Enhancing the biological hydrogen production from different biomass through individual pretreatment method

Enhancing the biological hydrogen production from different biomass through individual pretreatment 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 Enhancing the biological hydrogen production from different biomass through individual pretreatment method Chelladurai mumtha, Pambayan Ulagan Mahalingam This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3943615/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 Heat, ultrasonication, acid hydrolysis, and integrated treatment were the various pretreatment techniques employed by different substrates. For the two distinct biomass, integrated pretreatment techniques were used, including heat with acid hydrolysis in sugarcane bagasse and heat with ultrasonication in dairy whey (DW). A batch experiment with microorganisms was conducted to produce biohydrogen from dairy whey and sugarcane bagasse using three different pretreatment techniques. The heat-treated DW had a maximum cumulative hydrogen production of 153.4 ± 2.0 mL H 2 /L, which is 20% more than that of the untreated biomass. After pretreatment, FTIR, XRD, SEM, and EDAX were used to analyse the physicochemical changes in DW and SCB. Untreated and treated waste biomass were analyzed using FTIR spectroscopy to quantify their functional groups. According to EDX results, untreated SCB contains 30% Carbon, 13.71% Oxygen, and 0.50% Nitrogen. SCB was treated with acid using a hydrolysis time of 90mins at 121°C and H 2 SO 4 concentration 2 M the highest cumulative H 2 production of 189.6 ± 4.3 mL H 2 /L was obtained at 37℃ in co-culture. In the future, it may be possible to produce biomass biohydrogen that is both efficient and sustainable based on the findings of this study. Heat treatment Acid hydrolysis treatment FTIR XRD SEM and EDAX Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Environmental issues, climate change, limited resources, economic issues, and current fossil fuel-based energy systems are among the significant concerns (2019). For this reason, fuels are necessary for the future. According to the latest research on hydrogen, it is a clean, reusable, high-energy fuel that doesn't cause pollution (Bicer and Dincer 2018). Biological approaches to hydrogen productions most convenient processes operated efficiently are less energy intensive and more eco-friendly compared with conventional chemical methods (Chandrasekhar et al. 2020). Fermentative hydrogen production is another biological hydrogen generation method because it uses different carbon sources, including renewable biomass and industrial waste (Kotay and Das 2008). Focusing attention on raw materials such as starch, cellulose-containing biomass, and agricultural waste can be used as a potential substrates for biohydrogen production (Goula and Bereketidou 2014) . Industrial waste requires a higher energy input and yields less, so it is used for fermentative hydrogen production (Kapdan and Kargi 2006) . Therefore, Pretreatment is necessary to ensure the process's viability and sustainability. When substrates are pretreatment correctly, sugars can be converted into fermentable carbohydrates polymer such as starch, cellulose, and hemicellulose (Perez et al. 2023). Several pretreatment methods are available such as chemical methods (alkali, acid, and ionic liquids), physical methods (heating, size reduction, steaming, hot water, ultrasonication) and biological methods (Akhtar et al. 2016). Meanwhile, hydrolysis methods used to produce fermentable sugars are alternatives depending on the biomass (Trejo et al. 2022). Ultrasound has recently been employed to enhance the functionality of several bioprocesses. While alkali or acid pretreatments have been found to improve sugar recovery, they are associated with the formation of inhibitors (Sun et al. 2016). These inhibitors affect the hydrogen production negatively. The production of hydrogen from pretreated corn stover in alkaline treatment is favourable for producing fermentable sugar (Zhang et al. 2015). Optimizing the pretreatment process under the most appropriate and economical conditions is essential for improving product yields (Chin et al. 2013). The researchers have extensively investigated temperature and reaction time as optimization parameters (Tan et al. 2010). Organic residues in municipal solid waste, food waste, starch wastewater, kitchen trash, sweet sorghum and abattoir wastewater have all been effectively used to make hydrogen (Silva et al. 2017). Sewage sludge is a great source of organic waste for fermentative hydrogen production in consortiums (Bansal et al. 2012). One of the most widely studied pretreatment techniques, is acid hydrolysis were diluted sulfuric acid effectively hydrolyzes hemicellulose and cellulosic constituents into monomeric sugars (Sun and Cheng 2005). Furfural and acetic acid are produced as by-products. When these substances are present, fermentation is inhibited. Alternative to acid hydrolysis, one of the possible approaches is microbial hydrolysis of wastes by fungal species, which convert starch, hemicellulose, and cellulose into fermentable sugars (Zabed et al. 2016). Fermentation will be improved by selecting a suitable hydrolysis method and controlling inhibitor production (Sivagurunathan et al. 2017). Lignocellulosic biomass with its complex matrix makes it unsuitable for agricultural residue conversion into value-added products. Therefore, pretreatment is required to change the structure of biomass by, depending on the pretreatment methods applied, increasing the surface area, eliminating lignin, decreasing cellulose crystallinity, or depolymerizing the hemicellulose (Akhtar et al. 2016). Starch is almost entirely broken down into carbohydrate molecules by acid hydrolysis at high temperatures (90–150°C) (Cakır et al. 2010). Pure bacteria strains like Rhodobacter sp. M-19, Clostridium butyricum , and Enterobacter aerogenes can produce biohydrogen from starch. Hydrogen was produced from starch using a mixed culture of Rhodobacter sp M-19, E.aerogenes , and C.butyricum , a maximum hydrogen yield was achieved at 2 and 6.6 mol H 2 /mol glucose (Yang et al. 2019). Van Ginkel studied the effects of heat-treated inoculum at 100°C for two hours on hydrogen generation (Van Ginkel et al. 2001). Zhang (Zhang et al. 2020) investigated the effects of acid shock and heat shock combined treatment with sludge used for hydrogen production. In a batch reactor, the ratio between kitchen waste and inoculum has been varied to produce biohydrogen (Bansal et al. 2012). Therefore, In this study focuses on the evaluation of the pretreatment effect of substrates on biohydrogen production. The novelty of this study was to compare the effects of three individual pretreatments (Heat, Ultrasonication and Acid hydrolysis) and two combined pretreatments (Heat with ultrasonication in DW and Heat with acid hydrolysis in SCB). The pretreatment process was optimized by utilizing dairy whey, sugarcane bagasse, and liquid hydrolysis for producing biohydrogen from E.coli, S.bongori , and S.oneidensis and its consortium. Materials and Methodology 2.1) Substrates collection The previous research paper mentioned where two biomass were collected: dairy whey and sugarcane bagasse (Mumtha et al. 2023a). Then, this study used this waste biomass in different pretreatment methods. Dairy whey was stored in room temperature at 4.0°C used for further process. The collected sugarcane bagasse was dried in a hot-air oven at 40°C for 24hr and dried milled sample stored in room temperature. 2.2) Analysis of physio-chemical characteristics of Dairy whey and Sugarcane bagasse Physio-chemical characteristics include pH, VSS (Volatile Suspended Solids)COD (Chemical oxygen demand), TSS (Total Suspended Solid), VFA (Volatile fatty acid), TS (Total Solids), VDS (Volatile dissolved solids), Sodium, Phosphates, Nitrogen, Calcium, Moisture content and Ash content in the untreated substrates from both DW and SCB were analyzed in triplicates following standard method as described in APHA, (Federation 2012) manual. 