+usx5k4fQ1kiP0NBjcLf361xhv3d8FbV1jHkkVHUkubBpIQ1F3rz5XLHqqUoxP+jVmD6tJqpuGags5IT2bmzeZR4rScqLUtm7sT2HTPiSm9d8wcmTJnPpyjV8rjS21UpFLke1cmP/CetUkq/vX8XgtkO5+YdP2LhpFz5JVup/ZUWc3tuPbQctfesVhMr8FPZq6sQZ83/kzBGdaWwopq7IkCHdJvDui0x18VrJSAznd0s+oJ+zBQHQza81j1x7udqlSnHcBdoZanL+WtW2nnL/FB1FRly9955K+kvCTq6jqaYVD15RdYm7tm8xdfWteP6Zom7j7p/h6GETeCsyhzd2f0kjHVeef/jmVbO1H/agV9MxKuND2pPTDGkawo2b17KFXwPuvxxdlVPBr6cP4HsTv+D8ib3Yc8QXihXuKorT79Hf2pDLt96qSXwDRWlhDHG15fwVmzi2dwsa6Isp0jdmt6EfMSLpbZzU5UyNvMfP542nm40xAQ36Ne/Ny49q34oSfnoNDTVMue96jEr6tYOf00jkyAuPaxu35PxpTi+au7VirJrTxvIRIXT1G8ESks9ObaCpphUPXFYt+/r+JdQVW9bU1YOz1XV1c/cqGum48PzDdJVramPdhz3p1XS0io6T9lRRVxuq6mrfpaq6Kk/m+B7BnLN8Lcd0DeTM5TX99rV9q9im7WBu/v5TNmrUlU9SVP2gtyzoS+cGA1mHF3CtzB3QlN2HLeA3C8bSycqCero6tHPz5+bDiv6WJAtTn3HakPY0Mzaigb4+zaxdOG3hGubV5WQizeHYVvUIDS3q6emp/Glpgr7Nx1NpCFZh/9dj6FqvD9ev/YyN3OwoFunQ2NyR81buUnjiVBEXdoHv9QihmYkhxWJ9Wtl5cdayLcwtI8ksjm3TgACopa1FE5OmvBFTu9Z9++RmtmzoTkMDA+rrG9CrcRvuPFHzTW9f3J92Lr350kmzIOUppw/rpNDrDYwY0GYIv1g8hU4OgQzPIckyTmnvy74TPufnH46gg5UZdXW06egdxF9PPKwud/2c7nTxGcqcimJO7lJfoUdqalFb04SL1vzBxPtHaG/iyN+uJSgukJfz8MbP2MjDjvpiMcUGxmzRcQRvhVfpDvIyHtr8BZs2cKOhoSH1xXo0tnLijEVrWVD14lLv7KCViQ9/3PQzewY3pLGBiHoiA3YfOptx2RKyIovju1fdi4YmLV39eS38r3uMvSt/0uA04IfLj/Hklpm0smvJKi8IkuRn74Vw8KytDD34LcVKBmfak5OsbyVmcJ+JvHb/BeMiH3HFnKEUi6y44ehjkmRO1GX62eixSafRvHbvOZMSY3lk2zd0MQRHz9ulkIk5S2cDHZraOPPTb35ldFwCT+xYRXtdsN/E1TU3okbi/d/oKAbbD5rJu8+imRj7gnvWLqShljbnrj5Nkix6cYr2piKaWrpx4TfbGRmXwJO7VtFaFxwwda2iIEka32/vTTPnEO49eYtxUU/4+Yz+1AToXn9YnQZn6OFvaG5oyeUbTjAxOZlXf9/BBnbaDO69gBUkKwozuGx8O9p4deTdqDSWV5IxN/fRUkeL3YbP4/2IGMbHvuCO7z6ivqYeF29SDJIViVfpYWNAQ1Mnzlq+kc9jEnj+4I90NNBgl5GrFAq/PJdTejamoW0Atx+5yviYcH499z3qArRz6V1lcMr57Yxe1NSx4zdbjjE2KYnP7l/i2J6NaWjdjE8yFD3qV+PbEQBHzfyKT56G8eadZyzNjWYXPxu6NujC41ceMub5PX44ogMBsFmv6XUanKlhv9PNXMSWfaYzNCyKj2+dYv+WXgTA0R/vIElW5D5njyaOdPbtzMPnbjMpKYa/fjefdoY6nPG5osNODz9JZwM9rtx2p7rsopTbbGAp5pzvz5EkPx0cxEYdp7NETlbmPmd7bzO6N+nGk9cfMTkpnheObqS3jQE7jfyCFSSlZUX8bkZPWjt1YOjTGJZVkrEP9tFW14S7Lyna9O/r5lKkqc2RH3/NsBdxDAs9wyGdfSk28+X5B4rB6uymDwmAfs06c8exq4yPjeDyD/pRE4bcdCy8+n7V2bNyIgEjzv9qB6MSkvjiSSjnjm5PLZEbzz1TfHA7F48gAPYYNocPnzzl1Wv3KGclf/q4L/VN7Lhi/WHGJybx6ukdDPK1ZeN2k5hVh59Y6p1dNNQG7dza8OiV+7x77Qoziip54uePqC8y5IdfbGZkbCIf3vydfdvWo0O9bnyRoVAlj/yseEZX32BuPXKVMZEPuGBMV+pqmnLLccW3nRd9jY0dxKwX1JvHLj1gXEw4f/p0DA21tfjhyiMkybSHZ+liBFq7BfG3c7cZev0G41MzeXL9x9QRO/LAlSfMLygn5QWcO7gFTe3rc+2eP5iYlMjT+39iPQcTdhr+WZXCVc4VU7pS19SNX285wYSEGG5eMZOWeqDItAUfxdeuOFXkJrKjtzknLlQYnLf3LyUAejYI4Yb95xgfH8kfFr5PLejwqx1176sJP/Md9fRseVbJnbGiMJ6dXEzZf8J6kmRp+iO29rKgo28Pnr4exsiwm5wyMIQA2KTd2FoNTspllEgUWkdZUijrW+ly2pdKBmdlGvs2s2WH979QVfTlxZzZrwEbtJv42n2NpcmXWM+hIU/eqV05fMmzi7/QXEeH7fp/zNtPY/j03nn2beVOU+d2jMgiC2Ku0t/ekAGd3+eF2+GMj37GHxePoYG+KX/ce5t8jcH5PLmSLE9h/2bODOg0gXefxTI+8jE/GduJ0Lbjb6Fv3qZxYecS+jd4j7Wpqu/ComHNCGgzqMP7PH/7GZ+Enmbv1u40tm3BezF1D9jyimJeOrGTo/u1olhPTO9GIZyz7Hs+fpHCisq6Jyoubv6IugZuvKW2j/XhsW+oD3ueUprArUaaycEhjmw9ZBFVdb0yzh3qT5+Q0SwmmRd/id4iDWqJLdi6Qw8O7N+N9hb6NLVrzGNXXhowr5IbdZaOBnr8eu99lfS4x9d4/sYLysry+PvRo8woVu3o9345nnZurRiTWffzKiPNT2Uff0tC04h9Ry7gw+cxvHJ8Mxu5GbNe0Ghm1Pm6ZXx6+w8umD6Elsb6tHH245gZn/DczWcsKatbQ8+8v5+memL+XNVHvSQ/+ho9jQ25dMMFlfSXXPp1CU0NPRla1Q+/5PGFtTTVMeehGwqjUlpZycpKxTu5sn0ZDXVceeFNBqckkV18rTjoI0Uf8ZKC1Bc8fuIyK2XktdNH+Czppcu/lFdPn2BEcj5zkx/z+Klbqt99ZQEndvZm675L3rhH9yXF6WEMsRdTU2zJkR98yWdR8Ty56wd6WuuwZa/ZdY7nlJfz3qVjnDmqN80MRXTy8ueUuZ/xxsMolkvquoi8suMzGok9efWZqvb09NxGWurZ8Ni12uKAVHDpqCC6BQxmjlrz2jC3Jy3tujKb5LWdS2lq6MlbanvRwy6uo6mOOQ9er6WudnxGw7cxOCVJ7OpnzUEfqk7EFKS94PHjr9aVvDKfp4/9xswCGeMeXuSFWzWTVnFh13j+xjPKpfk8ceg3ZhWpTtM8OL2aVsYO/OPR22yJULBwcAChIWaPQdN55X4EIx5e5sBWHjS0a8EnaVKScq6a2JFWbq156voTJiXG8JevPqAOdLh440X14hTIKxl+P5QXL17m5UtXVP4uXbzE0LsRrKxj29Lh7ycoxraQXtx3+hbjo5/w0wldqalpzr0X4kiSxckPGOxhTNfGHbj/zG3Gx73gxuXTaKqnwTHzt1NOsiD1Ebs2smGH4QsZk5jD8lr605Lkewx0NmOvMcv4LDqRkU9ucnS3RhSZN+G9RIWVtm1RX1o79WQGFTry1F6NqWfagFsPX2Z8dDhXzR5BkRZoZt+cEVUG54xOfoS2AfuMmMtbjyL59M4f7B5oT0uXDoyuqpr1s7vSwXMgc0kWZSZx3tBAuvgO4tOkFBaWyRl7bQ9tjex55KrC4Ly463PqQpcDJy7nk+gE3rl4kCG+lnQLGM5sKXnrwCrqQYejPvqGz2LiGRMZxu8+eY+aMOaWs4o2lHpnF410tWjtUJ/L1x1gbHw8D6xbSCOA4xcptmLkJEVyQKA92/b7lImZWSyvqKOi/kH+pMGpz+mfn2BezGV6WVpz6/FnJElp5n028/bh7gvxvH/kK4pgy9NhihWj76Z0pZNvDybmKTeOUn7UrxF9QiaylGTCncN8773pDEuuUhPkFUyKuseuftZs0fkTyknmxpylra4Gh8xQ7pClXDjYny6Nh7K2+V+SfHB2B8dOmMXEDEVjk1VWMi78DJva6LP3ZEWHUfTiNC31waEfblK5dv6AALrWH8FSkpHnN9BY25Drj4UpSRRwSpf6dPAYWKfBuf+7yTQy9uCp0JrO89qhnzh26jJmVmkJa+f2oqNf7+o9nKG/b+GEiXOYmKV4Z5WVlYy+d5z1rHQ4ZM52ssrgdDbTYI/x31eXS5JfjGpDW+e+zCWZFLqHFnpifrNboegpKOWcAU1pade1yuDM51ezx/HrTWerJcqK8rj9y4nU0bPk7/cU6u/KMa1oaB3AZ0oxFa7sXUItbUsevVGzyV5WlMA+/hZs1KXuFc6NH/WmqXUIw9Nr1KScmEv0MdbliJmKFc5L2+dTpO/FS8o/SHLbkmG0dg7hiwySLOTUHr5s1u2j6n0P5zfMpolpI96OLSIp46dDFAZnGcms6FucPGoUrz2oUt7kcuamh3FEGy96tBjNvCpdb9eSYbR378P0YsWHGXN/L+30TLn7SjopT2EXHwu2GbxERckrSX3AAEcD9p38I0ny7MYPCBhy9x+KIAAkWZj8lI3MxZy4oK49WeXc/MWHXLhqF6VVfYKktJgnf1lMkZYuN/yumK3dsWgYtXQdefF5zWpJTuwl1jPW48K156vTSDL2xq801TPlxpNPVdJfknpnF0Va4NxvagLLSIuj2N7VmMNmb1GRzY+5Sk9zQ85brdgrdOTnGQSMuf1kzcouy1M4INCOzXrPJ0nuWD6CuibevBWtvLIj5TdTOtPYqjWjC8issD9opwNOX6W65+jB4c+pp+/Km1V7OGNC99BOZMD1x1Sf5fbBFRTp2vN0WDZLkm7S11iP0z9XLkvKNR/3odigCR++3FChhsLgtOCkKoMzdN9iAjpcs1/hcUCS5TkJbO1szAETqyahaqFWg7Monp1cTdl/oqLvOrVmOsUGHrygtFdIVhDL3o2sWL/VqDcaS7UanCXx7NjQhB3HrFBbWSrjJyNa0K/d+NcanLLyAkZFx7FM8uogXoOMi4YH0NFvIFOVjICUiEscP3I870dnc9vSobR2DuaLqomq6utGBNHTfwwLSVYkXKrV4HyRIqUk6xmb24g47MOfWSRRfASl+VH8aMx7PHAhUqnM2nl8aTdnzfjuNfvw3o6Fg/wptmrMR0k1ey6zIy/S3ViP05ar7p1V5tKOhQTAes178/ydSJa+9n3WcOz7cdQzcOedJNU7j7r4M421HXnyUS3Kd1kSezS1YOvhqn0RKeGyca1YL3gki0nmxFzl+wP7c+3uSyyrahxpkbfYtbENnZsOZlodBl3Ysa8p1rTmiYe1GLuv4e7h72kscua5R28XKEdakMru9Q3p1XIkq5yOSJKPTq6mvoYhN9XRd6U/OEgrQ9DAyovbjt5ifnEdSyxqxF3dSiMtXW44qTyWk5KU22xgbcYlG1T70Jf8vuFjGuh78XaE6hcaGforbUUWPFhlcCrztgZnUcxVeproc/EGxUTpX6eSX03vTOdGvZn2lsGDitPD2MxChy37zmWJkk56fusCirTtefx27ZNREWfX0lgHtHBszt8uPGTxa4x9ZU7/PIeGIm/eiFA1pqKv7aWjiS2PXq3t98o4Z4AvPZsPY56a3qyITdGVOSRPb5pFA7EXb0eoTt9F3d5JW5E5D1x/1ah8W4OzOOYaPU30uWj964Ox/R2kP7lAFyNj/rC7ZlL9Tczv34jGDs35Qkl1enj2expqWfDQlVSSEk5q48aG7SYyWbF8SLKI386bxFWbFYswfyeHvh1DaFjw2I2a+syJvkMvAxFnf3WCJPnbmmnUEjnyXJiqvrdp/kCKjfx5P7GMZA4HtnBiz8nf17mHM/nhSboY6HHBD0f5sutNj7vJie+P5JUnCi1926K+tHHuxRyScVe301zPkD/tVX6/ZVw8PIgGVv7VBufUdvVo5dqVCUqD6I39SyjSs+TJ+4qJhfWzu9HRcxBftuavJ7emV5Ox1bppzFWFwXn0ejLJQo5q7UT/jh+ySOlhwq8f5JixMxmdWczT21Zy+uyvmV81JFSUl/PemU201APnrlF4H6be2UWRJjjt88M1hcglHN/Wg41CZiq8iiqLOaa1C3uOVOim/xe8fkPTa5BWSmHqHogWPkY4dkqxR+L+1fMo0bRDm9YukFVU+SBraALIw4OoGKTE3UGfDs3h7+8Pf39/BAQE48DVCORmv0BKLuDcbADWrJyI3374GMFBzdGkcRO07TIQF8IzIa8shbTqwBANTU14+DVQuhsNiA10IJUWQ/qq6zMAwL/LKHw2ayjWLBmLls0D0aRJY3TsMQ6PMkohLy+vEdQAPP18lS+Fgb4OpJUSEMCzsDBQ3xXBfm5KEsbo0DEAZN0+0Z36jkAjh1L0aO2Lxs2CMXLKPJRYt8P6nxbBqirytlxOEKze99qi53h8OqM/vv9kKIKaB8K/SRN0GTAVkVmVqJTU3DNJePrVr/4/ABgY6EAqVdzzi2dhkGjao1VjLyUJMTp2ag7FMSwAYILZK35GQ4NI9O7SBoEBTeEf0ALzv94LuaYmyssU/u8kYWzoAhulEyOe338AI7P6aNxUERIcADQNHdEqKABaUqmK/30N+bjxMByu/i3gZlkTetzcqQmCAp1QWbXn8NHtpygvTcIHIzqj6ct2ExiA5VvPITMpHYkpGQCM8N7wQYgKPYuwmEIABdh54AQad+mHxm6GAGp8/GVSwNIjCKu+XIo7x1ahXUgL+DdphBateuHozShQKgWrxOUECDnkak+gqaUNWXIUXqQWok2n1lAOnK5v543WzbzwPCIcEgAaJDQNjeHiXtNedLR1INLVgERa174aPYxf8A26+8oxvG9HNAsMQBP/AEz65GeUy4Dy0lIAACmHvsgRjvaK/YYAkPbiEWKKJNj94ywEBDRVfGtNAzBg0mfIl+TjeXSK0u+ooqEJOHg4Vv+/IC4cz3JLcPHAN4r24O8Pf/+m6NB/EhJyixEVEwsAkEulsHb0RnCQYh8UAEDPHl1bN0f07btIL5Pi+YMncPNujYYeRjUy0EL7bu1RWvAQkYnl0NIkQDFcnGraEQDIqPgu5HLF9xUf/hBp5SX4fsFIBDRVtAn/pgGYsGgDyitSERmXi9SEp0go0kFQcIBSSVpo0y4YhgZar+wtrwuSgK4Y7t6e1WnaWtoQ62miorKu+nsTim733u0w2NcPRlPvmiNNNI1d0K59M6BSVsd38wZEIpjoG0Kufj2lKC4qhJam9muPqtDUM4anh6vaPmU1mI77d+LRuHlb2ClOxwAA2Pu0w5Zft6CpmxiPHz9FZvozDOneovq7bRrQDNvPPEJO5kMkFwBadfyEXCaFrqU3Ro8biN9+mgFvr/ro2HUg1u1/gI9XrcPgDsr9WO00avcevlszC2L1jHdEKpOjUdPuaORYE77ewtMP/r7WCAsLg+qO7Rq8m3dBv7YBKEx4hB+//wk3H4S/dq/RS0xNzUDIIFXbc12QXwypjLXvQdYTwdjAEHKpepuRoaiwEJqa2pADMHdvjZ2HjmDae+0gqjpiwdYrCHM+HI+MRzfxKCJV5eqX5GQXQEffEtYmSpX9FlhamwIVeUjMyFXPqh0Scg0R2rbpAcuaLg0NWwTD2aICDx/HKEtXY+raFKMG9YEpirDxp29w8uJt5Bcrjel1YGRsAm1tTVSqbcguLS1HWVkFtDRrj0FgZGgKHS1CKlPby16Yj1J5HXX0lhQXlkAi04Wd1avHHP05tGFtbYOizGwUFtShHKlBEtDRR6dOPaGv1Fm0aNUaRnqpeBARpyxejb1fCIb1aQ9dSRJ++uZ7nL/5CMVqMQ1qw8jUFJoaMkjlqu+ztDQXZWWEZq3vUwvGJqaQSitRNSxUk5dXCI2qujN+WVdqG/xLCvNQKn/znv7XUVRUgoq/ta7qxtjEEAZ6lYhPqf0brQ25nLC2qg97JV1NW0cfWnIZpBXlAHQxYvoUZNzaAj9vbwS36YL5n/2CLhOWYM6EbspF1SAvwBcTB6NZiyCEhISo/AW1aIahY5ajsI4ql8vl0DU3g5NLjY6ho6MNXR1AUqnYFx9+7yHsXYMQ0FD1SLK2XTtAXv4MEXH5ADQUbVS94pVwaNASw/sH46uP+6OeTyP06DMCx2/lYcWajWjTwEJFVgtAxNPHoIE72gY1VMoRoWOPdtBljQYol8vh4OwHe8MaKS2RASCTo6Ki9gennAD4yniuoaEJeWkCwsKyEdSqLQyVmmL9kEHY9stP8LAyQLcxn2BKPz/MHtcLzZsFwN/fHwPHL0a2BJCUKXRBANDQAjz9lHQwDU0Y6OugsrIc1dE4CMjfVvn5B/jzXxulAAwwdHBn3Pr9GFJLpDhz7Bh8Og6Dow4gfbmjVUOh5ZeXlsC5XgDGT56GyZMnYdKkSZgwYRKWf78VG79bBDtjIOH2YbRo1gI7LyajW99hmPrRPOzY/RtGtvOARG1QUFeaSEADmq+kv+TR2S0IahqEcxHl6D34fcyctRB7ju5BF29rVKgrAmqGI6vOrdEAIJVWAhpar3SCWto6NbZbLZh7tMbpy1ex9ceFcDfTwNkDG9CtTWN07P8BUuoIBHHnxBoEBrTDtRg5+g8djQ9nfYr9h