Lactobacillus delbrueckii ssp. indius AG1: Potential candidate for Degradation of agriculture wastes and production of biogas by orsat instrument method

Lactobacillus delbrueckii ssp. indius AG1: Potential candidate for Degradation of agriculture wastes and production of biogas by orsat instrument method | 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 Research Article Lactobacillus delbrueckii ssp. indius AG1: Potential candidate for Degradation of agriculture wastes and production of biogas by orsat instrument method Ankita Suvagiya This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4102508/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 The anaerobic digestion of organic materials, which is necessary to add chemicals to the continuous biochemical process, is largely mediated by microorganisms. Through a sequence of metabolic events, the microorganisms efficiently break down complex organic compounds to produce simple compounds like methane. In this investigation, inoculums used for biogas production was isolated from agriculture waste sources— L. delbrueckii ssp. Indius AG1 at mesophilic temperature (35ºC)—to assess their suitability for anaerobic digestion of agricultural residues (AR) and fruit and vegetable waste (FVW). The highest biogas production of 215 ml/2 kg waste was achieved for a mixture of inoculums, and biogas characterization by orsat instrument method showed 47.7% of the methane content. The morphological, biochemical, and molecular techniques were used to identify the microbial flora present in the high-yield reactor. The abundance of L. delbrueckii ssp. indius AG1 . To get a high methane yield from organic waste it is necessary to maintain the equilibrium and availability of efficient microbial communities like firmicutous, hydrogenotrophic, and acetoclastic methanogens. Agriculture waste Biogas L. delbrueckii ssp. indius AG1 Molecular identification Orsat instrument method Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction India produces 108.34 million tons of fruit annually, and it produces 212.91 million tons of vegetables annually, the second-highest amount in the world. The lack of storage, transportation, and physical deformations results in the annual loss of almost thirty percent of this total crop (Indian Horticulture Annual Report, 2019). After processing, both local vendors and the industries produce wastes including peel, pomace, seeds, and so forth. Waste buildup at disposal locations causes a bad stench and a protracted natural decomposition, which raises health concerns. Thus, it is strongly advised against using this strategy and instead to focus on finding a workable solution that may deal with the present waste management and energy crisis issues. In our nation, almost 80% of people are either directly or indirectly dependent on agriculture. Massive volumes of vegetables are grown throughout the winter, but because they are not efficiently transported or preserved, they are lost. This might potentially be used to produce biogas (Iqbal, et.al. , 2024)The breakdown of organic waste in the absence of oxygen produces biogas. Because it comes from biological material (biomass), it is also known as biogas. With the aid of enzymes, anaerobic bacteria break down organic waste into smaller molecules like methane, carbon dioxide, and hydrogen sulfide, which combine make up biogas. Since lignocelluloses makes up the majority of the biomass in agricultural waste composting, its breakdown is crucial to the process's success (Tuomela et et.al. , 2000; Dixon and Langer, 2006). The amount and type of material introduced to the system determines how much methane is produced during the anaerobic digestion of biologically degradable organic materials. Consequently, there are numerous methods for producing energy through anaerobic digestion using leftover food, vegetable and fruit wastes, and cow dung. These days, dry fermentation, co-digestion, single-phase digestion, and two-phase digestion are the most popular ideas (Chanakya, et.al. ,2006).Features and Constituency of Biogas depends on the kind of substrate utilized to create biogas. It greatly influences its composition. Methane (50–70%), carbon dioxide (30–40%), hydrogen, nitrogen, and hydrogen sulfide are the main components of biogas. After it was determined that thermo-chemical pre-treatment and co-digestion were the most effective methods for optimizing biogas production from plant wastes, it was suggested that more research be done on the subject and that AD(Anaerobic digesters) digesters adopt this technology on a larger scale. Moisture content, C: N ratio, and TS were found to have an impact on the gas production (Rahmat, B., Hartoyo, T., & Sunarya, Y. ( 2014 ). Material and Methods 2.1. ISOLATION OF LACTIC ACID BACTERIA Sample collection: Lactic acid bacteria were isolated from vegetable waste or fruit waste. After collection, the samples were stored aseptically in low temperature (4°C) refrigerator to protect from contamination and spoilage. Molecular Identification of LAB Species Identification of the isolates AG1 was carried out on the basis of 16S rRNA analysis after identifying the genera of the isolated bacteria AG1 by basic characterization procedure described in Bergey’s Manual of Systemic Bacteriology, for 16S rRNA sequencing, the bacteria were sent to “Genexplore Diagnostics & Research Centre Pvt. Ltd.” Ahmedabad, Gujarat, India. 2.3 TO PRODUCE BIOGAS FROM WASTE BY L. delbrueckii ssp. indius AG1 In this work, various types of agriculture wastes, from students’ cafeteria of Noble University, Junagadh mixed L. delbrueckii ssp. indius AG1 were used as substrates for biogas production in 5 Litters sterile cylindrical plastic anaerobic digesters. In the above stated biogas digester one of the sterile cylindrical plastic anaerobic digester prepared for sterile agriculture wastes of students’ cafeteria and lactic acid bacteria and the other digester contained sterile agriculture wastes without lactic acid bacteria as a control. 