3) Substrate pretreatment 3.1) Pretreatments of dairy whey (DW) The Dairy whey substrates (DW) was pre-treated by heat treatment, ultrasonication and combined heat treatment and ultrasonication as per standard procedures (Karadag et al. 2009; Elbeshbishy et al. 2011a). The pretreated samples were analysed for carbon, protein, reducing sugar and COD. 3.2) Heat treatment of DW Pretreatment of dairy whey of 150 ml of the was done at 90℃ in a hot air oven at different time intervals (15, 35, 55, and 75 min). After treatment of DW, allowed to cool until in the temperature and then used for further studies (Karadag et al. 2009). The pretreated samples were analysed for carbon, protein, reducing sugar and COD. 3.3) Ultrasonication of DW Ultrasonication of 200ml of DW was conducted using ultrasonicator (make) with the high frequency 440w and 24 kHz at 37C for different time intervals of 15, 35, 55, and 75 mins (Elbeshbishy et al. 2011b). The sonicator samples analysed for futher studies. The pretreated samples were analysed for carbon, protein, reducing sugar and COD. 3.4) Combination heat treatments with ultrasonication of DW The DW was pretreated with combined heat treatment and ultrasonication (Karadag et al. 2009; Elbeshbishy et al. 2011b).The initial heat treatments of DW was done at 90°C in a hot air oven at different time intervals (10,25,40 and 55). Followed by the heat treatment DW substrate was subjected for ultrasonication with high frequency of 440w and 24 kHz at 37°C for different time intervals of 15, 35, 55, and 75. The pretreated samples were analysed for carbon, protein, reducing sugar and COD. 4) Sugarcane bagasse (SCB) The sugarcane bagasse (SCB) substrates was pretreated by heat treatment, acid hydrolysis and combined heat treatment and acid hydrolysis as per standard procedures (Ivanova 2009; Reddy et al. 2017). 4.1) Heat treatment of SCB Pretreatment of SCB was done with heat treatment by standard procedure (Ivanova 2009). 100ml of distilled water was added to 50 g of blended sample and subjected for heat treatment at 121℃ using autoclave for at different intervals (30, 60, 90,120 min). After cooling the substrates, the solid residue was separated on Whatman filter paper, and liquid hydrolyzed samples were used for biohydrogen production in batches in dark fermentation. The pretreated samples were analysed for carbon, protein, reducing sugar and COD. 4.2) Acid hydrolysis of SCB Sugarcane bagasse SCB of 10g sample was acid hydrolyzed with 2M H 2 SO 4 in an autoclave at different time intervals of 30, 60, 90, and 120mins at 121°C (Reddy et al. 2017; Mumtha et al. 2023a). The biomass was filtered under vacuum using Whatman filter paper, and a sample was collected on filter paper. The solid residues were washed thrice with deionized water to remove inhibitory compounds formed during acid pretreatment. Solid hydrolyzed and liquid hydrolyzed sample were adjusted to pH 7.0 using 1N NaOH. and subsequently used as raw material for H 2 production. Carbon, protein, reducing sugar and COD were analyzed in the hydrolysed SCB sample according to the standard method. 4.3) Combined heat treatment and acid hydrolysis of SCB Sugarcane bagasse of 100g was transferred to the conical flask and poured 150ml of deionized water then the sample was placed in an autoclave and heat-treated at 121°C for different intervals (10, 15, 20,and 25 mins). After filtering, the substrate completely dried a solid residue, and each liquid hydrolysis was separately collected and stored at room temperature. Additionally, 100g of SCB was mixed with 2M H 2 SO 4 and autoclaved at 121°C for different intervals (15, 30, 60 and 120 mins). The samples were allowed to cool to room temperature after being treated, and the solid residues were collected by filtering them using Whatman filter paper. Before the hydrogen fermentation process, the sample was neutralised to pH 7.0 by adding 1 N HCl or 1 N NaOH solution (Ramprakash and Muthukumar 2015). Carbon, protein, reducing sugar and COD were analyzed in the hydrolysed SCB sample according to the standard method. 5) Structural characteristics of pretreated and untreated substrates 5.1) FTIR analysiss Fourier Transform - Infrared Spectroscopy was used to analyze the different functional charactesitic present in organic molecules. The structural changes from sugarcane bagasse and dairy whey pre-treated samples were identify by FT-IR spectroscopy (Jascob FT-IR Thermoscientific, USA) spectrophotometer. Potassium bromide (KBr) in a 1:5 ratio is mixed with liquid and solid samples and pressed into discs. The samples were then scanned between 4000 and 450 cm1 at a resolution of 4 cm -1 (Moretti et al. 2014a). 5.2) SEM and Edax analysis In a scanning electron microscope (SEM), the morphology of treated and untreated SCB were analyzed to determine their external structure changes. The pretreated sample was dried for room temperature at 64hrs and the dried sample was placed in a conductive glue and then coated with a thin layer of gold to increase conductivity and improve image quality. The element concentration and chemical identity of both pretreated and untreated SCB were analysed by EDX. X-ray absorption spectroscopy, which generates energy differences in the form of peaks, is performed by energy-dispersive X-ray spectrometers (Bruker EDX) (Badiei et al. 2012). 5.3) XRD analysis X-Ray diffraction (XRD) is used to analyze a sample's chemical properties, crystallographic properties, and physical properties. The crystallinity of the native sugarcane bagasse after the pretreatment of the solid residue crystalline index (CrI) was quantified according to the empirical formula followed by Sun (Sun et al. 2015). The test was conducted using an XRD using a ( Jambes bolt/3) operating at at 40 kV and 30 mA and scanning at 25C per minutes The XRD patterns were obtained over a 2θ = 5–60 o angular range (Bouramdane et al. 2022) . For the amporphus region, the crystalline index was calculated by estimating the intensity of the fitted peak after removing the background and determining the peaks from crystalline and non-crystalline region 6) Batch experiment Batch experiment were performed in 150 mL of serum bottles with a standard volume of 50mL. Each fermentation bottle added the 5 mL of nutrient medium, which included (3g/L NH 4 HCO 3 , 0.125g/L KH 2 PO 4 , 0.015g/L MnSO 4 . 6H 2 O, and 0.100 g/L MgCl 2 .6H 2 O, Trace element 0.001 g/L CoCl 2 .5H 2 O, 5.73 g/L NaHCO 3, 0.005 g/L CuSO 4 .5H 2 O, and 0.025g/L FeSO 4 . 7 H 2 O,). The raw and pretreated sample was used as substrate. Batch test, the inoculum was added to a monoculture and its bacterial consortium in microbial fermentation for biohydrogen production. After the N 2 and CO 2 gas had been set for 5 minutes, the fermentation serum bottles were sealed with a rubber corck and blocked with an aluminium led using squeezing forceps. The fermentation bottles were maintained at 150 rpm in a (Remi Instrument) orbital shaking incubator for 62hr. GC-TCD was used to determine the production of H 2 gas (Murugan et al. 2021). 7) Analytical methods The physiochemical characteristics include pH, VSS (Volatile suspended solids), TSS (Total suspended solid ), COD (Chemical oxygen demand), TS (Total solids), Sodium, Phosphates, Calcium, and Nitrogen were determined by the standard method (Federation 2012). GC-FID (Shimadzu GC 2014) was used to evaluate volatile fatty acids (VFAs), and centrifuged fermented sample at 5,000 rpm for 5°C. The supernatant was collected and passed through a membrane filter with a pore size of 0.2 mm (2% H 3 PO 4 80/100 mesh) and a capillary column coated with 10% PEG-20 M. The temperatures of the programmed column, injection port, and detector were set to 220°C, 240°C, and 130-175°C, respectively. The gases H 2 and CO 2 were analysed by gas chromatography-thermal conductivity detector (Shimadzu GC 2014). Nitrogen gas served as the carrier gas and The packaging material utilised was Propak Q tube, which has an 80/100 mesh. The temperature range of the oven, injection port, and detector was 150°C to 100°C to 80°C. The bio-H 2 was manually injected into a volume of around 1 ml. The modified Gompertz equation was used to determine maximum H 2 production rates from cumulative H 2 production. (Mumtha et al. 2022). Result and Discussion 8.1) Physicochemical characteristics of substrates A physiochemical parameter was determined for both substrates in the previous study. A moisture content of 6.7 x 0.5 was recorded in an earlier study for sugarcane bagasse. Sugarcane bagasse contains a high amount of soluble carbohydrates and lignocellulosic components. Solids presence in dairy whey expressed as volatile suspended solids (VSS), total suspended solids (TSS), and total solids (TS) were recorded at 16.69 g/L, 18.4 g/L, and 38.74 g/L respectively. The sugarcane bagasse was determined for a variety of characteristics, such as ash content, total solids (TS), Chemical Oxygen Demand (COD), volatile solids (VS), etc, according to Standard methods. 