7ejpYcZKivV7lltp7bilqvuWVYJQBOaagOolrZyoIhCLBrbA73Hfg4L72CMGjMRK79bhw0/zIautBJUjraj9AECgLRSBk1oqymQmtDR1KmzPgA5KmVyaGprq1amhgZ0dLSq32VJUQmMLephzLhJmDx5MiZNmoTx4yZgwRffYc/u71HfSfHlN+8xGN6GKThy8h7yYu7i6r10DB8+DOqnHGlpA/nxt9A9qAm+33sHwV0HYtK0D7Fl7z5M6xcMqaTi1cZVC3KZDJRrQVdXPdiGFrS1dQCVwZOKCnn5Pyo6H0Xt1EYFVs97H+37TofM3A8jRo3HkhU/4Nety2Gqo6EWeIIqwQ0qSosBDR10GzACU6re16SJ4zHlwwXYtXsX3uukOpmizkujDgAqy0shrZCjVdcBmDJlqqKsSRMwcdos/LJzJz4a0V5xDQFdPRFEatq9jq4WNDQ0QQBSqRw62qKqc6lq0NbRgRY0lJovXztxAwCSkkJoaJug/3vjqtvEpAnjMWPOZ9izdy+6B9qhrFwCmYYGtNQCV+noaCkCR73mW60V5fqr+qu7/lDVQ1GlLSnkaxKkMhm0tLXVjsTSgJa2LjTe+Qar0DSCg6UlMpLSoTL+SSqRnVYIK3On6mAUfx4pKqVyaKpbjJQiLy8PpcXlKC4qhb1rE4yf+rLdTMKE8RPw+XebsGXdCtiKoBTgSg0SgDamrdiOS38cwag+QUiPDsWiD4bD168xth65r37FP4qBialqTWtoQUtLE2Td9W/v0xa/XbyFA1tXgslX0LllU7TuMgS7j19HUXntigkAmNg5QFOSi9Q01UAUWSnZ0DGygKWBkqbzEg1DOFpaISspHRLlOZAKKbLS8mFp5gRDAGWFuUiIT30lyJSluR205CXIL6kjOgsITS1taGu8m7qgraUFDU35K8p+XRCAhqYWDE1Uzyeklja0NDQhr+N70zNzx7fbjuHy2b1obF2Esf3aoWlQR6xaux/p+TUKmTpiSxsY61UiMTFHJb0gNxdlUl3YWtduSFhbO0CjNA9JmarX5acnQ6KrBysr5Um1d0PxDjShrVW7sftn0NHSBmVSsM4P7lU0NTUhNlE9Y11LSwsaGlpAHe3e2KExNh66gLO/bYKzRhQGdGqJ5m17YO2vvyOvsO7JOWtbS1CSjWS1oHjZ2amo0BXBzLK2c0R1YGlri8KcNOSpxadLT8yDmb0DDABYWjlAo6yWukpLUdSV9Z+vK1TVlc5bBkf8K2hqaUJTE5DWtaJSF2p1pa55tBkyD9dvXcG8KYOgURSPdV/PQ5MG3pi3cr/KdTVow9HTB00a+6NxoyYqf00a+6Oet2OtQR+rIVXH3qp/a1QNgjKpHDpategJ2jrQelXNrRttM6zYegKnDm1Hr1beePHwHKaP7IkGAW1wJjQBUBqJNQDIZFJoaGq9oiNr6ui+YiRpaKqNzqT6a35r5JRCKiM01b93eQVycnIhgxRH185D89YDEFtmiaEjxmHep8uxf99auJvoQqb+QtSMSZLQeIOm8v8T9Xf5zjTvOgAWkqc4vH8fTt/MxcCBHVQF5AS0TOBsbwdZpQGGvTcOU6ZMxdSpUzF16iTUt9FBYQlgoA0c2LoeGfKmOHnqGJYsmIXJ40YjxN8C6cl51Q2yhrdteQBQih1bf0aFa1ecOP4bFs79CBPGjUCAgxHSCgtqKbs2CCkAb5/60ChNRnhiukru82cxrymHuH3+AH47H43R0+bjtzPXkJqZgSNr5+LeqX24cCOqSkwODQ0NaGoCYBl2bloLkV83/H76MObP/gATxg2Hn60hsgry3/LgVkIGwMOzPnRlGXgaqxoZ8fkzRQRBLW0gO+wafjlyGTO/3o7tP6/CzA+mon+vjtAuzkKZvK6hXoFno/ooKopCVJTSgMECvIiNhlSzrkkAczT1cUdqxBNkF9UYZxXFmYh6kQmtqh7Hx9cFleWlCO45EpOnTMHUqVMxbdpUtGpoj+LyMoj0FCalrrkvhvVriXOHdmP/3v2QWIWgV6d61eW+RAfAucObEJpjhF1HLmDlknmYOnkC2gR4Iys3EzIlpYokwFenMSiXQcfWCTZmWrh/56lqXlEmHoVHw87JHnovy3hHSpLCsHHnQQyY9Q0Ob1+Nj2ZOw7CB3WFcWYD8yso63qcCS3cvGFEOZ48QTJw0ueo7m4ZRfVqjLL8Y2rpvc5C1AhNnN1jqacPUwg8TJk6sLmvSe72gWV4CaiqMbU0tDWSnpyI5QTmicSHuPIyAU6OGsBFrw9nHBXEJdxGfpfo+ntx5CGo7w8lO9FoDRKNKCQIAW09v6EqlaOjfFZMmK9rE1GnTMKhDI+TnlEBbVxd2zvVhr1eBiCcRKkVFRsSivEz2dp/PX0ADGqiU5iM1teadVJZmICOvFJoaindQ38cNGVFPkZivFD1PVoLnT8Mh09J6bT3XjT7atmqC5GehiMmsWeXJT3uM+y+y0ax10J8sVwkNK/h4WyE+MhzKJkpezG10CAzArnNR8PLzgESmiT4DxmPKVEV/P23aVDSw10dBoRb09V4ZG6vR1NJCeW4sNq9bC5P63bBqzXaERSUiPvwmWlgXYf2mX6Eab/CfQ1MDiIl4hGwlPa88LRWRsbmoV8/r9QeGa+qgVc/3ceLyXVw6ugkWZRF4v29rBLbuh7vRqsr1S5zrBcFdvxJ3bykb1WW4FnoN5l6ecLarTUEWoXWIP9Ke30F0ek2NFGWE4e6zTDRr0xIaAK7tXQlfn+Y4+zhL5eqHD25C09wZ3k41Kw/KGBrpQ1KUhYxC1YjlbyIrMx9yGsPeStWArAsNDQ1AKsGzZ2HVM/IAkPr8OVLytdDAx0kp9VU8/dthw74zeHTrFDr6iLB0xjDUq98Me849UxcFAOjb+SHY0wZ3r1xX0SaePLiIQl1jNGrgrpRag2uDBrAwLcONe+Eq6aGXQ2Hh0BLezurTnG+PWKwHLVkZ0rJU6+jPI0NqRhrEFhYwMH67vl9DQwMySTmehz1WSX8eHoGyCis08Ky9nSjQQMOQXthx6iruXtyPRpaF+GB0b9Rv0hZn7serCwMAnBr6w964FLduqNbT3as3oW/jCy+32qfIAgOCIU+Pw/MoJb2mJAqXH4ajYYg/9AC4NPCDpVkZrt9TLbu6rpyUltLfEZFID5ryMqRm/l11VTeF+cUoLtGEg62NetYbqKOT1dAEUIT9v6xGstwZC7/4HtfuhyMzNRYf9fXF1p9XI/bVgLaApgHGzv8Cmzetx7r1a1X+NmzahM8XjoH+X7C/Xeu7Izn1PqKSVCOBP733CBVye7g4GACQQ05C4zWr02kx97Bp6y74dRyFNb8cRFRCGiJCj8E0Pwxb95wAlMxuGQBXN0/IC5MRkaCmIz8MQ7mScaqgjnf6jpByaIvt4eFqiMgX4SoLCfG3jyGoaRscO3MHO3Zsh1/vafj96HbM/mg6Ro0YACexHDmlFe+mx1Stbmr8dbPvT/OXf9na0x/BTR3x+cezUOrQAO0CalzPAEDhn6iLkaPHoCD8CKZ/8g0Ss0oAaSkuHVqL/r2H4+jFFwAAYxNjlBWlILSqI89KCseSD2bgdFQu9HSqXMH+VF1rwcTIGEVZ8Xj4VGFkpUbfw0czPsbjdAl03xAS/iVSAI3a90XbBrpYOHcB7kVmAnIJ/tj9Fb7ZdRU6r6x2vUQD0XfOYuKEMdh6JBSV1IQGK5CTmQkdkQg21ooZPB19MXKzEpGaLgM0tGBgYoi89Fg8fpIIAEh6HoqPZs5GVDahp6c+B1Q7UjngGdIdXZqZY9kn83HzaQpAKa4eWYPlW09DS1sXBKCnL4KeCIh4dAdZRTLIKopx+fA6zP58G6Ct+xpjGgjuPhJNrIow76N5CE/KB+Ul2PfTF9h2+sVr323/sWMhz/gDcxb9hIyCClQUp+HbRbNxOT4fulWrJ20GvA8XvUTMmDgLj2IyAMjx7MZvGD10AL7dfBrQefkeNNF38CgUxh/Dki8PoeewIXBU9lGoggCMjEyA0nzcf3APlQSKsuPx/eLZ2H0xGnriGmVBLNZFXmYs4tWOsaBMCoi8MHZ4d5zcvAyr915EaQVRmpeMr5fNwflnUgwfMhB4lxk5JXRFehAb6CI2/AFiM8oAuQT3zu3B9Pk/KFaq1VeWlHDy7Ywhnb3x9dKp2Hv6EWQE8lLDMX/qWEyf8w1yS+uuR3VE1oGYMKw9dq6Zg9W7LqOsUo7y/GR8s2gmRk6ch7hMxUqNlpYuyktisXjpYkSlFYHSYhxY/Tn2XIrHeyOHQBNAz4GjIM5+gDnzPkdcRhEok+D6sXX45IeDaNvvfTS0BGR1uMhoi8WokGQhJiYTANCwZX90bqiPBXOn4vSNSBBAevRtTBk5HAuWb0WxhLD0aon+fRrh56/n4+ilcABE2OW9WPLDVpRB57Xt+W9BQwNymQTrvv4KMVllkJZmYu2XX+JJgQTaVfXXYdB7sMJjfLroeyTnlEEuyceuNUvxy9lIiHTf7vuujTb934c9wjF3/pdIzZegNDcWy+d/ghxDfwzp31Jd/E8gxoD3ByMm9Fd8tfooisplKMtNxKoVSxFdYo6WwYEYNHgkGH8JH877DHHpivq+dXIj+vQfjAPn7ivc0OuoAg1NTVCShfXL5mHaRysQn1kILU0tFBfkoai0Ata2jorjXv4iqTEPsHvXIcSn1aZZKdDW1UHCkyP4YsUO5JXJUJaXiC+WzEV4oT1GDe2hLl4rGloitO07Br9fvo3zhzbAWSMOjyNrV1LN3Bqjf19/bPzuUxy/FgmgEmd3/oQtB+9i2KhJsKryIkh4ehv79hxHVrFCMWvVbwRcdGMwZ95KJOeWoyw/His/+QRpun4YNlBxvE1Ap55wM87Gwo8X4GliDigvx/n9P2Lpt3vRbdgENHav3QHZub4vxDr5iE9QPo7qzcTHRELPygEedgol+emdi9i7/zRyi+te4dXVA84d+hk//3oRFXIgM/YuPp6/FGK3DujTtpG6eC1oon7zrth84Awe3j6Dng30cO++6qRTNZpmGD5hEEJP/ohvt19EJYnIO8ex7PPNCO49EYEeilYmLc7E8UO7EfpEsTIisgvAuAEh2LxiIY5cfQ5SisuHfsA3++9g+MSxsK57yHsjpo5OsLUVITZeMeb/ZeSlSIhNhJ1TPVgaA5CX4+a53/Dbmeuo5eSFKjSgoy3F/u2rsP3oHcgBJDy5hLlLv4JD825oH+iqfkEt6KBpu4HYe/wK7l4+iKb2MjwMq/2IHJF1AMYOboftX8/B4cvhIKW4cuQHrNp2E8MnToVNlZ2cmfgU+3cdRmKGYuIjoGMvBHhKsHDBEkQkF0JWlo0fli7CwzRDjBneFwCgZxuIcQNaYcvKhThyRVFXVw79iG8O3MawCWOqy/4zmDo6wtZG/PfV1WvITItGidwAvt6KSZCU6PvYvesQEtLr7rtejwYAGc7+8h1Gjp6FOxHJ0NTURkVZMXLzCmFm5QTj2ruDfwiFotS570jYVMRgzqxP8SI5H2Al7p7djtmrfkFAt6EIcDMCoAl9XT0kxcShqI6F84rcGHzx8Qws+Hw9Mosk0NTUQn5uDsqlGrC1sQOUTAkpgAZte6JlfeCLxcvwODobkFfg+vENWL7xd2jr/IVG8hpIOaBpjUHDeuDasdVYu/syJDIgL/kJlnyxAmXGnmjexAMisR7S4p4iPCoHgBzP75zBtA8Xo6AS0K7Wf98CTQ2I9TSREP8cdThV/vOob+p8E4Up99nISIfjPtlfnfb7TzMJgGOqIsmS5I2dy6gNS554/DLIQTn3fD+XjuaGNLOyp7urA/X1jdh50IeMr4qjnBV9nd2aulJbV48u7p709PRkr4ETOHFoazr6dWN6JZkfc5qWepqc9/OV6t8ipfzsfX/a+XZleh371BMf/s4gb1tqaxvRzcOLXh5eHDB6Pkd2bUjfllNYRrL4+e+01Ac/Wa843PolS4c2pY1Hv+qARPEPT7J1Y1caGlnSzdWZzs712DqwHh29B1B1q3MNFXlxnNY/mGKRAZ1c3OnibEcLC0cu+GF/9cbnB2c20lpHk4bGblyz5xYTw04zwNOGWnomdPdU3POwSfM4rJUnm3aey8qqwBuOpuD0bxTHBbzkmwmtaWrXiSlVMTtSwy+wUzNPGhhY0M3VmU6Onmwb5Ecr525MqiTJCm75bBLFOjo0s3Kkl5cH/RqF8JMF8+huZsrvdioiFH4xohktXXtRPUj9wwu/0t/VmqbmNnR1caSHb0M283FiYJfJzK8zLr6Uv29eQmdLE1pYO9DZyY5+fsGs72rBIVNrAtXcPLaWvm7WNDa1oru7K0309dmwZX/efKIWgEGWwfdbOBEiR/5xT7kmJJzfL5C+7aayREZKsp/zvY6NqAldOrq408vTg627j+bciX1pbhPMZ6mK3dmRV7fR2liH+mYOXLnlGpPCDtBK04i/XlC06YqiZC4Y25NmxkZ0cHano60FzW08+Pmao9VHP5xaO5kaBna8ElnzEkpSwxlgqcP35iiO+KiNo+sX0kxfj0ZmdvTy8qRP/aactWAxm9gaceaKkyTJbZ/0p8gwgBEFqkEyMmNCOaR9Y4rFhnRxc6eNpQktbH348/5rKnLKpIZup1gH/OaAamCOstwYzhjcjvoiMR2d3ehoa05DEwcu+vFQddTDw2tmUKxvwY7t2tLdxYXOjnY0MbHk1CUbWVK9EV7O07tW0cPWjGZWDnRzcaKpsSm7DJrD+ExFwIKU+ydoranNFTtV7zMn/iabO5hSW8eUo6f/TJKMvX+KnfzdKTYwoZu7Gy1NDeng0Zz7z9fcf0HSQ47s0pT6+iZ0cXWhvb0H2wf708SqGe/F1B6SpyInge09jDlmnqJ/u7X7E0LXmMfv18hX5iWxo5s+u6odCaVMxNkfKNLRZ0N/Pzo5OdPJ3poNGrZnCz8b9hlTE2zo+oHv6GZtTDNLO7q7uTKweSe2DfSgb6uRzH9DELmyhJv0sdDglBWvBp+6vO8bulkb09zKgfZWprRyqM9tJ2oCH/1lKou4ZuEYWhgb0cHRlQ62ZrRybMQdx1+Gh5fy8PrFdLEwopmlPd1dHWmgr8/WPaYwKlMRnaY87jztjUVcuUtR36dWf0wzK38+i1e0hz9+/ZIu1iY0s7Chh4c7rcxNGdhhFB/F1Rq/9505uH46AUNuO1F3tOiFQwJpZtmQ7TsE0MXZjQ625rS09+XaPdfVRd+a8sIc5hfVBCFSpzD5MUd0bECRvimdnR1pIDbmkGkrmKf0mW+bN4SALS/F1PTE1w//SA9bE5pZ2tPe2oyWdt7c9JtqkJHbv6+nr4sljUyt6ObqSAN9Y/YYMZ/JBXVEdiMpL01h/8a2fK8qkNvbUcq5QwPZtPOHVYGbyjl/fGtCw5e3o15GWFVFWpDKng2s6ekbwhbNfenq4kprC2O6+rbjyZuqR5C8LdLyYubk1v6tkyQluVw+tTcNRPp0cHahqYE+A9q/xwilCEoFz8/S1QgcvnBPdVpJZjhHdG5KsciQLi5ONNA3ZP9xXzCnjsA8F7d+ShEcefb+mwIoyblydCs2CJnK15y69NaUZYcz2NGEc76tCmxTksqhLYxp1XAgU+t4LcXpYQyyM2ajgPYMbOhFVxdXWpoa069FX16vCgL5rkhKi5iXX3e4stKs5xzdOYD6YgO6uDhTX9+QfUYuY65S1KJzuxYRMOKO0zXH3Dy/tp+BHpY0NLOls701DY3t+MVGRQCal5RkRfD9LgEqddVvbN11demXRYq6uvemZ5XzyzGt6Rc85W+pq9exd+UIWrt0YlLVPe//eSoBI/56qq7jmeSc3dOHLo3eVzka6tEfa6gPY+4+qwg8GH37KIPqO1Df0Izunp60szKna/3W/O1qTZDDv4sDq96jtrkH7yXWpBXE3qOPkRanLFcE6SPJy0dWs76TJU0t7Ojm6kwzYxO26TmNz5NqnmTnZ+OpC9DBNZBnaw1KJuGOVR/QwkifVrYOdHd3pbmZGbsOnc/EPIV2tm1BT5o7dmN6VRNLuHeCLX3sqG9kSXc3V/r4tWS/Ls1pZteU4dkkWcoJLV3pF/JRtX5HkjeOrKCWlhkP3VD0xes+7khr9xp7YdX4ILo0GlkdrC/60q+0FNvw4CVFZF5pUTqXTOpNEwNjOjm70dbKhE7erXnypiL/9rF1dLM2pp6hJT08Pent5ctJsxazU31L9hqn0CVq198qOb2TO72aja8KWCTnrq/GEwAd64Xwwv3XBzD7J9DgO/r8ScsLcO3CFZi5B6JJfXsAQFlOPP649hB+zTrB00Hh8pOb/ALX78aiRYf2sDGpmY/OiH+GW/efoqwCsHP1QcsWjaCnWTPVXZSbgls3Q5FdWAkHNz+EtGyI3ISnuPM0Ha07dIIBMnHu3E24NGwNX7eXm3+J5/evID5fD+07tIReHTPnhRkxuHbzIQrKpXCt54/gpvWQ/OIOHsaWo1PXNtAty8CZCzfg1rCtUtnA83uXEZ+nj46dm1e7UJUXpuPq5ZvILpGhQdNg2Ihy8PBFKTp0CVIJIqOMrLIUD0JvICY5CxraYvj6t0BDT8U7VFCJR7ev4VlkBnwCWiHA1wn5qVG4GvoQJRLCrX4Agpp4IuHpTTxL0kTH7kHQLcvB2QtXYV8vGI28atwtoh9dR2SaJjp0C4ao6n1UlGTh2uUbyCiQwKdxEJxNS3AnLA8duoZApKnwG392/xaeRKdAR2yK5sFt4WRGXLlwCSbOAWjia4/I+1cRk62Ljl1ffc6izARcu3EbBRWaaBrcHrr5sYgv0ELrkKbQrqNOACA97ilu3HkCua4pWrcJRnb0fZSKPNC8sUu1THFuEm7cvIucQglMrVwQHNICprX4bsQ+vobn