2.4 Biogas production: Anaerobic co-digestion of agriculture wastes was carried out with L. delbrueckii ssp. indius AG1culture. Biogas was started to form within a day of incubation and was measured using orset instrumentation method. Biogas formation took place only in the first stage. Biogas was collected from agriculture waste. The biogas formation reached its maximum in 20 days. The setup was continued for few more days for experimental observation. No more biogas was formed after that. The methods used in the construction of bio-digester, feeding of bio-digester and mode of biogas collection are as discussed as follows; Design Consideration The requirements for designing of a Bio digester are volume of digester (Vol digester ), storage capacity of the gas, volume of gas holder (Vol Holder ), retention period and the amount and type of organic waste to be disposed in the digester. In order to determine the unit size of a biogas unit, Eq. 1 must be achieved: Volume of digester (liters) = Daily feed-in (liters/day) × Retention time (day) Where the volume of digester is volume occupied by the fermented material and the volume of gas storage. The digesters were fed at once but the calculation was based on daily feeding with the design criteria of retention period of 20 days, daily feeding of 0.24 kg and 0.24 kg of water for feeding i.e. 1:1 of waste and water Computation of the Bio-digester 1 kg is equivalent to 1 liter; hence the total volume of digester’s feed per day is given as: Vol total of digester feed/day = 0.24 l of waste + 0.24 l of water = 0.48 l/day From Eq. 1, Vol digester = vol total of digester feed/day × retention period (day) = 0.48 × 20 = 9.6 liters Also the volume of the gas holder is given as one-fifth of the volume of the digester: Vol holder =1/5× 9.6 = 1.92 Liters Hence the total volume of digester is given as: Total digester volume = volume of digester + volume of gas holder = 9.6 + 1.92 = 11.52 ~ 12 liters Feeding of Digester The mode of feeding used was a discontinued feeding (batch feeding). This simply means loading the digester at once and maintaining a closed environment throughout the retention period. Two different digesters were prepared for loading. These one digester is for the wastes (sterile agriculture waste & L. delbrueckii ssp. Indius AG1 ) and the control (only sterile agriculture waste). The procedures followed during feeding of the digester are as below; 1. 2 kg of each of the wastes (sterile agriculture waste & L. delbrueckii ssp. Indius AG1 ) was weighed and 2 liters of sterile water was mixed thoroughly with each of the waste in the ratio of 1:1 (Table 1 ). 2. The mixture of each of the wastes were poured into digester. 3. 2 kg of sterile agriculture waste without L. delbrueckii ssp. Indius AG1 were weighed and mixed thoroughly with 2 liters of sterile water each for the co-digestion (Table − 1) Table 1 Ratio of Waste and Water Used Waste used Weight of waste Inoculum Liter of sterile water used Sterile agriculture waste 2 kg - 2 liters Co-digestion Sterile agriculture waste & L. delbrueckii ssp. Indius AG1 2 kg 10 ml 2 liters Anaerobic digester setup: The experimental set up for the study using anaerobic digestion consists of plastic cane with a plastic cover and all the three anaerobic digesters were constructed at bench-scale experiments where the degradation of the agriculture waste and lactic acid bacteria and one as control without lactic acid bacteria were accomplished in sealed serum bottles with a capacity of 2 liters. Each bottle was sealed with its cover having two outlets. The first outlet was attached to an 8 mm internal diameter hose gas pipe and immersed up to a little above the bottom of the solution level in order to take samples without introducing air into the digester and indicate the quantity of gas produced inside the digester. Thus, a plastic cane was extended from the bottom of the substrate up to the plastic cane cover to prevent out flow of the substrate from the inside of the digester. The second outlet was above the top of the solution for gas collection. The whole cover and the hose gas pipe were sealed with gasket to protect air leakage from the environment. It was operated at ambient temperature in the hemophilic range (27℃-31°C), yet the temperature and moisture were monitored daily using thermo-hygrometer. A gas collector was provided for collection and determination of the amount of biogas. The content of methane concentration produced in the reactor was monitored daily. In the digesters’ internal working temperature was maintained at the ambient temperature of the room using thick cover of sand and pH was regularly measured (every three days) throughout the digestion process. Biogas yield and its quality The volume and methane content of the gas produced in the anaerobic reactors were measured by an indirect method and determination of the composition of biogas gas was done by gas chromatography analysis. The indirect method was employed to estimate both the amount of biogas produced and the methane content of the gas. First, the volume of water displaced by the gas was measured by down ward displacement of water for each digester which corresponds to the amount of biogas produced. Subsequently, the methane content in the biogas was estimated corresponding to the amount of CO 2 produced from the digesters. The gas was allowed to pass through a 10% NaOH solution as the CO 2 dissolves in it and form carbonate. Thus, the amount of NaOH displaced is approximately equal to the amount of methane in the gas. Other types of gases were dissolved in the solution Results and Discussion 3.1. Isolation and identification of lactic acid bacteria Isolation and Characterization of potential lactic acid bacteria: Identification of the isolate AG1 on the basis of 16s rRNA analysis After identifying the genera of isolated bacteria AG1 by basic characterization described in Bergey's Manual of Systemic Bacteriology, the isolates was further identified to species level by 16s rRNA sequencing. For this, the bacteria were sent to “Genexplore Diagnostics & Research Centre Pvt. Ltd.” Ahmedabad, Gujarat, India. Alignment report - 16S rRNA sequencing of isolate L. delbrueckii ssp. Indius AG1 TTCCTTCGGGATGATTTGTTGGACGCTCGCGGCGGATGGGTGAGTAACACGTGGGCAATCTGCCCTAAAGACTGGGATACCACTT GGAAACAGGTGCTAATACCGGATAACAACATGAATCGCATGATTCAAGTTTGAAAGGCGGCGCAAGCTGTCACTTTAGGATGAG CCCGCGGCGCATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCAATGATGCGTAGCCGAGTTGAGAGACTGATCGGCCACAT TGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACG CCGCGTGAGTGAAAAAGGTTTTCGGATCGTAAAGCTCTGTTGTTGGTGAAAAAGGATAGAGGCAGTAACTGGTCTTTATTTGAC GGTAATCAACCAGAAAGTCACGGCTAACTACGTGCCAGCACCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGG GCGTAAAGCGAGCGCAGGCGGAATGATAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGAAACTGCATCGGAAACTGTCATT CTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCG GCTCTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAAC GATGAGCGCTAGGTGTTGGGGACTTTCCGGTCCTCAGTGCCGCAGCAAACGCATTAAGCGCTCCGCCTGGGGAGTACGACCGCA AGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTA CCAGGTCTTGACATCCTGCGCTACACCTAGAGATAGGTGGTTCCCTTCGGGGACGCAGAGACAGGTGGTGCATGGCTGTCGTCA GCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCTTTAGTTGCCATCATTAAGTTGGGCACTCTAGA GAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGACCTGGGCTACACACGTGCTAC AATGGGCAGTAC ACGAGAAGCGAACCCGCGAGGGTAAGCGGATCTCTTAAAGCTGTTCTC 3.2 VOLUME OF BIOGAS PRODUCED FOR EACH CO-DIGESTION WASTE Figure 5 shows the volume biogas produced from sterile agriculture waste & L. delbrueckii ssp. indius AG1, control within the retention period 20 days. Production of gas from sterile agriculture waste & L. delbrueckii ssp. indius AG1, started on day 9 of the retention period by producing average biogas of 45 ml, thereafter increases to 97.5 ml on day 10 and reduces to 185 ml on day 12. At day 13, the biogas produced was 255 ml in which decreases back to 185 ml on the next day and increases thereafter until it reached the peak on day 16 with 575 ml biogas production after which it begins to reduce till the completion of the retention period which is similar to the work of Aremu and Agarry, 2012). Temperature of Slurry for Co-digestion waste Figure 6 indicates the temperature of the co-digestion of sterile agriculture waste & L. delbrueckii ssp. indius AG1, control. The temperature varies from 25.1 - 27.4 for agriculture waste & L. delbrueckii ssp. Indius AG1 , 25.2 – 27.8°C and remained stable on day 15 to day 17. These temperature ranges also signifies a mesophilic thermal stage of biogas production (25 - 45°C. The maximum biogas produced for each co-digestion was attained at day 20, day 14 and day 19 respectively in which the temperature for these days was 27.1°C, 26.4°Cand 26.9°C respectively. Temperature has been observed by most biogas researchers to be quite critical for anaerobic digestion, since lactic acid bacteria – L. delbrueckii ssp. Indius AG1 operate most efficiently at temperatures 30.0 – 40.0°C. For this study, the four digesters operated under mesophilic condition which is similar to the temperature 30.0 – 40.0°C .The temperature of below 30 in which this experiment was operated, could have contributed to the slow development of methanogens and consequently low methane production. This is similar to the report of (Ilori et al., 2007) that the recovery time for biogas production as well as the quality and quantity of biogas produced from agricultural materials are a function of the nature, and composition of the digester feedstock. Conclusion The study on the production of biogas from the co-digestion of sterile agriculture waste L. delbrueckii ssp. Indius AG1 has shown that biogas can be produced from these wastes through anaerobic digestion for biogas generation. These wastes are always available in our environment and can be used as a source of fuel if managed properly. The study revealed further that L. delbrueckii ssp. Indius AG1 has great potentials for generation of biogas if only one type of waste is to be used and co-digestion of agriculture waste if co-digestion is to be used. The utilization should be encouraged due to high volume of biogas yields. Moreover, it has been found that temperature variation that affected the volume yield of biogas production and the temperature ranges also signifies a mesophilic thermal stage of biogas production (25– 40°C The temperature in which the production of biogas was at the peak for each waste ( L. delbrueckii ssp. Indius AG1 ) was attained at day 22, day 24 and day 30 with the temperature for these days was 27.7°C, 26.9°C and 27.1°C respectively and for each co-digestion (agriculture waste) was attained at day 24, day 14 and day 19 in which the temperature for these days was 27.1°C, 26.4°C and 26.9°C respectively. Declarations CONFLICT OF INTEREST The authors declare no conflict of interest. FUNDING SOURCES There is no funding or financial support for the above research work. Author Contribution Ankita Suvagiya was corresponding author have made a substantial contribution to the concept or design of the article; or the acquisition, analysis, or interpretation of data for the article; drafted the article or revised it critically for important intellectual content; Dr. Gira Mankad approved the version to be published. ACKNOWLEDGMENT The author is sincerely thankful and highly indebted to the Department of Biotechnology and Food Testing Laboratory, Junagadh Agricultural University for their valuable support and research facilities provided and all faculties. I prompt my deep sagacity of gratitude and indebtedness to Dr. Gira Mankad , Assistant Professor, Department of Microbiology, M.V.M. science, and home science college, Rajkot- Gujarat, for allowing me to carry out my project and for allowing me to do my project work under her supervision. References Aremu, M. O., &Agarry, S. E. (2012). Comparison of Biogas production from Cow dung and Pig dung under Mesophilic condition. International Refereed Journal of Engineering and Science, 1 (4), 1–6. Morales-Polo, C., Cledera-Castro, M. D. M., &MoratillaSoria, B. Y. (2019). Biogas production from vegetable and fruit markets waste—Compositional and batch characterizations. Sustainability, 11 (23), 6790. Al Mamun, M. R., & Torii, S. (2014, October). Anaerobic co-digestion of cafeteria, vegetable and fruit wastes for biogas production. In 2014 International Conference on Renewable Energy Research and Application (ICRERA) (pp. 369–374). IEEE. Morales-Polo, C., Cledera-Castro, M. D. M., Revuelta-Aramburu, M., &Hueso-Kortekaas, K. (2021). Enhancing energy recovery in form of biogas, from vegetable and fruit wholesale markets by-products and wastes, with pretreatments. Plants, 10 (7), 1298. Scano, E. A., Asquer, C., Pistis, A., Ortu, L., Demontis, V., &Cocco, D. (2014). Biogas from anaerobic digestion of fruit and vegetable wastes: Experimental results on pilot-scale and preliminary performance evaluation of a full-scale power plant. Energy conversion and management, 77 , 22–30. Navickas, K. (2007). Biogas for farming, energy conversion and environment protection. Bioplin, Tehnologija in Okolje: mednarodnisimpozij, Rakičan, MurskaSobota, 29. November 2007. Maribor, 2007 . Ilori, M. O., Adebusoye, S. A., Iawal, A. K., &Awotiwon, O. A. (2007). Production of biogas from banana and plantain peels. Advances in Environmental Biology, 33–39. Chanakya, H. N., Ramachandra, T. V., &Vijayachamundeeswari, M. (2006). Anaerobic digestion and reuse of digested products of selected components of urban solid