8.2) FT-IR analysis of treated and untreated biomass FTIR spectroscopy to determine functional group characteristics of two different biomass, dairy whey and sugarcane bagasse. The intensity of strong and broad peaks was attained at 3326 cm -1 , indicating that carbohydrates, proteins, and lipids were present. Ekka (Ekka and Mierin 2022) observed a broad and strong peak intensity in this wavelength of 3450-3285 cm -1 corresponding with lipids, protein and carbohydrates. There is a peak at 3326 cm- 1 linked with O–H stretching caused by either carbohydrates or hydroxyl groups from dairy whey (Figure 1) . Following the treatment of dairy whey with heat and ultrasonication for 55 minutes, the peaks of carbohydrates and proteins achieved a higher intensity than those in untreated dairy whey. A similar reduction in peak intensity was observed after heat treatment for 15 min and ultrasonication treatment for 15, 35, 55 min and it was assumed to be carbohydrates and protein degradation. During the combined treatment of Heat+Ultrasonication treatment (HT+UT) for 10+15 min, an increase in peak intensity was observed for both carbohydrates and proteins. 2349 cm −1 is corresponding O=C=O stretching carbon dioxide group (Figure 1) . CO 2 bands may cause the O=C=O stretching group at 669 and 2600 cm −1 (Tan and Lebron 2012). The strong peak is 1633 cm −1 in this C=O bond on protein-relevant peaks observed after pretreatment. Tang (Tang et al. 2017) dairy effluent contains proteins, and the peak at this wavelength shows if it decreased or increased before and after pretreatment. After heat treatment, The C=C bending alkene group is represented by the band at 676 cm -1 . (Figure 1). FTIR analysis was used to determine the functional group of SCB biomass before and after acid hydrolysis. The FTIR analysis of raw sugar cane bagasse (SCB), liquid fraction and solid fraction after acid hydrolysis of SCB revealed significant functional group changes (Figure 2 A) . Cellulose was determined to be the band assigned at 4,000–2,995 cm -1 (Morán et al. 2008; Vukoja et al. 2021). In both treated and untreated samples, crystalline cellulose is indicated by the OH group, which is shown by the peak at 3785 cm -1 . After heat treatment, the peak at 3347 cm -1 indicates that the hydroxyl (OH) group has been solubilized in the liquid fraction of acid hydrolysis. The peak intensity of 2360 and 2342 cm-1 for the hydrolysis of SCB in both the liquid and solid fractions was attributed to the -CH stretching of the methyl and methylene groups. The peak intensity of 1589–1635 cm –1 in both liquid and solid hydrolysis was attributed to C–H bond deformations, and lignin was linked to aromatic ring vibration. The vibration of aromatic rings (1600, 1635, 1510 cm-1) and the stretching of carbonyl groups (1728 cm-1) are responsible for the strong peak seen at 1516–1598 cm -1 ; lignin compounds are present in all of these (de et al ., 2014). The bands corresponding to the symmetric CH 2 bending are located at 1439 cm -1 and 1352 cm -1 (Cao and Tan 2004; Bouramdane et al. 2022). The peak intensity at 1297 cm -1 , which is attributed to the C-O ether group, is similar in treated and untreated SCB. According to liquid hydrolysis, the ring C-O-C in hemicellulose is vibrating at the highest intensity of 1102 cm -1 (Ju et al. 2011; Vijayan and Prabhu 2022).Therefore, increasing the time intervals in solid acid hydrolysis has proven to broken down cellulose's intramolecular and extra molecular hydrogen bonds at 120 min. Peak intensity of 3333 was ascribed to the OH group because cellulose breaks down hydrogen bonds (Figure 2 B b) . After the solid acid hydrolysis treatment, the carbon chain is partially destroyed, resulting in cellulose loss. Thus the band at 2889 is assigned at -CH and -CH stretching; this peak is disappeared in integrated treatment. The band at 1646 represents the C=C stretching of the vibration aromatic ring and the C=C aromatic skeleton vibration ring. As increasing time interval, a large portion of lignin was removed in this solid acid hydrolysis treatment. Furthermore, a similar peak at 1634 cm -1 was observed, indicating that lignin is soluble in liquid hydrolysis (Figure 2 B f). The peak intensity at 1156-1027 cm -1 , linked to C-O stretching, was observed in the cellulose and hemicellulose following the integrated and acid hydrolysis treatment. As a result of acid hydrolysis, a stronger band at 872-880 cm-1 was observed, which was characteristic of cellulose II or amorphous cellulose, characterized by C–O–C stretching at the 1,4-glycosidic bonds. Because the SCB contains cellulose, hemicellulose, and lignin, these bands indicate the existence of a lignocellulose matrix (Naik et al. 2010) 8.3) SEM analysis: According to an SEM analysis of treated and untreated SCB, pretreatment resulted in physical alterations to the biomass. Compared with raw SCB, treated SCB exhibited significant morphological differences. The surface of untreated sugarcane bagasse was smooth and continuous, but the surface of heat-treated sugarcane bagasse revealed surface disturbances at different time intervals ranging from 30 to 120 minutes Figure 4 A(i) to A(iv) . In fibres appear rough and even damaged, but fragments with fibrous structures were observed while in 60 min heat treatment Figure 4 A(ii) . The SEM evaluations confirmed the reduction of fiber, and 60 min deformed of particles and cracks were apparent on the surface of SCB. The treated material surface was covered in powdery debris after 90 min, and it appeared as though a thin layer of deposits covered the whole surface. About 120min of heat treatment was observed in SCB with more structural collapse and more powdery debris Figure 4 A (iv) . As compared to the untreated SCB, H 2 SO 4 removed external fibres from the surface and increased the surface area was observed in 30 min Figure 5 B (i) . In acid hydrolysis treatment, cellulose was observed, while 60 to 90 min treatment lignin and hemicellulose separated and it was observed in SEM (Mumtha et al. 2023b). About 120 min of acid hydrolysis treatment could identify the unstructural formation of SCB Figure 5 B (iv) . Combined treatment of heat and acid hydrolysis disrupted the cellular bond, which resulted in the degradation of cellulose, lignin and hemicellulose as unstructural formation in the SEM analysis Figure 6 . A relatively more irregular and porous structure of pretreated sugarcane bagasse was observed in higher magnification. Similar structural changes were reported in the literature for SCB pretreated with hydrothermal carbonization (Naik et al. 2010). 8.4) XRD analysis The X-ray diffraction profile of untreated and pretreated sugarcane bagasse (SCB) showed profiles of native, Heat pretreated and acid hydrolysis pretreated and integrated treatment SCB. The crystalline structure of cellulose observed in SCB is due to hydrogen bonding and van der Waals interactions between adjacent molecules, in contrast to hemicellulose and lignin, cellulose area amorphous in nature (Jose et al. 2014). The intensity and peaks were more defined and sharpened with the increases of acid hydrolysis treatment (Figure 7c) . The separated cellulose after 90mins acid hydrolysis treatment may be the reason for the increased crystallinity index. After being heat treated at various intervals, the crystallinity of SCB increased linearly with an increase in cellulose content, corresponding to 43% and 58% of cellulose with crystallinity index (CrI) values of 60% and 69%, respectively. Pretreated biomass had a higher crystallinity degree than untreated biomass. A high crystallinity index was observed in treated samples compared to integrated and acid hydrolysis samples, an indication of lignin removal by H 2 SO 4 . Amorphous zone crystallinity increased more than the crystalline zone due to acid hydrolysis treatment. In that order, the cellulose crystals display characteristic plane assignments of 110, 200, and 004 (Wada et al. 2004; Cheng and Zhu 2013; Kumar and Sreekrishnan 2013). The samples' crystallinity index was determined using the amorphous subtraction method (Park et al. 2010). As a result of removal of lignin and hemicelluloses, the crystallinity index of SCB increased from 35.6% to 63.5% for 60 minutes (Kumari and Das 2019), and 72.5% for 90 minutes in acid hydrolysis treatment. The data matching the crystalline size also demonstrated the effect of acid hydrolysis treatment on the amorphous zone. 8.5) EDAX analysis The elemental phases and chemical makeup of the treated and untreated