6Zro2DVEaf+mHOF3ryFdYoo2IY2hrQFUFmfg+rXbSMsrhoW9J9q2ag5ZbhTO3YxCyw4dYWOsB0CKiHvX8fBFKjwbhKChhy4uX7iPhiEd4WhZNf1HKcIfhOJJZBI0dfXRpFkreDnXtJ2MuMe49TQVrdt3g4VhlQuHpBjXLl6EkVMTBDRwrpZVRYaYJ3fx4FkcqK0P/xZt4OVogDuXz0NqVA/BgR5IDL+DxzGlaN+tDQx1VFc95RXFuBt6HbHJudDVN0HTFsFws1Mcql0b5XlJ+OPqPfgGdoSng+reHcgq8OT+DYTHpEFDWwy/pi3g51HTbg+vnobRn13BxdBb0Ei5hsjUEvg0aYGmvi6vLGLlpEbjRugDFEsIZ8+GaB7gi5fxacoLMnDp0m14BrSCl5Pi0PuXJEQ+QOi95zC3r4/27fyhDaCiJBeht24gJbMIYmMrtAgOhp25amATuaQQt65fQ3x6AVzqBaKxqzau301Ey7atYKr/6uwgK8tw8/JF6Ds0gb+vA3KTI3DlfjSC23aHjalCnpXluHn5PLQsfBDUVM2jo4qIs9+jaf/v8dv1yzDMeoqkAg20btcGRYmPUKDpgqCm7igrykVRhQ5YkoKbtx9BqmOOTq0D8eXENjiX3xm3Lv7w2pU8WVkeLl26CivPQDT2dlDPRmb8U1y78wQyTUM0bdkGng5v59r49sgQ//Qe7j2NBXUMEBjUBm4OqvutMpNeIPROGIolUtg4eaNVy6bQq+oM5KXZ+OPiLXg0CYGXozky457h3vMstG7XGkZixbedmRCB0HtPUCyRwdzWHSEtm8NIrN6q/hy5qRGYM34W2k3/DqN61b63+ZOBTXAkpilCb32Fu2cvIV+qh2Yt28Jd7Tn/bqSlubhx/TqSs0rh5NUQQc38oKv02MnPH+BeeBZad20PC4Oanjg7MRxXQx+jUsMATYNaw8vp1W++MCse167fRUG5DI4eDdCyWQPovOGV7lk5Fkv3ZOBy6Ck41LKNVJ2SjPto07gThn1zDnNHBgIgIh+cw+yZq7Fo2y608Hr1/ckK09CzmR9EIcuw/YfeuHTmFjQMrRES0hpWpn/eTfWNyCR4dPc6wmMyYGLtglatg2AiqhlbpCXZuHDpCiw9WiCgfo07qUxSgJtXriApuxj2rg0Q3KIR1M5yryY78TluPUxAULt2sDJ5/bM8u7AB3Qavwo5rT9DBrzYX6rfnwdFV6Dp5H06FXkUzN2OAMtw8tQZLf3qEHYe2w16tqweAkownaOvXEi2m/orl0+rj3KX70LdwQatWwTA1qOMB/wak5UUIvXYJCVklsHetj1YtmkBH6eeyU57jRmgMAtu0g4NVTT9fmBGLyzfuoaRCC34BLdHIS1mnUiCrKMDNy4q6snP1Q0jzxqjLiSQn8TluvmVdhV/YiK6DV2L7tSfo6FfLy/xbyMaQgADod1qO7V+NBADkpIRj9vhZ6PThj3i/u1KgGCWehl5AWqkp2ncIqN4TWZgdh6vXnsG/VQc4WClclUtyknHz1l1kF5ZBZGSFoODWsLN43cjz50iJuo97kTlo16ELTKrUJ2lpPq5cvAIrr2ZoVK+m3goy43H91j3kl0jh4OaLoGaNqgOdAYBUUoAbV64gJacUQe17wt22tu9EjoSIh7j/NArlUsDexRctmzfCS+fAhPBQPEskOnZrCe3yYmQXVUIPJQi9FYq8Mk0Et+mM21vHYcraXDyKuwBnkRyPrl1AnoYd2rZqUO0empcWiat3otC8dSfYmesh7ulNvEjVQscuLaADIPLhVSTkGVTXQ0lOMq7efILGwe1gb/FyGbkCEfdCERaVBB0DS7QMbg07yxpX8rSYMNx6EI4KuTZ8/YPRyNse4XcuILXUEh3aNUZlXhLOvqK/EWE3zyOzwgrt2zWBFgBK8nDtylUk58oR0rYTXGrdovEPom6BCggICLwLh36aSn3TerwT83bHEfwbqO1YFHUe/LaCFlbePBdWM9OY/vwSfW1EHDVnp4qswN9PWeYTThj0Hu/F1n14yvwBjenRcKTKKsG/kdKUO2zh4sK1R1TPrKyL31fPoF/gIKYpvbiEe0c4YvhHdZ6nKS1IZVdvU/Yc9Z161r+LymyO79iQo+e+y4pybRTyg97NOWzGOio7S1zd+y0nzvxO7QidGorTwxhoYcAJC2q82ATqoDKbEzo15KjXeCv9VV5c3EJvD3/ejqxZIS5Nf8zxg0bwQZzS+UECf5r8iJN0s7XmjwdqPEKkRQns19yBTbvMqfNbEXg36t4MJiAgIPAWyCiDVE7I/8yG1f9V5HIQ0leO1FGmXqs+CLAuxuAu7fHB3KVYPG862nXsiwLDYHwwvY+6uMDfTFZmIYZMm4MAt7o3K1EqRYVc/biRfx9i+2aY91FvbP/xe+SqxvN4hfLMJ/hx2wVM+WQ+lBcecgt1MHPBLFjXcboKKYdcJkXly3Op/q1oW2Du4tm4d2wD7kT/+d1WTy/sx6UoXcyZNUbJ06Qc5WIvzJk76RUPpZeQcshklW8dXfhfTVVd3T++EXeUgyb+XUjz8OP3G9F+5Bw096pxLcjKKsLQ6XPg7/r3r0T+GzGpF4x+wR6YM7IXRk2dh8+XfIIOrdvg7DM9fPLp5Dq/FYF3Q2vZsmXL1BMFBAQE3pb8zCTklInRq28vWNQSqOnfSHlRJqJTK9Gzbx/Y1eEOqKNvjV4De8NYlokbt24jIbUQ7fpPxdq1y9HA6VWXQ4G/FxNrJ3i4KQJI1EVKbAR0LOujV4/g10ek/Rfg06gxUp7dh9i+Mdxe44r17NZpZIr8MXdqX5WtFLZu3nCweY1bt1yK6MhIuDRsg3YtVc+V/rdh6eoL/bJ4JBWZIrBhXdsuXocUZw8cQ5P+U9CnlXK0XW14+NSDxWsi1sql5YiKjIdfyy5o0aRmW4tA7Vi4+MLgZV01+jN1VTeZUaG4EaOFTz+ZBiMlN28Ta2d4uKqeVy3wF9AQoX3vPvC2IkJv3sSLmGR4BPTE2k1r0KVZzRnqAn+Nd97DKSAgICAgICAgICAgICDwNgjLEQICAgICAgICAgICAgL/CILBKSAgICAgICAgICAgIPCPIBicAgICAgICAgICAgICAv8I/w9T1kwlad8eBwAAAABJRU5ErkJggg==\" style=\"width: 692px; height: 119.078px;\" width=\"692\" height=\"119.078\"\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eLipid content increased 324% in the N- TAP \u003cem\u003eC. vulgaris\u003c/em\u003e monoculture compared to its N\u0026thinsp;+\u0026thinsp;TAP counterpart. Since these groups were identical in all other experimental parameters, the increased lipid accumulation can be attributed to nitrogen deprivation. Stress induction through nitrogen deprivation was the intended result of changing the media to N- TAP on day five. This observation is consistent with previous literature, which identifies nitrogen deprivation as a key stressor that redirects algal metabolic energy from growth and biomass accumulation toward lipid storage (Yaakob et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Chen and Wang \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This method for inducing lipid production has been extensively validated as a standard technique for microalgal metabolic manipulation. Our findings further support this well-established approach.