waste. Center for Ecological Sciences & Centre for Sustainable Technologies . Tuomela, M., Vikman, M., Hatakka, A., &Itävaara, M. (2000). Biodegradation of lignin in a compost environment: a review. Bioresource technology, 72 (2), 169–183. Herout, M., Malaťák, J., Kučera, L., &Dlabaja, T. (2011). Biogas composition depending on the type of plant biomass used. Res AgricEng 57: 137–143. Rahmat, B., Hartoyo, T., &Sunarya, Y. (2014). Biogas production from tofu liquid waste on treated agricultural wastes. American Journal of Agricultural and Biological Sciences, 9 (2), 226–231. Rao, M. S., Singh, S. P., Singh, A. K., &Sodha, M. S. (2000). Bioenergy conversion studies of the organic fraction of MSW: assessment of ultimate bioenergy production potential of municipal garbage. Applied energy, 66 (1), 75–87. Ukpai, P. A., &Nnabuchi, M. N. (2012). Comparative study of biogas production from cow dung, cow pea and cassava peeling using 45 litres biogas digester. Advances in Applied Science Research, 3 (3), 1864–1869. Kinyua, M. N., Rowse, L. E., & Ergas, S. J. (2016). Review of small-scale tubular anaerobic digesters treating livestock waste in the developing world. Renewable and Sustainable Energy Reviews, 58 , 896–910. Iqbal, S. A., Rahaman, S., Rahman, M., & Yousuf, A. (2014). Anaerobic digestion of kitchen waste to produce biogas. Procedia Engineering, 90 , 657–662. 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-4102508","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":280434217,"identity":"e1b0742b-7d4d-4c21-a515-243bed51d0b4","order_by":0,"name":"Ankita Suvagiya","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCklEQVRIie2PMUvEMBiGK4WAEOhaOGl+gZBScDz/hfNXhGS5gxs7OHTqJM6FDP4FD+HmrwRuOp0LddB/EBfH0zTqpO3pdnB5hvAN78ObNwg8nr2EBGhf2p9oivfEHc+7FPxUjpp6E2ZOgR2Kq7GE+rgK87I/x5RIVbk2V6uTSGnUASHy9kK/2JZpclr+rsRPa0RcdzR+FKAXlM6XneBWuczOcKCmlSUi6SinlOs6judLBb2C+WpAYU7ZfimUc5kqaUYV3grEpvpWAIBNZuMtaSsAH27slg2BpkZM7yazBQIf3pK0IjPFW3ceXYfavG6RMSXvjSmmyeD8n191Sf7XeA8r/5P2eDyeQ+ADBc9tKtNHbKgAAAAASUVORK5CYII=","orcid":"","institution":"Saurashtra University","correspondingAuthor":true,"prefix":"","firstName":"Ankita","middleName":"","lastName":"Suvagiya","suffix":""}],"badges":[],"createdAt":"2024-03-14 16:58:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4102508/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4102508/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53033464,"identity":"b4034a67-0fc5-4a3f-82d0-569fa6804be4","added_by":"auto","created_at":"2024-03-19 20:25:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1240928,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eSchematic presentation of vegetable waste management proposed in this study to produce Biogas.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4102508/v1/66524b764f5a9e415f2a3fe9.png"},{"id":53033465,"identity":"a2d19bc0-df21-4b1d-9866-8b4b024e02e5","added_by":"auto","created_at":"2024-03-19 20:25:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":503506,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eDigester setup of the experiment\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4102508/v1/ba8c99e689da9c6837f89bbc.png"},{"id":53033744,"identity":"92d25130-db38-477e-8e99-84f255fd8db7","added_by":"auto","created_at":"2024-03-19 20:33:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":541891,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eOrset Setup\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4102508/v1/7ddf50317cc767021cc43d69.png"},{"id":53033460,"identity":"719495a9-0ba4-4b89-b313-26e6a53401ab","added_by":"auto","created_at":"2024-03-19 20:25:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1176129,"visible":true,"origin":"","legend":"\u003cp\u003eCultural and Morphological characteristics of bacteria\u003c/p\u003e\n\u003cp\u003e(A)Microscopic view showing gram positive long rods\u003c/p\u003e\n\u003cp\u003e(B and C) Growth on MRS agar plates\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4102508/v1/b7eae44817702d3bff29f8e0.png"},{"id":53033461,"identity":"63d81548-2481-4e7d-a714-530b8687b93b","added_by":"auto","created_at":"2024-03-19 20:25:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":27100,"visible":true,"origin":"","legend":"\u003cp\u003eVolume of Biogas of Co-digestion Waste against Number of Days\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4102508/v1/4006b5154341c76bd48638ff.png"},{"id":53033462,"identity":"470a8d85-659c-4b8d-98d9-f1641330a260","added_by":"auto","created_at":"2024-03-19 20:25:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":21002,"visible":true,"origin":"","legend":"\u003cp\u003eTemperature of Slurry for Co-digestion Waste against Number of Days\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4102508/v1/bd3ac5bf0424b15aa01cb2dd.png"},{"id":53268498,"identity":"e6e51c08-71c1-4c39-af7d-4f8719440e19","added_by":"auto","created_at":"2024-03-22 16:00:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3036136,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4102508/v1/f56131f3-4d1f-4631-8f13-733095ff8d32.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Lactobacillus delbrueckii ssp. indius AG1: Potential candidate for Degradation of agriculture wastes and production of biogas by orsat instrument method","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIndia produces 108.34\u0026nbsp;million tons of fruit annually, and it produces 212.91\u0026nbsp;million tons of vegetables annually, the second-highest amount in the world. The lack of storage, transportation, and physical deformations results in the annual loss of almost thirty percent of this total crop (Indian Horticulture Annual Report, 2019). After processing, both local vendors and the industries produce wastes including peel, pomace, seeds, and so forth. Waste buildup at disposal locations causes a bad stench and a protracted natural decomposition, which raises health concerns. Thus, it is strongly advised against using this strategy and instead to focus on finding a workable solution that may deal with the present waste management and energy crisis issues. In our nation, almost 80% of people are either directly or indirectly dependent on agriculture. Massive volumes of vegetables are grown throughout the winter, but because they are not efficiently transported or preserved, they are lost. This might potentially be used to produce biogas (Iqbal, \u003cem\u003eet.al.