SCB were examined using energy-dispersive X-ray (EDX) analysis.. The EDX spectrum of untreated SCB was attributed to the presence of mainly carbon, oxygen and nitrogen. Untreated SCB contained 30% carbon, 13.71 % oxygen, and 0.5% nitrogen as dominant compositions, according to EDX results. About 30 min of the acid hydrolysis pretreatment in SCB, the EDX spectra showed a higher percentage of carbon than oxygen, and sulfur was predominant relative to their binding energies. The relatively high metal content was commonly found in SCB, and the value that it is composed of 5.6 wt% Hydrogen, 45.5 wt% Carbon, 0.3 wt% Nitrogen and 45.2 wt% Oxygen which were consistent with other reports sun (Sun and Cheng 2005). The sugarcane bagasse waste also contains some elements, such as 3.87 wt% Calcium, 3.89 wt% Aluminum, 1.32 wt% Magnesium, 27.0 wt% Silicon and 0.97 wt% Sodium (Jústiz-smith et al. 2008). Carbon, Oxygen, Nitrogen, Sulfur, and Silicon are the most prominent elements in the SCB and these elements have weight percentages of 41.39%, 36.28%, 2.30%, 10.81% and 9.91% at 60 min respectively. This reduction may be associated with lignin removal by heat pretreatment since silica is complex with lignin moieties. About 90 min acid hydrolysis pretreatment, the percentages of carbon increased, while oxygen and nitrogen also increased. The weight percentage of carbon (51.7% and 69.42%), oxygen (36.06% and 37.26%) and nitrogen (3.12% and 3.25%) were higher at 90 and 120 min by acid hydrolysis treatment of SCB. Acid hydrolysis pretreatment sulphur is present in the sample because sulfuric acid can be used in the hydrolysis activity. This interpretation is consistent with earlier reports (Cheng and Liu 2012). However, the N element was not identified in EDAX analyses after pretreatment at HT20+AT60min in integrated treatment, here confirming nitrogen has been removed in the SCB. Also, the amount of the element was changed in the case of after-pretreatment. The SCB sample showed an overall increase in oxygen, carbon, nitrogen and phosphorus, which is explained by the fact that with acid hydrolysis treatment, there might be a chance of oxygen bonding on the adsorbent surface. Nitrogen peaks were found to be significantly higher in EDX analysis with the sulfuric acid treatment experiments. The sulphur peaks could only be observed using sulfuric acid in this experiment lasting 120 minutes. After integrated treatment, showed a lower percentage of carbon and oxygen, in this study was found possible metal reduction for HT25+AT120 min (Figure 7 c) . 8.6) Effect of pretreatments on hydrogen fermentation A batch experiment was conducted with dairy whey and Sugarcane bagasse to evaluate two heat and acid hydrolysis pretreatment and an untreated sample (as the control). The cumulative H 2 production achieved 96.2 mL/H 2 /L for heat treatments at 75mins by using pure culture in S.bongori respectively. An evaluation of selective pretreatment for enriching H 2 production from dairy wastewater using mixed cultures was conducted by Mohan (Mohan 2008). Pretreatment with 2-Bromoethanesulfonic acid (0.22g/l for 24 h) possible higher H 2 yields along with high substrate degradation efficiency. Compared to untreated grass, acid and alkaline pretreatments increased hydrogen production, with acid pretreatment outperforming alkaline pretreatments regarding hydrogen yield from grass (Cui and Shen 2012). Bio-H 2 production from heat treated (FVW) fruit and vegetable waste through dark fermentation the maximum HPR and biomass removal efficiency was obtained 63.0 mL/g VS and 372.6 mL/L/d (Pascualone et al. 2019). Heat treatment is most suitable for hydrogen production as a positive effect on the substrates Figure 8 . The effects of the Acid hydrolysis treatment on biohydrogen production were linked to the changes of the physicochemical characteristics of the liquid hydrolysis of pretreated SCB and Solid hydrolysis of pretreated SCB. Fig. 5 illustrates the variations of cumulative H 2 production with time interval for different pretreatment methods. The average amount of hydrogen produced from from acid hydrolysis pretreated sugarcane bagasse of 189.6±4.3 mL/H 2 /L at 90mins by using a co-culture. Five different pretreatment techniques were evaluated, including autoclaving, acid pretreatment, alkali pretreatment, aeration, and fungal pretreatment, which used pretreated food waste for biohydrogen production (Khan et al. 2018; Bhurat et al. 2023). The results showed that the cumulative H 2 yields at the various interval times of 120mins declined to 121 mL/H 2 /L in acid liquid hydrolysis pretreated sample by using co-culture, respectively, indicating that the too high starting times were all favorable for bio-H 2 production. Conclusion The dark fermentation by bacterial monocultures and consortium using Dairy whey (DW) and Sugarcane bagasse (SCB) feedstocks was performed and the findings revealed that pretreatment of the feed stock improves hydrogen yield and its production. Through pretreatment methods, microorganisms can easily digest and convert hydrogen and methane, simplifying the structural compounds and dissolving them into monomers. Compared to other methods, this one is more straightforward and less energy-intensive, and the end-products produced are non-toxic and non-polluting. Individual pretreatment can be paired with other pretreatment techniques to increase hydrogen production. The experiment successfully assessed the relative effectiveness of the various pretreatment techniques applied in this investigation. In comparison with the control experiment, all pretreatment methods improved hydrogen and biogas production. Maximum cumulative hydrogen production of 153.4 ± 2.0 mL H 2 /L were achieved using the heat-treated DW, which is 20% higher than that of the untreated biomass. The optimum acid hydrolysis pretreatment process of SCB substrate with 2 M H 2 SO 4 in an autoclave condition at 90 min for 121°C, showed the maximum specific hydrogen production of 189.6±4.3 mL H 2 /L. The results clearly revealed that acid hydrolysis pretreatment significantly promoted hydrogen production by SCB compared with other pretreatments. Declarations Acknowledgment We sincerely thank GRI-DTBU, the Head of the Biology Department, for supplying the required materials. We acknowledge that the Grammarly and Ithenticate software were provided by the GRI General Library. Authors’Contribution: Chelladurai mumtha (conceptualization, data curation, methodology, formal analysis, writing – original draft), Pambayan Ulagan Mahalingam ( correcting the whole manuscript) . Each writer has contributed significantly, directly, and intellectually to the work and has given permission for it to be published . Funding: No governmental, private, or nonprofit entities provided funding for this research. Data availability : This published paper contains all of the data created or analysed during this investigation. Ethics Statement : Neither humans nor animals were used in any of the authors' studies. Consent to participate : Not applicable. Consent for publication: Not applicable. 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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-3943615","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":316432685,"identity":"0c08b6e7-3ce7-4c08-b855-fd62ddf43e27","order_by":0,"name":"Chelladurai mumtha","email":"","orcid":"","institution":"Gandhigram Rural University: The Gandhigram Rural Institute Deemed University","correspondingAuthor":false,"prefix":"","firstName":"Chelladurai","middleName":"","lastName":"mumtha","suffix":""},{"id":316432686,"identity":"64ffbe6a-cd01-42d0-bb53-83689363c927","order_by":1,"name":"Pambayan Ulagan 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16:50:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3943615/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3943615/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60598357,"identity":"11872126-8777-42ec-912c-0a042aac04db","added_by":"auto","created_at":"2024-07-18 15:53:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":147575,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe functional group characteristic on treated and untreated Dairy whey Substrates sample by FTIR analysis: a) Heat treatment, b) Ultrasonication treatement, and c) Integrated treatment\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3943615/v1/93e09504fb17d36b3b9c0d0e.png"},{"id":60598360,"identity":"ed37fb0e-1158-42d3-b764-2625561015ff","added_by":"auto","created_at":"2024-07-18 15:53:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":329848,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A): The functional group characteristic on treated and untreated SCB substrates by FTIR analysis: a) Heat treatment of SCB, b) Acid hydrolysis treatment of SCB, c) Integrated treatment of SCB (HT+AT).