\u003c/p\u003e \u003cp\u003eCo-culture of \u003cem\u003eC. vulgaris\u003c/em\u003e with \u003cem\u003eA. chroococcum\u003c/em\u003e under nitrogen-deficient conditions for seven days yielded a 730% increase in lipid content per cell relative to the N\u0026thinsp;+\u0026thinsp;TAP \u003cem\u003eC. vulgaris\u003c/em\u003e monoculture. This value represents the difference between a wild-type microalgal culture and the nitrogen-fixing co-culture model. When compared directly to the N- TAP \u003cem\u003eC. vulgaris\u003c/em\u003e monoculture, a statistically significant 90% enhancement in lipid content per cell was observed (t-test: t(4)\u0026thinsp;=\u0026thinsp;4.74, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). These results indicate that co-culture with \u003cem\u003eA. chroococcum\u003c/em\u003e enables nearly a doubling of lipid yields beyond what is achieved through nitrogen deprivation alone.\u003c/p\u003e \u003cp\u003eIn contrast, the nitrogen-deprived \u003cem\u003eC. vulgaris\u0026ndash;E. coli\u003c/em\u003e co-culture exhibited no statistically significant difference in lipid content compared to the N- TAP \u003cem\u003eC. vulgaris\u003c/em\u003e monoculture (t-test: t(4)\u0026thinsp;=\u0026thinsp;0.39, p\u0026thinsp;=\u0026thinsp;0.72). This finding suggests that the lipid increase observed in the \u003cem\u003eE. coli\u003c/em\u003e co-culture relative to the N\u0026thinsp;+\u0026thinsp;TAP control was solely due to nitrogen deprivation. The lipid yields were not enhanced by the presence of \u003cem\u003eE. coli\u003c/em\u003e. This result is notable, as it underscores the differing effects of bacterial co-culture partners. Moreover, this supports the idea that co-culture with \u003cem\u003eA. chroococcum\u003c/em\u003e is beneficial for reasons not typically associated with \u003cem\u003eE. coli.\u003c/em\u003e\u003c/p\u003e \u003cp\u003ePrevious literature has identified several merits to bacterial co-culture that make it a promising method to increase microalgal lipid content. These include the exchange of hormones, growth factors, vitamins, or stress induction from resource competition (Magdouli et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Tong et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In this experiment, lipid content was observed to be 110% greater in the \u003cem\u003eA. chroococcum\u003c/em\u003e co-culture compared to the \u003cem\u003eE. coli\u003c/em\u003e co-culture. These groups were otherwise identical in cultivation conditions. Given this increase and the lack of statistical difference between \u003cem\u003ethe E. coli\u003c/em\u003e co-culture and the N- TAP \u003cem\u003eC. vulgaris\u003c/em\u003e monoculture, it is evident the \u003cem\u003eA. chroococcum\u003c/em\u003e provided some benefit that the \u003cem\u003eE. coli\u003c/em\u003e could not. Further work must be done to explain specific pathways and break down the molecular mechanisms involved. However, this result supports the idea that the observed lipid increase was not due to the previously mentioned standard bacterial co-culture benefits, but rather nitrogen fixation, a unique ability of \u003cem\u003eA. chroococcum\u003c/em\u003e and the primary reason for its selection in this design.\u003c/p\u003e \u003cp\u003eNitrogen fixation is the process by which atmospheric N\u003csub\u003e2\u003c/sub\u003e, which is not actively usable by algae, is converted into bioavailable ammonia (Walker and Yates \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1978\u003c/span\u003e). Xu et al (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) found that when \u003cem\u003eC. vulgaris\u003c/em\u003e is co-cultured with nitrogen-fixing \u003cem\u003eA. chroococcum\u003c/em\u003e in nitrogen-deplete conditions, this nitrogen supplementation was beneficial to biomass and lipid accumulation. Although it may be intuitive to assume supplemental nitrogen would be deleterious to lipid accumulation, this has not been observed. Instead, findings suggest that \u003cem\u003eA. chroococcum\u003c/em\u003e nitrogen fixation is just enough for the algae to be enhanced photosynthetically without compromising the effects of stress induction (Xu et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The nutrient deprived microalgae may see the provided ammonia as a finite resource, perhaps due to its method or rate of production, and use it to facilitate increased lipid production without exiting its stressed state. Xu et al (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) also showed that co-culture with \u003cem\u003eA. chroococcum\u003c/em\u003e leads to upregulation of several important genes in triacylglycerol synthesis in \u003cem\u003eC. reinhardtii.