\u003c/em\u003e, 2024)The breakdown of organic waste in the absence of oxygen produces biogas. Because it comes from biological material (biomass), it is also known as biogas. With the aid of enzymes, anaerobic bacteria break down organic waste into smaller molecules like methane, carbon dioxide, and hydrogen sulfide, which combine make up biogas. Since lignocelluloses makes up the majority of the biomass in agricultural waste composting, its breakdown is crucial to the process's success (Tuomela\u003cem\u003eet et.al.\u003c/em\u003e, 2000; Dixon and Langer, 2006). The amount and type of material introduced to the system determines how much methane is produced during the anaerobic digestion of biologically degradable organic materials. Consequently, there are numerous methods for producing energy through anaerobic digestion using leftover food, vegetable and fruit wastes, and cow dung. These days, dry fermentation, co-digestion, single-phase digestion, and two-phase digestion are the most popular ideas (Chanakya, \u003cem\u003eet.al.\u003c/em\u003e,2006).Features and Constituency of Biogas depends on the kind of substrate utilized to create biogas. It greatly influences its composition. Methane (50\u0026ndash;70%), carbon dioxide (30\u0026ndash;40%), hydrogen, nitrogen, and hydrogen sulfide are the main components of biogas. After it was determined that thermo-chemical pre-treatment and co-digestion were the most effective methods for optimizing biogas production from plant wastes, it was suggested that more research be done on the subject and that AD(Anaerobic digesters) digesters adopt this technology on a larger scale. Moisture content, C: N ratio, and TS were found to have an impact on the gas production (Rahmat, B., Hartoyo, T., \u0026amp; Sunarya, Y. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. ISOLATION OF LACTIC ACID BACTERIA\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003eSample collection:\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eLactic acid bacteria were isolated from vegetable waste or fruit waste. After collection, the samples were stored aseptically in low temperature (4\u0026deg;C) refrigerator to protect from contamination and spoilage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMolecular Identification of LAB Species\u003c/h2\u003e \u003cp\u003eIdentification of the isolates AG1 was carried out on the basis of 16S rRNA analysis after identifying the genera of the isolated bacteria AG1\u003c/p\u003e \u003cp\u003eby basic characterization procedure described in Bergey\u0026rsquo;s Manual of Systemic Bacteriology, for 16S rRNA sequencing, the bacteria were sent to \u0026ldquo;Genexplore Diagnostics \u0026amp; Research Centre Pvt. Ltd.\u0026rdquo; Ahmedabad, Gujarat, India.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.3 TO PRODUCE BIOGAS FROM WASTE BY\u003c/b\u003e \u003cb\u003eL. delbrueckii ssp. indius AG1\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn this work, various types of agriculture wastes, from students\u0026rsquo; cafeteria of Noble University, Junagadh mixed \u003cem\u003eL. delbrueckii ssp. indius\u003c/em\u003e AG1 were used as substrates for biogas production in 5 Litters sterile cylindrical plastic anaerobic digesters. In the above stated biogas digester one of the sterile cylindrical plastic anaerobic digester prepared for sterile agriculture wastes of students\u0026rsquo; cafeteria and lactic acid bacteria and the other digester contained sterile agriculture wastes without lactic acid bacteria as a control.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.4 Biogas production:\u003c/h2\u003e \u003cp\u003eAnaerobic co-digestion of agriculture wastes was carried out with \u003cem\u003eL. delbrueckii ssp. indius\u003c/em\u003e AG1culture. Biogas was started to form within a day of incubation and was measured using orset instrumentation method. Biogas formation took place only in the first stage. Biogas was collected from agriculture waste. The biogas formation reached its maximum in 20 days. The setup was continued for few more days for experimental observation. No more biogas was formed after that.\u003c/p\u003e \u003cp\u003eThe methods used in the construction of bio-digester, feeding of bio-digester and mode of biogas collection are as discussed as follows;\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eDesign Consideration\u003c/h2\u003e \u003cp\u003eThe requirements for designing of a Bio digester are volume of digester (Vol\u003csub\u003edigester\u003c/sub\u003e), storage capacity of the gas, volume of gas holder (Vol\u003csub\u003eHolder\u003c/sub\u003e), retention period and the amount and type of organic waste to be disposed in the digester. In order to determine the unit size of a biogas unit, Eq.\u0026nbsp;1 must be achieved:\u003c/p\u003e \u003cp\u003eVolume of digester (liters)\u0026thinsp;=\u0026thinsp;Daily feed-in (liters/day) \u0026times; Retention time (day)\u003c/p\u003e \u003cp\u003eWhere the volume of digester is volume occupied by the fermented material and the volume of gas storage. The digesters were fed at once but the calculation was based on daily feeding with the design criteria of retention period of 20 days, daily feeding of 0.24 kg and 0.24 kg of water for feeding i.e. 1:1 of waste and water\u003c/p\u003e \u003cp\u003eComputation of the Bio-digester\u003c/p\u003e \u003cp\u003e1 kg is equivalent to 1 liter; hence the total volume of digester\u0026rsquo;s feed per day is given as:\u003c/p\u003e \u003cp\u003e \u003cem\u003eVol\u003c/em\u003e \u003csub\u003e \u003cem\u003etotal\u003c/em\u003e \u003c/sub\u003e \u003cem\u003eof digester feed/day\u0026thinsp;=\u0026thinsp;0.24 l of waste\u0026thinsp;+\u0026thinsp;0.24 l of water\u0026thinsp;=\u0026thinsp;0.48 l/day\u003c/em\u003e\u003c/p\u003e \u003cp\u003eFrom Eq.