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B): The functional group characteristic on treated liquid sugarcane bagasse substrates by FTIR analysis: a) Heat treatment of LHTSCB, b) Acid hydrolysis treatment of LATSCB, and c) Integrated treatment of LSCB (HT+AT).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3943615/v1/3f6758b31761dc44aa6bb81a.png"},{"id":60598352,"identity":"01bb1459-00bc-4309-8562-76d300dfd137","added_by":"auto","created_at":"2024-07-18 15:53:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":531708,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSEM images of treated and untreated sugarcane bagasse at different times interval heat treatment: A(i) 30 min, A(ii) 60 min, A(iii) 90 min, A(iv)120 min and A(v) Control.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3943615/v1/d58437c72abbd6e17c60ad20.png"},{"id":60598353,"identity":"a63dd4cd-4736-46c1-941b-0c86fcbb0cd9","added_by":"auto","created_at":"2024-07-18 15:53:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":496779,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSEM images of treated and untreated sugarcane bagasse at different times Interval acid hydrolysis treatment: B(i) 30 min, B(ii) 60 min, B(iii) 90 min, B(iv)120 min and A(v) Control.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3943615/v1/66ceed85717f1ec917b8345b.png"},{"id":60598354,"identity":"437b51da-de71-4d5d-ae29-175a602ad19c","added_by":"auto","created_at":"2024-07-18 15:53:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":511631,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSEM images of treated and untreated sugarcane bagasse at different time interval integrated treatment C(i) 10+15 min, C(ii) 15+30 min, C(iii) 20+60min, C (iv) 25+120min and C(v) Control.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3943615/v1/6a6f4121bab2529da6967e30.png"},{"id":60598355,"identity":"8203b2f1-92ce-4c92-b7f3-01eca629be30","added_by":"auto","created_at":"2024-07-18 15:53:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":222972,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eXRD diffraction patterns of treated and untreated sugarcane bagasse: a) Heat treatment, b) Acid treatment, and c) Integrated treatment.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3943615/v1/2a054df029149cf0f2c1f090.png"},{"id":60598359,"identity":"c80f7e49-a628-4262-b877-d6d28cca9a2a","added_by":"auto","created_at":"2024-07-18 15:53:46","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":176177,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEDX Spectra treated and untreated of sugarcane bagasse treated and untreated: a) Heat treatment, b) Acid hydrolysis treatment, and c) Integrated treatment.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-3943615/v1/a2d68c4ea7cb4e66c5dcdbb0.png"},{"id":60598356,"identity":"e38a0174-f3b3-4f09-a836-60709528c952","added_by":"auto","created_at":"2024-07-18 15:53:44","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":443965,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCumulative hydrogen production of dairy whey, SCB and LSCB by pure bacterial culture and Co-culture\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-3943615/v1/0d2b5bed220f551ba72dd2c5.png"},{"id":70271149,"identity":"7c5ab97b-65c3-4a74-8444-390103ed2452","added_by":"auto","created_at":"2024-12-01 08:54:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3963389,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3943615/v1/9a8fa307-01ba-4f92-b5ab-2bb7d0e4e456.pdf"}],"financialInterests":"","formattedTitle":"Enhancing the biological hydrogen production from different biomass through individual pretreatment method","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEnvironmental issues, climate change, limited resources, economic issues, and current fossil fuel-based energy systems are among the significant concerns\u0026nbsp;(2019). For this reason, fuels are necessary for the future.\u0026nbsp;According to the latest research on hydrogen, it is a clean, reusable, high-energy fuel that doesn\u0026apos;t cause pollution\u0026nbsp;(Bicer and Dincer 2018).\u0026nbsp;Biological approaches to hydrogen productions most convenient processes operated efficiently are less energy intensive and more eco-friendly compared with conventional chemical methods\u0026nbsp;(Chandrasekhar et al. 2020).\u0026nbsp;Fermentative hydrogen production is another biological hydrogen generation method because it uses different carbon sources, including renewable biomass and industrial waste\u0026nbsp;(Kotay and Das 2008).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eFocusing attention on raw materials such as starch, cellulose-containing biomass, and agricultural waste can be used as a potential substrates for biohydrogen production\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(Goula and Bereketidou 2014)\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eIndustrial waste requires a higher energy input and yields less, so it is used for fermentative hydrogen production\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(Kapdan and Kargi 2006)\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eTherefore, Pretreatment is necessary to ensure the process\u0026apos;s viability and sustainability.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eWhen substrates are pretreatment correctly, sugars can be converted into fermentable carbohydrates polymer such as starch, cellulose, and hemicellulose\u0026nbsp;(Perez et al. 2023).\u0026nbsp;Several pretreatment methods are available such as chemical methods (alkali, acid, and ionic liquids), physical methods (heating, size reduction, steaming, hot water, ultrasonication) and biological methods\u0026nbsp;(Akhtar et al. 2016).\u0026nbsp;Meanwhile, hydrolysis methods used to produce fermentable sugars are alternatives depending on the biomass\u0026nbsp;(Trejo et al. 2022). Ultrasound has recently been employed to enhance the functionality of several bioprocesses.\u0026nbsp;While alkali or acid pretreatments have been found to improve sugar recovery, they are associated with the formation of inhibitors\u0026nbsp;(Sun et al. 2016).\u0026nbsp;These inhibitors affect the hydrogen production negatively.\u0026nbsp;The production of hydrogen from pretreated corn stover in alkaline treatment is favourable for producing fermentable sugar\u0026nbsp;(Zhang et al. 2015).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOptimizing the pretreatment process under the most appropriate and economical conditions is essential for improving product yields\u0026nbsp;(Chin et al. 2013). \u0026nbsp;The researchers have extensively investigated temperature and reaction time as optimization parameters\u0026nbsp;(Tan et al. 2010). Organic residues in municipal solid waste, food waste, starch wastewater, kitchen trash, sweet sorghum and abattoir wastewater have all been effectively used to make hydrogen\u0026nbsp;(Silva et al. 2017). Sewage sludge is a great source of organic waste for fermentative hydrogen production in consortiums\u0026nbsp;(Bansal et al. 2012). One of the most widely studied pretreatment techniques, is acid hydrolysis were diluted sulfuric acid effectively hydrolyzes hemicellulose and cellulosic constituents into monomeric sugars\u0026nbsp;(Sun and Cheng 2005). Furfural and acetic acid are produced as by-products.\u0026nbsp;When these substances are present, fermentation is inhibited. Alternative to acid hydrolysis, one of the possible approaches is microbial hydrolysis of wastes by fungal species, which convert starch, hemicellulose, and cellulose into fermentable sugars\u0026nbsp;(Zabed et al. 2016). Fermentation will be improved by selecting a suitable hydrolysis method and controlling inhibitor production\u0026nbsp;(Sivagurunathan et al. 2017). Lignocellulosic biomass with its complex matrix makes it unsuitable for agricultural residue conversion into value-added products.