\u003c/em\u003e These genes include acetyl-CoA carboxylase (ACC), diacylglycerol acyltransferase 1 (DGAT), phospholipid diacylglycerol acyltransferase (PDAT), and the 5 genes that encode the various amino acid chains of DGAT2. They also observed a downregulation of phosphoenolpyruvate carboxylase (PEPC2), promoting generation of acetyl-CoA which can be used in fatty acid and therefore triacylglycerol production (Deng et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Further experimentation must be performed to elucidate the pathways that allow nitrogen fixation to further increase lipid accumulation when paired with nitrogen-deplete conditions.\u003c/p\u003e \u003cp\u003eOne limitation of this study is that success of the \u003cem\u003eA. chroococcum\u003c/em\u003e co-culture over the \u003cem\u003eE. coli\u003c/em\u003e co-culture control is not sufficient to conclude that nitrogen fixation is solely responsible for the observed high lipid content. There are other possible ways by which \u003cem\u003eA. chroococcum\u003c/em\u003e may induce lipid production, such as by the secretion of a nutrient or growth factor. As both \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eA. chroococcum\u003c/em\u003e are members of the class \u003cem\u003eGammaproteobacteria\u003c/em\u003e, they share some potentially beneficial secretions, including indole-3-acetic acid (El-Essawy et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Heo et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The \u003cem\u003eE. coli\u003c/em\u003e co-culture successfully ruled out any of these as the dominant factor. The observed upregulation of genes involved in lipid synthesis and general increase in lipid content could have been caused by nitrogen fixation or by some unknown secretion unique to \u003cem\u003eA. chroococcum\u003c/em\u003e in this study.\u003c/p\u003e \u003cp\u003eUltimately, this work provides a baseline for enhancing lipid yield in nitrogen deprived \u003cem\u003eC. vulgaris\u003c/em\u003e through co-culture with the nitrogen-fixing bacteria \u003cem\u003eA. chroococcum\u003c/em\u003e. The results are encouraging for the prospect of pushing lipid accumulation past the limits of environmental modifications. By proving that co-culture with \u003cem\u003eA. chroococcum\u003c/em\u003e can significantly increase lipid content in \u003cem\u003eC. vulgaris\u003c/em\u003e, this experiment represents a key next step in increasing the feasibility of microalgal based biofuel. Further work would be necessary to explore the scalability of the system from the laboratory setting. For example, this model could be tested in a photobioreactor or raceway pond. Dynamics of biomass harvesting must be also studied. The growth rate of both species in the co-culture after medium renewal or harvesting should be explored such that bacterial overgrowth is limited. Expression or metabolomic profiling could offer insights into the exact pathways and molecular mechanisms being targeted by this co-culture pairing.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded entirely by the Northeastern University Department of Bioengineering.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare they have no financial or non-financial interests or associations relevant to the content of the report.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during the current study are available upon reasonable request to the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe MATLAB code generated during this study is available upon reasonable request to the authors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contributions \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation was performed by all authors. G.B. and K.F. performed bacterial preparation and culture. Data collection and analysis were performed by A.K., C.A, K.F, G.B. and T.C. Figures were prepared by C.A. The first draft of the manuscript was written by C.A., A.K., G.B., K.F., and T.C. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors would like to thank Professors Samuel Chung and Esin Sozer for their valuable insight throughout the conception and execution of the study. This work was supported by the Northeastern University Department of Bioengineering. We also acknowledge the Northeastern University Wet Lab Makerspace for providing access to equipment, lab space, and common reagents. We extend our gratitude to Helen Kurkjian and team for their help with materials acquisition and experimental support. We additionally thank Professor Timothy Lannin for his thoughtful review of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAhmad MT, Shariff M, Md. Yusoff F, et al (2020) Applications of microalga \u003cem\u003eChlorella vulgaris\u003c/em\u003e in aquaculture. Reviews in Aquaculture 12:328\u0026ndash;346. https://doi.org/10.1111/raq.12320\u003c/li\u003e\n\u003cli\u003eAl‐Hammadi M, G\u0026uuml;ng\u0026ouml;rm\u0026uuml;şler M (2024) New insights into \u003cem\u003eChlorella vulgaris\u003c/em\u003e applications. Biotech \u0026amp; Bioengineering 121:1486\u0026ndash;1502. https://doi.org/10.1002/bit.28666\u003c/li\u003e\n\u003cli\u003eAsase RV, Okechukwu QN, Ivantsova MN (2024) Biofuels: present and future. 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Cells 10:393. https://doi.org/10.3390/cells10020393\u003c/li\u003e\n\u003cli\u003eYamada R, Yokota M, Matsumoto T, et al (2023) Promoting cell growth and characterizing partial symbiotic relationships in the co‐cultivation of green alga \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e. Biotechnology Journal 18:2200099. https://doi.org/10.1002/biot.202200099\u003c/li\u003e\n\u003cli\u003eYang L, Chen J, Qin S, et al (2018) Growth and lipid accumulation by different nutrients in the microalga Chlamydomonas reinhardtii. Biotechnol Biofuels 11:40. https://doi.org/10.1186/s13068-018-1041-z\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Climate change, microalgae, co-culture, biofuel, Chlorella vulgaris, Azotobacter chroococcum","lastPublishedDoi":"10.21203/rs.3.rs-6604448/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6604448/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eReliance on fossil fuels significantly contributes to climate change. Sustainable biofuels are a promising alternative. The high lipid content and environmental benefits of microalgae lend themselves strongly to biofuel production. This study focuses on taking an industrially relevant, lipid-rich microalgae strain and engineering a unique solution to further increase the lipid content. Although lipid yield is greater than terrestrial biofuel feedstocks, it remains a key limiting factor preventing microalgal biofuel from becoming a positive investment. Both microalgal-bacterial co-culture and nitrogen deprivation have individually increased lipid yields in prior art. Nitrogen depletion forces microalgae into a stressed state in which lipids accumulate rapidly, while certain bacteria have been shown to have symbiotic relationships with algae increasing lipids or biomass. Co-culture with a nitrogen-fixer supplies low levels of bioavailable nitrogen to the algae, allowing for higher photosynthetic efficiency while keeping the algae in a stressed state. These effects enhance lipid production. However, the combination of nitrogen-fixing \u003cem\u003eAzotobacter chroococcum\u003c/em\u003e and lipid-rich \u003cem\u003eChlorella vulgaris\u003c/em\u003e under nitrogen-deplete conditions has yet to be tested. This study combined co-culture of \u003cem\u003eC. vulgaris\u003c/em\u003e with nitrogen-fixing \u003cem\u003eA. chroococcum\u003c/em\u003e under nitrogen-deplete conditions and found a significant 8.3-fold increase in lipid content per cell compared to nitrogen-replete monoculture algae. This was paired with a nearly 2-fold increase in algal lipids compared to a nitrogen-deprived control as well as an \u003cem\u003eEscherichia coli\u003c/em\u003e co-culture nitrogen-deprived control.\u003c/p\u003e","manuscriptTitle":"Co-culture of Chlorella vulgaris with nitrogen-fixing Azotobacter chroococcum in nitrogen-deplete medium to increase algal lipid content","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-07 11:25:59","doi":"10.21203/rs.3.rs-6604448/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"af58b1e3-a72d-412d-a372-8ab9be637de4","owner":[],"postedDate":"May 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-08T15:23:36+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-07 11:25:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6604448","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6604448","identity":"rs-6604448","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}