\u0026nbsp;1,\u003c/p\u003e \u003cp\u003e \u003cem\u003eVol\u003c/em\u003e \u003csub\u003e \u003cem\u003edigester\u003c/em\u003e \u003c/sub\u003e \u003cem\u003e= vol\u003c/em\u003e \u003csub\u003e \u003cem\u003etotal\u003c/em\u003e \u003c/sub\u003e \u003cem\u003eof digester feed/day\u003c/em\u003e \u0026times; \u003cem\u003eretention period (day)\u003c/em\u003e\u003c/p\u003e \u003cp\u003e=\u0026thinsp;0.48 \u0026times; 20\u0026thinsp;=\u0026thinsp;9.6 liters\u003c/p\u003e \u003cp\u003eAlso the volume of the gas holder is given as one-fifth of the volume of the digester:\u003c/p\u003e \u003cp\u003e \u003cem\u003eVol\u003c/em\u003e \u003csub\u003e \u003cem\u003eholder\u003c/em\u003e \u003c/sub\u003e=1/5\u0026times; 9.6\u0026thinsp;=\u0026thinsp;1.92 Liters\u003c/p\u003e \u003cp\u003eHence the total volume of digester is given as:\u003c/p\u003e \u003cp\u003eTotal digester volume\u0026thinsp;=\u0026thinsp;volume of digester\u0026thinsp;+\u0026thinsp;volume of gas holder\u003c/p\u003e \u003cp\u003e=\u0026thinsp;9.6\u0026thinsp;+\u0026thinsp;1.92\u0026thinsp;=\u0026thinsp;11.52\u0026thinsp;~\u0026thinsp;12 liters\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFeeding of Digester\u003c/h2\u003e \u003cp\u003eThe mode of feeding used was a discontinued feeding (batch feeding). This simply means loading the digester at once and maintaining a closed environment throughout the retention period. Two different digesters were prepared for loading. These one digester is for the wastes (sterile agriculture waste \u0026amp;\u003cem\u003eL. delbrueckii ssp. Indius AG1\u003c/em\u003e) and the control (only sterile agriculture waste). The procedures followed during feeding of the digester are as below;\u003c/p\u003e \u003cp\u003e1. 2 kg of each of the wastes (sterile agriculture waste \u0026amp;\u003cem\u003eL. delbrueckii ssp. Indius AG1\u003c/em\u003e) was weighed and 2 liters of sterile water was mixed thoroughly with each of the waste in the ratio of 1:1 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e2. The mixture of each of the wastes were poured into digester.\u003c/p\u003e\u003cp\u003e3. 2 kg of sterile agriculture waste without \u003cem\u003eL. delbrueckii ssp. Indius AG1\u003c/em\u003e were weighed and mixed thoroughly with 2 liters of sterile water each for the co-digestion (Table \u0026minus;\u0026thinsp;1)\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRatio of Waste and Water Used\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWaste used\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWeight of waste\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eInoculum\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLiter of sterile water used\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSterile agriculture waste\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 liters\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCo-digestion\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSterile agriculture waste \u0026amp;\u003cem\u003eL. delbrueckii ssp. Indius AG1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 ml\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 liters\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAnaerobic digester setup:\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe experimental set up for the study using anaerobic digestion consists of plastic cane with a plastic cover and all the three anaerobic digesters were constructed at bench-scale experiments where the degradation of the agriculture waste and lactic acid bacteria and one as control without lactic acid bacteria were accomplished in sealed serum bottles with a capacity of 2 liters. Each bottle was sealed with its cover having two outlets. The first outlet was attached to an 8 mm internal diameter hose gas pipe and immersed up to a little above the bottom of the solution level in order to take samples without introducing air into the digester and indicate the quantity of gas produced inside the digester. Thus, a plastic cane was extended from the bottom of the substrate up to the plastic cane cover to prevent out flow of the substrate from the inside of the digester. The second outlet was above the top of the solution for gas collection.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe whole cover and the hose gas pipe were sealed with gasket to protect air leakage from the environment. It was operated at ambient temperature in the hemophilic range (27℃-31\u0026deg;C), yet the temperature and moisture were monitored daily using thermo-hygrometer. A gas collector was provided for collection and determination of the amount of biogas. The content of methane concentration produced in the reactor was monitored daily. In the digesters\u0026rsquo; internal working temperature was maintained at the ambient temperature of the room using thick cover of sand and pH was regularly measured (every three days) throughout the digestion process.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eBiogas yield and its quality\u003c/h2\u003e \u003cp\u003eThe volume and methane content of the gas produced in the anaerobic reactors were measured by an indirect method and determination of the composition of biogas gas was done by gas chromatography analysis. The indirect method was employed to estimate both the amount of biogas produced and the methane content of the gas. First, the volume of water displaced by the gas was measured by down ward displacement of water for each digester which corresponds to the amount of biogas produced. Subsequently, the methane content in the biogas was estimated corresponding to the amount of CO\u003csub\u003e2\u003c/sub\u003eproduced from the digesters. The gas was allowed to pass through a 10% NaOH solution as the CO\u003csub\u003e2\u003c/sub\u003e dissolves in it and form carbonate. Thus, the amount of NaOH displaced is approximately equal to the amount of methane in the gas. Other types of gases were dissolved in the solution\u003c/p\u003e\u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003ch2\u003e3.1. Isolation and identification of lactic acid bacteria\u003c/h2\u003e\n\u003cp\u003eIsolation and Characterization of potential lactic acid bacteria:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of the isolate AG1 on the basis of 16s rRNA analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter identifying the genera of isolated bacteria AG1 by basic characterization described in Bergey\u0026apos;s Manual of Systemic Bacteriology, the isolates was further identified to species level by 16s rRNA sequencing. For this, the bacteria were sent to \u0026ldquo;Genexplore Diagnostics \u0026amp;amp; Research Centre Pvt. Ltd.\u0026rdquo; Ahmedabad, Gujarat, India.