\u0026nbsp;Therefore, pretreatment is required to change the structure of biomass by, depending on the pretreatment methods applied, increasing the surface area, eliminating lignin, decreasing cellulose crystallinity, or depolymerizing the hemicellulose\u0026nbsp;(Akhtar et al. 2016). Starch is almost entirely broken down into carbohydrate molecules by acid hydrolysis at high temperatures (90\u0026ndash;150\u0026deg;C)\u0026nbsp;(Cakır et al. 2010). Pure bacteria strains like \u003cem\u003eRhodobacter\u003c/em\u003e sp. M-19, \u003cem\u003eClostridium butyricum\u003c/em\u003e, and \u003cem\u003eEnterobacter aerogenes\u0026nbsp;\u003c/em\u003ecan produce biohydrogen from starch. Hydrogen was produced from starch using a mixed culture of \u003cem\u003eRhodobacter\u003c/em\u003e sp M-19, \u003cem\u003eE.aerogenes\u003c/em\u003e, and \u003cem\u003eC.butyricum\u003c/em\u003e, a maximum hydrogen yield was achieved at 2 and 6.6 mol H\u003csub\u003e2\u003c/sub\u003e/mol glucose\u0026nbsp;(Yang et al. 2019). Van Ginkel studied the effects of heat-treated inoculum at 100\u0026deg;C for two hours on hydrogen generation\u0026nbsp;(Van Ginkel et al. 2001). Zhang\u0026nbsp;(Zhang et al. 2020)\u0026nbsp;investigated the effects of acid shock and heat shock combined treatment with sludge used for hydrogen production. In a batch reactor, the ratio between kitchen waste and inoculum has been varied to produce biohydrogen\u0026nbsp;(Bansal et al. 2012).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTherefore, In this study focuses on the evaluation of the pretreatment effect of substrates on biohydrogen production. The novelty of this study was to compare the effects of three individual pretreatments (Heat, Ultrasonication and Acid hydrolysis) and two combined pretreatments (Heat with ultrasonication in DW and Heat with acid hydrolysis in SCB). The pretreatment process was optimized by utilizing dairy whey, sugarcane bagasse, and liquid hydrolysis for producing biohydrogen from \u003cem\u003eE.coli, S.bongori\u003c/em\u003e, and \u003cem\u003eS.oneidensis\u003c/em\u003e and its consortium.\u003c/p\u003e"},{"header":"Materials and Methodology","content":"\u003cp\u003e\u003cstrong\u003e2.1) Substrates collection\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe previous research paper mentioned where two biomass were collected: dairy whey and sugarcane bagasse (Mumtha et al. 2023a). Then, this study used this waste biomass in different pretreatment methods. Dairy whey was stored in room temperature at 4.0\u0026deg;C used for further process. The collected sugarcane bagasse was dried in a hot-air oven at 40\u0026deg;C for 24hr and dried milled sample stored in room temperature.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2) Analysis of physio-chemical characteristics of Dairy whey and Sugarcane bagasse\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhysio-chemical characteristics include pH, VSS (Volatile Suspended Solids)COD (Chemical oxygen demand), TSS (Total Suspended Solid), VFA (Volatile fatty acid), TS (Total Solids), VDS (Volatile dissolved solids), Sodium, Phosphates, Nitrogen, Calcium, Moisture content and Ash content in the untreated substrates from both DW and SCB were analyzed in triplicates following standard method as described in APHA,\u0026nbsp;(Federation 2012)\u0026nbsp;manual.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3) Substrate pretreatment\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1) Pretreatments of dairy whey (DW)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Dairy whey substrates (DW) was pre-treated by heat treatment, ultrasonication and combined heat treatment and ultrasonication as per standard procedures\u0026nbsp;(Karadag et al. 2009; Elbeshbishy et al. 2011a). The pretreated samples were analysed for carbon, protein, reducing sugar and COD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2) Heat treatment of DW\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePretreatment of dairy whey of 150 ml of the was done at 90℃ in a hot air oven at different time intervals (15, 35, 55, and 75 min). After treatment of DW, allowed to cool until in the temperature and then used for further studies (Karadag et al. 2009). The pretreated samples were analysed for carbon, protein, reducing sugar and COD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3) Ultrasonication of DW\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUltrasonication of 200ml of DW was conducted using ultrasonicator (make) with the high frequency 440w and 24 kHz at 37C for different time intervals of 15, 35, 55, and 75 mins (Elbeshbishy et al. 2011b). The sonicator samples analysed for futher studies. The pretreated samples were analysed for carbon, protein, reducing sugar and COD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4) Combination heat treatments with ultrasonication of DW\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe DW was pretreated with combined \u0026nbsp;heat treatment and ultrasonication (Karadag et al. 2009; Elbeshbishy et al. 2011b).The initial heat treatments of DW was done at 90\u0026deg;C in a hot air oven \u0026nbsp;at different time intervals (10,25,40 and 55). Followed by the heat treatment DW substrate was subjected for ultrasonication with high frequency of 440w and 24 kHz at 37\u0026deg;C for different time intervals of 15, 35, 55, and 75. The pretreated samples were analysed for carbon, protein, reducing sugar and COD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4) Sugarcane bagasse (SCB)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sugarcane bagasse (SCB) substrates was pretreated by heat treatment, acid hydrolysis and combined heat treatment and acid hydrolysis as per standard procedures\u0026nbsp;(Ivanova 2009; Reddy et al. 2017).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.1) Heat treatment of SCB\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePretreatment of SCB was done with heat treatment by standard procedure (Ivanova 2009). 100ml of distilled water was added to 50 g of blended sample and subjected for heat treatment at 121℃ using autoclave for at different intervals (30, 60, 90,120 min). After cooling the substrates, the solid residue was separated on Whatman filter paper, and liquid hydrolyzed samples were used for biohydrogen production in batches in dark fermentation. The pretreated samples were analysed for carbon, protein, reducing sugar and COD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2) Acid hydrolysis of SCB\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSugarcane bagasse SCB of 10g sample was acid hydrolyzed with 2M H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e in an autoclave at different time intervals of 30, 60, 90, and 120mins at 121\u0026deg;C (Reddy et al. 2017; Mumtha et al. 2023a). The biomass was filtered under vacuum using Whatman filter paper, and a sample was collected on filter paper. The solid residues were washed thrice with deionized water to remove inhibitory compounds formed during acid pretreatment. Solid hydrolyzed and liquid hydrolyzed sample were adjusted to pH 7.0 using 1N NaOH. and subsequently used as raw material for H\u003csub\u003e2\u003c/sub\u003e production. Carbon, protein, reducing sugar and COD were analyzed in the hydrolysed SCB sample according to the standard method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.3) Combined heat treatment and acid hydrolysis of SCB\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSugarcane bagasse of 100g was transferred to the conical flask and poured 150ml of deionized water then the sample was placed in an autoclave and heat-treated at 121\u0026deg;C for different intervals (10, 15, 20,and 25 mins). After filtering, the substrate completely dried a solid residue, and each liquid hydrolysis was separately collected and stored at room temperature. Additionally, 100g of SCB was mixed with 2M H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and autoclaved at 121\u0026deg;C for different intervals (15, 30, 60 and 120 mins). The samples were allowed to cool to room temperature after being treated, and the solid residues were collected by filtering them using Whatman filter paper. Before the hydrogen fermentation process, the sample was neutralised to pH 7.0 by adding 1 N HCl or 1 N NaOH solution (Ramprakash and Muthukumar 2015). Carbon, protein, reducing sugar and COD were analyzed \u0026nbsp;in the hydrolysed SCB sample according to the standard method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;5)\u003c/strong\u003e \u003cstrong\u003eStructural characteristics of pretreated and untreated substrates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.1) FTIR analysiss\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFourier Transform - Infrared Spectroscopy was used to analyze the different functional charactesitic present in organic molecules. The structural changes from sugarcane bagasse and dairy whey pre-treated samples were identify by FT-IR spectroscopy (Jascob FT-IR \u0026nbsp;Thermoscientific, USA) spectrophotometer. Potassium bromide (KBr) in a 1:5 ratio is mixed with liquid and solid samples and pressed into discs. The samples were then scanned between 4000 and 450 cm1 at a resolution of 4 cm\u003csup\u003e-1\u003c/sup\u003e(Moretti et al. 2014a).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;5.2) SEM and Edax analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn a scanning electron microscope (SEM), the morphology of treated and untreated SCB were analyzed to determine their external structure changes. The pretreated sample was dried for room temperature at 64hrs and the dried sample was placed in a conductive glue and then coated with a thin layer of gold to increase conductivity and improve image quality. The element concentration and chemical identity of both pretreated and untreated SCB were analysed by EDX. X-ray absorption spectroscopy, which generates energy differences in the form of peaks, is performed by energy-dispersive X-ray spectrometers (Bruker EDX) (Badiei et al. 2012).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.3) XRD analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eX-Ray diffraction (XRD) is used to analyze a sample\u0026apos;s chemical properties, crystallographic properties, and physical properties. The crystallinity of the native sugarcane bagasse after the pretreatment of the solid residue crystalline index (CrI) was quantified according to the empirical formula followed by Sun (Sun et al. 2015). The test was conducted using an XRD \u0026nbsp; using a ( Jambes bolt/3) operating \u0026nbsp; at at 40 kV and 30 mA and scanning at 25C per minutes \u0026nbsp;The XRD patterns were obtained over a 2\u0026theta; = 5\u0026ndash;60\u003csup\u003eo\u003c/sup\u003e\u0026nbsp; angular range (Bouramdane et al. 2022) .\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"293\" height=\"73\"\u003e\u003c/p\u003e\n\u003cp\u003eFor the amporphus region, the crystalline index was calculated by estimating \u0026nbsp;the intensity of the fitted peak after removing the background and determining the peaks from crystalline and non-crystalline region\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6) \u0026nbsp;Batch experiment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBatch experiment were performed in 150 mL of serum bottles with a standard volume of 50mL. Each fermentation bottle added the 5 mL of nutrient medium, which included (3g/L NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e3\u003c/sub\u003e, 0.125g/L KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 0.015g/L MnSO\u003csub\u003e4\u003c/sub\u003e. 6H\u003csub\u003e2\u003c/sub\u003eO, and 0.100 g/L MgCl\u003csub\u003e2\u003c/sub\u003e.6H\u003csub\u003e2\u003c/sub\u003eO, Trace element\u003csub\u003e\u0026nbsp;\u0026nbsp;\u003c/sub\u003e0.001 g/L CoCl\u003csub\u003e2\u003c/sub\u003e.5H\u003csub\u003e2\u003c/sub\u003eO, 5.73 g/L NaHCO\u003csub\u003e3,\u0026nbsp;\u003c/sub\u003e 0.005 g/L CuSO\u003csub\u003e4\u003c/sub\u003e.5H\u003csub\u003e2\u003c/sub\u003eO, and 0.025g/L FeSO\u003csub\u003e4\u003c/sub\u003e.\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003eO,). The raw and pretreated sample was used as substrate. Batch test, the inoculum was added to a monoculture and its bacterial consortium in microbial fermentation for biohydrogen production. After the N\u003csub\u003e2\u003c/sub\u003e and CO\u003csub\u003e2\u003c/sub\u003e gas had been set for 5 minutes, the fermentation serum bottles were sealed with a rubber corck and blocked with an aluminium led using squeezing forceps. The fermentation bottles were maintained at 150 rpm in a (Remi Instrument) orbital shaking incubator for 62hr. GC-TCD was used to determine the production of H\u003csub\u003e2\u0026nbsp;\u003c/sub\u003egas\u0026nbsp;(Murugan et al. 2021).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7) Analytical methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe physiochemical characteristics include pH, VSS (Volatile suspended solids), TSS (Total suspended solid ), COD (Chemical oxygen demand), TS (Total solids), Sodium, \u0026nbsp;Phosphates, Calcium, and Nitrogen were determined by the standard method (Federation 2012). GC-FID (Shimadzu GC 2014) was used to evaluate volatile fatty acids (VFAs), and \u0026nbsp;centrifuged fermented sample at 5,000 rpm for 5\u0026deg;C. The supernatant was collected and passed through a membrane filter with a pore size of 0.2 mm (2% H\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4\u0026nbsp;\u003c/sub\u003e80/100 mesh) and a capillary column coated with 10% PEG-20 M.\u0026nbsp;The temperatures of the programmed column, injection port, and detector were set to 220\u0026deg;C, 240\u0026deg;C, and 130-175\u0026deg;C, respectively. The gases H\u003csub\u003e2\u003c/sub\u003e and CO\u003csub\u003e2\u003c/sub\u003e were analysed by gas chromatography-thermal conductivity detector (Shimadzu GC 2014). Nitrogen gas served as the carrier gas and The packaging material utilised was Propak Q tube, which has an 80/100 mesh. The temperature range of the oven, injection port, and detector was 150\u0026deg;C to 100\u0026deg;C to 80\u0026deg;C. The bio-H\u003csub\u003e2\u003c/sub\u003e was manually injected into a volume of around 1 ml. The modified Gompertz equation was used to determine maximum H\u003csub\u003e2\u003c/sub\u003e production rates from cumulative H\u003csub\u003e2\u003c/sub\u003e production. (Mumtha et al. 2022).\u003c/p\u003e"},{"header":"Result and Discussion","content":"\u003cp\u003e\u003cstrong\u003e8.1) Physicochemical characteristics of substrates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA physiochemical parameter was determined for both substrates in the previous study. A moisture content of 6.7 x 0.5 was recorded in an earlier study for sugarcane bagasse. Sugarcane bagasse contains a high amount of soluble carbohydrates and lignocellulosic components. Solids presence in dairy whey expressed as volatile suspended solids (VSS), total suspended solids (TSS), and total solids (TS) were recorded at \u0026nbsp;16.69 g/L, 18.4 g/L, and 38.74 g/L respectively. The sugarcane bagasse was determined for a variety of characteristics, such as ash content, total solids (TS), Chemical Oxygen Demand (COD), volatile solids (VS), etc, according to Standard methods.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8.2) FT-IR analysis\u003c/strong\u003e \u003cstrong\u003eof treated and untreated biomass\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFTIR spectroscopy to determine functional group characteristics of two different biomass, dairy whey and sugarcane bagasse. The intensity of strong and broad peaks was attained at 3326 cm\u003csup\u003e-1\u003c/sup\u003e, indicating that carbohydrates, proteins, and lipids were present. Ekka\u0026nbsp;(Ekka and Mierin 2022)\u0026nbsp;observed a broad and strong peak intensity in this wavelength of 3450-3285 cm\u003csup\u003e-1\u003c/sup\u003e corresponding with lipids, protein and carbohydrates. There is a peak at 3326 cm-\u003csup\u003e1\u003c/sup\u003e linked with O\u0026ndash;H stretching caused by either carbohydrates or hydroxyl groups from dairy whey \u003cstrong\u003e(Figure 1)\u003c/strong\u003e. Following the treatment of dairy whey with heat and ultrasonication for 55 minutes, the peaks of carbohydrates and proteins achieved a higher intensity than those in untreated dairy whey. A similar reduction in peak intensity was observed after heat treatment for 15 min and ultrasonication treatment for 15, 35, 55 min and it was assumed to be carbohydrates and protein degradation. During the combined treatment of Heat+Ultrasonication treatment (HT+UT) for 10+15 min, an increase in peak intensity was observed for both carbohydrates and proteins. 2349 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e is corresponding O=C=O stretching carbon dioxide group \u003cstrong\u003e(Figure 1)\u003c/strong\u003e. CO\u003csub\u003e2\u003c/sub\u003e bands may cause the O=C=O stretching group at 669 and 2600 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e (Tan and Lebron 2012). The strong peak is 1633 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e in this C=O bond on protein-relevant peaks observed after pretreatment. Tang (Tang et al. 2017) dairy effluent contains proteins, and the peak at this wavelength shows if it decreased or increased before and after pretreatment. After heat treatment, The C=C bending alkene group is represented by the band at 676 cm\u003csup\u003e-1\u003c/sup\u003e. \u003cstrong\u003e(Figure 1).