\u003c/p\u003e\n\u003cp\u003eAlignment report - 16S rRNA sequencing of isolate \u003cem\u003eL. delbrueckii ssp. Indius AG1\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTTCCTTCGGGATGATTTGTTGGACGCTCGCGGCGGATGGGTGAGTAACACGTGGGCAATCTGCCCTAAAGACTGGGATACCACTT\u003cbr\u003eGGAAACAGGTGCTAATACCGGATAACAACATGAATCGCATGATTCAAGTTTGAAAGGCGGCGCAAGCTGTCACTTTAGGATGAG\u003cbr\u003eCCCGCGGCGCATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCAATGATGCGTAGCCGAGTTGAGAGACTGATCGGCCACAT\u003cbr\u003eTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACG\u003cbr\u003eCCGCGTGAGTGAAAAAGGTTTTCGGATCGTAAAGCTCTGTTGTTGGTGAAAAAGGATAGAGGCAGTAACTGGTCTTTATTTGAC\u003cbr\u003eGGTAATCAACCAGAAAGTCACGGCTAACTACGTGCCAGCACCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGG\u003cbr\u003eGCGTAAAGCGAGCGCAGGCGGAATGATAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGAAACTGCATCGGAAACTGTCATT\u003cbr\u003eCTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCG\u003cbr\u003eGCTCTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAAC\u003cbr\u003eGATGAGCGCTAGGTGTTGGGGACTTTCCGGTCCTCAGTGCCGCAGCAAACGCATTAAGCGCTCCGCCTGGGGAGTACGACCGCA\u003cbr\u003eAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTA\u003cbr\u003eCCAGGTCTTGACATCCTGCGCTACACCTAGAGATAGGTGGTTCCCTTCGGGGACGCAGAGACAGGTGGTGCATGGCTGTCGTCA\u003cbr\u003eGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCTTTAGTTGCCATCATTAAGTTGGGCACTCTAGA\u003cbr\u003eGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGACCTGGGCTACACACGTGCTAC\u003cbr\u003eAATGGGCAGTAC\u003c/p\u003e\n\u003cp\u003eACGAGAAGCGAACCCGCGAGGGTAAGCGGATCTCTTAAAGCTGTTCTC\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 VOLUME OF BIOGAS PRODUCED FOR EACH CO-DIGESTION WASTE\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 5 shows the volume biogas produced from sterile agriculture waste \u0026amp; \u003cem\u003eL. delbrueckii ssp. indius\u0026nbsp;AG1,\u003c/em\u003e control within the retention period 20 days. Production of gas from sterile agriculture waste \u0026amp;\u003cem\u003eL. delbrueckii ssp. indius AG1,\u0026nbsp;\u003c/em\u003estarted on day 9 of the retention period by producing average biogas of 45 ml, thereafter increases to 97.5 ml on day 10 and reduces to 185 ml on day 12. At day 13, the biogas produced was 255 ml in which decreases back to 185 ml on the next day and increases thereafter until it reached the peak on day 16 with 575 ml biogas production after which it begins to reduce till the completion of the retention period which is similar to the work of Aremu and Agarry, 2012).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTemperature of Slurry for Co-digestion waste\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 6 indicates the temperature of the co-digestion of sterile agriculture waste \u0026amp;\u003cem\u003eL. delbrueckii ssp. indius AG1,\u0026nbsp;\u003c/em\u003econtrol. The temperature varies from 25.1 - 27.4 for agriculture waste \u0026amp;\u003cem\u003eL. delbrueckii ssp. Indius AG1\u003c/em\u003e, 25.2 \u0026ndash; 27.8\u0026deg;C and remained stable on day 15 to day 17. These temperature ranges also signifies a mesophilic thermal stage of biogas production (25 - 45\u0026deg;C. The maximum biogas produced for each co-digestion was attained at day 20, day 14 and day 19 respectively in which the temperature for these days was 27.1\u0026deg;C, 26.4\u0026deg;Cand 26.9\u0026deg;C respectively. Temperature has been observed by most biogas researchers to be quite critical for anaerobic digestion, since lactic acid bacteria \u0026ndash;\u003cem\u003eL. delbrueckii ssp. Indius AG1\u003c/em\u003e operate most efficiently at temperatures 30.0 \u0026ndash; 40.0\u0026deg;C. For this study, the four digesters operated under mesophilic condition which is similar to the temperature 30.0 \u0026ndash; 40.0\u0026deg;C .The temperature of below 30 in which this experiment was operated, could have contributed to the slow development of methanogens and consequently low methane production. This is similar to the report of (Ilori\u003cem\u003eet al.,\u003c/em\u003e 2007) that the recovery time for biogas production as well as the quality and quantity of biogas produced from agricultural materials are a function of the nature, and composition of the digester feedstock.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe study on the production of biogas from the co-digestion of sterile agriculture waste \u003cem\u003eL. delbrueckii ssp. Indius AG1\u003c/em\u003e has shown that biogas can be produced from these wastes through anaerobic digestion for biogas generation. These wastes are always available in our environment and can be used as a source of fuel if managed properly. The study revealed further that \u003cem\u003eL. delbrueckii ssp. Indius AG1\u003c/em\u003ehas great potentials for generation of biogas if only one type of waste is to be used and co-digestion of agriculture waste if co-digestion is to be used. The utilization should be encouraged due to high volume of biogas yields. Moreover, it has been found that temperature variation that affected the volume yield of biogas production and the temperature ranges also signifies a mesophilic thermal stage of biogas production (25\u0026ndash; 40\u0026deg;C The temperature in which the production of biogas was at the peak for each waste (\u003cem\u003eL. delbrueckii ssp. Indius AG1\u003c/em\u003e) was attained at day 22, day 24 and day 30 with the temperature for these days was 27.7\u0026deg;C, 26.9\u0026deg;C and 27.1\u0026deg;C respectively and for each co-digestion (agriculture waste) was attained at day 24, day 14 and day 19 in which the temperature for these days was 27.1\u0026deg;C, 26.4\u0026deg;C and 26.9\u0026deg;C respectively.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCONFLICT OF INTEREST\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003ch2\u003eFUNDING SOURCES\u003c/h2\u003e \u003cp\u003eThere is no funding or financial support for the above research work.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAnkita Suvagiya was corresponding author have made a substantial contribution to the concept or design of the article; or the acquisition, analysis, or interpretation of data for the article; drafted the article or revised it critically for important intellectual content; Dr. Gira Mankad approved the version to be published.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGMENT\u003c/h2\u003e \u003cp\u003eThe author is sincerely thankful and highly indebted to the Department of Biotechnology and Food Testing Laboratory, Junagadh Agricultural University for their valuable support and research facilities provided and all faculties. I prompt my deep sagacity of gratitude and indebtedness to \u003cb\u003eDr. Gira Mankad\u003c/b\u003e, Assistant Professor, Department of Microbiology, M.V.M. science, and home science college, Rajkot- Gujarat, for allowing me to carry out my project and for allowing me to do my project work under her supervision.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAremu, M. O., \u0026amp;Agarry, S. E. (2012). Comparison of Biogas production from Cow dung and Pig dung under Mesophilic condition. International Refereed Journal of Engineering and Science, \u003cem\u003e1\u003c/em\u003e(4), 1\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorales-Polo, C., Cledera-Castro, M. D. M., \u0026amp;MoratillaSoria, B. Y. (2019). Biogas production from vegetable and fruit markets waste\u0026mdash;Compositional and batch characterizations. Sustainability, \u003cem\u003e11\u003c/em\u003e(23), 6790.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl Mamun, M. R., \u0026amp; Torii, S. (2014, October). Anaerobic co-digestion of cafeteria, vegetable and fruit wastes for biogas production. In \u003cem\u003e2014 International Conference on Renewable Energy Research and Application (ICRERA)\u003c/em\u003e (pp. 369\u0026ndash;374). IEEE.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorales-Polo, C., Cledera-Castro, M. D. M., Revuelta-Aramburu, M., \u0026amp;Hueso-Kortekaas, K. (2021). Enhancing energy recovery in form of biogas, from vegetable and fruit wholesale markets by-products and wastes, with pretreatments. Plants, \u003cem\u003e10\u003c/em\u003e(7), 1298.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScano, E. A., Asquer, C., Pistis, A., Ortu, L., Demontis, V., \u0026amp;Cocco, D. (2014). Biogas from anaerobic digestion of fruit and vegetable wastes: Experimental results on pilot-scale and preliminary performance evaluation of a full-scale power plant. Energy conversion and management, \u003cem\u003e77\u003c/em\u003e, 22\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNavickas, K. (2007). Biogas for farming, energy conversion and environment protection. \u003cem\u003eBioplin, Tehnologija in Okolje: mednarodnisimpozij, Rakičan, MurskaSobota, 29. November 2007. Maribor, 2007\u003c/em\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIlori, M. O., Adebusoye, S. A., Iawal, A. K., \u0026amp;Awotiwon, O. A. (2007). Production of biogas from banana and plantain peels. Advances in Environmental Biology, 33\u0026ndash;39.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChanakya, H. N., Ramachandra, T. V., \u0026amp;Vijayachamundeeswari, M. (2006). Anaerobic digestion and reuse of digested products of selected components of urban solid waste. \u003cem\u003eCenter for Ecological Sciences \u0026amp; Centre for Sustainable Technologies\u003c/em\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTuomela, M., Vikman, M., Hatakka, A., \u0026amp;It\u0026auml;vaara, M. (2000). Biodegradation of lignin in a compost environment: a review. Bioresource technology, \u003cem\u003e72\u003c/em\u003e(2), 169\u0026ndash;183.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHerout, M., Malať\u0026aacute;k, J., Kučera, L., \u0026amp;Dlabaja, T. (2011). Biogas composition depending on the type of plant biomass used. Res AgricEng 57: 137\u0026ndash;143.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRahmat, B., Hartoyo, T., \u0026amp;Sunarya, Y. (2014). Biogas production from tofu liquid waste on treated agricultural wastes. American Journal of Agricultural and Biological Sciences, \u003cem\u003e9\u003c/em\u003e(2), 226\u0026ndash;231.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRao, M. S., Singh, S. P., Singh, A. K., \u0026amp;Sodha, M. S. (2000). Bioenergy conversion studies of the organic fraction of MSW: assessment of ultimate bioenergy production potential of municipal garbage. Applied energy, \u003cem\u003e66\u003c/em\u003e(1), 75\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUkpai, P. A., \u0026amp;Nnabuchi, M. N. (2012). Comparative study of biogas production from cow dung, cow pea and cassava peeling using 45 litres biogas digester. Advances in Applied Science Research, \u003cem\u003e3\u003c/em\u003e(3), 1864\u0026ndash;1869.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKinyua, M. N., Rowse, L. E., \u0026amp; Ergas, S. J. (2016). Review of small-scale tubular anaerobic digesters treating livestock waste in the developing world. Renewable and Sustainable Energy Reviews, \u003cem\u003e58\u003c/em\u003e, 896\u0026ndash;910.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIqbal, S. A., Rahaman, S., Rahman, M., \u0026amp; Yousuf, A. (2014). Anaerobic digestion of kitchen waste to produce biogas. Procedia Engineering, \u003cem\u003e90\u003c/em\u003e, 657\u0026ndash;662.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"Agriculture waste, Biogas, L. delbrueckii ssp. indius AG1, Molecular identification, Orsat instrument method","lastPublishedDoi":"10.21203/rs.3.rs-4102508/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4102508/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe anaerobic digestion of organic materials, which is necessary to add chemicals to the continuous biochemical process, is largely mediated by microorganisms. Through a sequence of metabolic events, the microorganisms efficiently break down complex organic compounds to produce simple compounds like methane. In this investigation, inoculums used for biogas production was isolated from agriculture waste sources\u0026mdash;\u003cem\u003eL. delbrueckii ssp. Indius AG1\u003c/em\u003e at mesophilic temperature (35\u0026ordm;C)\u0026mdash;to assess their suitability for anaerobic digestion of agricultural residues (AR) and fruit and vegetable waste (FVW). The highest biogas production of 215 ml/2 kg waste was achieved for a mixture of inoculums, and biogas characterization by orsat instrument method showed 47.7% of the methane content. The morphological, biochemical, and molecular techniques were used to identify the microbial flora present in the high-yield reactor. The abundance of \u003cem\u003eL. delbrueckii ssp. indius AG1\u003c/em\u003e. To get a high methane yield from organic waste it is necessary to maintain the equilibrium and availability of efficient microbial communities like firmicutous, hydrogenotrophic, and acetoclastic methanogens.\u003c/p\u003e","manuscriptTitle":"Lactobacillus delbrueckii ssp. indius AG1: Potential candidate for Degradation of agriculture wastes and production of biogas by orsat instrument method","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-19 20:24:56","doi":"10.21203/rs.3.rs-4102508/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":"2c1fdacd-d98a-44e6-826e-8e7034e27868","owner":[],"postedDate":"March 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-08T11:33:15+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-19 20:24:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4102508","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4102508","identity":"rs-4102508","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}