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFTIR analysis was used to determine the functional group of SCB biomass before and after acid hydrolysis. The FTIR analysis of raw sugar cane bagasse (SCB), liquid fraction and solid fraction after acid hydrolysis of SCB revealed significant functional group changes \u003cstrong\u003e(Figure 2 A)\u003c/strong\u003e.\u0026nbsp;Cellulose was determined to be the band assigned at 4,000\u0026ndash;2,995 cm\u003csup\u003e-1\u003c/sup\u003e (Mor\u0026aacute;n et al. 2008; Vukoja et al. 2021). In both treated and untreated samples, crystalline cellulose is indicated by the OH group, which is shown by the peak at 3785 cm\u003csup\u003e-1\u003c/sup\u003e.\u0026nbsp;After heat treatment, the peak at 3347 cm\u003csup\u003e-1\u003c/sup\u003e indicates that the hydroxyl (OH) group has been solubilized in the liquid fraction of acid hydrolysis. The peak intensity of 2360 and 2342 cm-1 for the hydrolysis of SCB in both the liquid and solid fractions was attributed to the -CH stretching of the methyl and methylene groups. The peak intensity of 1589\u0026ndash;1635 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e in both liquid and solid hydrolysis was attributed to C\u0026ndash;H bond deformations, and lignin was linked to aromatic ring vibration. The vibration of aromatic rings (1600, 1635, 1510 cm-1) and the stretching of carbonyl groups (1728 cm-1) are responsible for the strong peak seen at 1516\u0026ndash;1598 cm\u003csup\u003e-1\u003c/sup\u003e; lignin compounds are present in all of these (de \u003cem\u003eet al\u003c/em\u003e., 2014).\u0026nbsp;The bands corresponding to the symmetric CH\u003csub\u003e2\u003c/sub\u003e bending are located at 1439 cm\u003csup\u003e-1\u003c/sup\u003e and 1352 cm\u003csup\u003e-1\u003c/sup\u003e (Cao and Tan 2004; Bouramdane et al. 2022).\u003c/p\u003e\n\u003cp\u003eThe peak intensity at 1297 cm\u003csup\u003e-1\u003c/sup\u003e, which is attributed to the C-O ether group, is similar in treated and untreated SCB. According to liquid hydrolysis, the ring C-O-C in hemicellulose is vibrating at the highest intensity of 1102 cm\u003csup\u003e-1\u003c/sup\u003e (Ju et al. 2011; Vijayan and Prabhu 2022).Therefore, increasing the time intervals in solid acid hydrolysis has proven to broken down cellulose\u0026apos;s intramolecular and extra molecular hydrogen bonds at 120 min. Peak intensity of 3333 was ascribed to the OH group because cellulose breaks down hydrogen bonds \u003cstrong\u003e(Figure 2 B b)\u003c/strong\u003e. After the solid acid hydrolysis treatment, the carbon chain is partially destroyed, resulting in cellulose loss.\u0026nbsp;Thus the band at 2889 is assigned at -CH and -CH stretching; this peak is disappeared in integrated treatment. The band at 1646 represents the C=C stretching of the vibration aromatic ring and the C=C aromatic skeleton vibration ring. As increasing time interval, a large portion of lignin was removed in this solid acid hydrolysis treatment. Furthermore, a similar peak at 1634 cm\u003csup\u003e-1\u003c/sup\u003e was observed, indicating that lignin is soluble in liquid hydrolysis \u003cstrong\u003e(Figure 2 B f).\u003c/strong\u003e The peak intensity at 1156-1027 cm\u003csup\u003e-1\u003c/sup\u003e, linked to C-O stretching, was observed in the cellulose and hemicellulose following the integrated and acid hydrolysis treatment.\u0026nbsp;As a result of acid hydrolysis, a stronger band at 872-880 cm-1 was observed, which was characteristic of cellulose II or amorphous cellulose, characterized by C\u0026ndash;O\u0026ndash;C stretching at the 1,4-glycosidic bonds.\u0026nbsp;Because the SCB contains cellulose, hemicellulose, and lignin, these bands indicate the existence of a lignocellulose matrix\u0026nbsp;(Naik et al. 2010)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8.3) SEM analysis:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to an SEM analysis of treated and untreated SCB, pretreatment resulted in physical alterations to the biomass. Compared with raw SCB, treated SCB exhibited significant morphological differences. The surface of untreated sugarcane bagasse was smooth and continuous, but the surface of heat-treated sugarcane bagasse revealed surface disturbances at different time intervals ranging from 30 to 120 minutes \u003cstrong\u003eFigure 4 A(i) to A(iv)\u003c/strong\u003e. In fibres appear rough and even damaged, but fragments with fibrous structures were observed while in 60 min heat treatment \u003cstrong\u003eFigure 4 A(ii)\u003c/strong\u003e. The SEM evaluations confirmed the reduction of fiber, and 60 min deformed of particles and cracks were apparent on the surface of SCB. The treated material surface was covered in powdery debris after 90 min, and it appeared as though a thin layer of deposits covered the whole surface. About 120min of heat treatment was observed in SCB with more structural collapse and more powdery debris \u003cstrong\u003eFigure 4 A (iv)\u003c/strong\u003e. \u0026nbsp;As compared to the untreated SCB, H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e removed external fibres from the surface and increased the surface area was observed in 30 min \u003cstrong\u003eFigure 5 B (i)\u003c/strong\u003e. In acid hydrolysis treatment, cellulose was observed, while 60 to 90 min treatment lignin and hemicellulose separated and it was observed in SEM (Mumtha et al. 2023b). About 120 min of acid hydrolysis treatment could identify the unstructural formation of SCB \u003cstrong\u003eFigure 5 B (iv)\u003c/strong\u003e. Combined treatment of heat and acid hydrolysis disrupted the cellular bond, which resulted in the degradation of cellulose, lignin and hemicellulose as unstructural formation in the SEM analysis \u003cstrong\u003eFigure 6\u003c/strong\u003e. A relatively more irregular and porous structure of pretreated sugarcane bagasse was observed in higher magnification. Similar structural changes were reported in the literature for SCB pretreated with hydrothermal carbonization (Naik et al. 2010).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8.4) XRD analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe X-ray diffraction profile of untreated and pretreated sugarcane bagasse (SCB) showed profiles of native, Heat pretreated and acid hydrolysis pretreated and integrated treatment SCB. The crystalline structure of cellulose observed in SCB is due to hydrogen bonding and van der Waals interactions between adjacent molecules, in contrast to hemicellulose and lignin, cellulose area amorphous in nature (Jose et al. 2014). The intensity and peaks were more defined and sharpened with the increases of acid hydrolysis treatment \u003cstrong\u003e(Figure 7c)\u003c/strong\u003e. The separated cellulose after 90mins acid hydrolysis treatment may be the reason for the increased crystallinity index. After being heat treated at various intervals, the crystallinity of SCB increased linearly with an increase in cellulose content, corresponding to 43% and 58% of cellulose with crystallinity index (CrI) values of 60% and 69%, respectively. Pretreated biomass had a higher crystallinity degree than untreated biomass. A high crystallinity index was observed in treated samples compared to integrated and acid hydrolysis samples, an indication of lignin removal by H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e. Amorphous zone crystallinity increased more than the crystalline zone due to acid hydrolysis treatment. In that order, the cellulose crystals display characteristic plane assignments of 110, 200, and 004 (Wada et al. 2004; Cheng and Zhu 2013; Kumar and Sreekrishnan 2013). The samples\u0026apos; crystallinity index was determined using the amorphous subtraction method (Park et al. 2010). As a result of removal of lignin and hemicelluloses, the crystallinity index of SCB increased from 35.6% to 63.5% for 60 minutes (Kumari and Das 2019), and 72.5% for 90 minutes in acid hydrolysis treatment. The data matching the crystalline size also demonstrated the effect of acid hydrolysis treatment on the amorphous zone.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8.5) EDAX analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe elemental phases and chemical makeup of the treated and untreated SCB were examined using energy-dispersive X-ray (EDX) analysis.. The EDX spectrum of untreated SCB was attributed to the presence of mainly carbon, oxygen and nitrogen. Untreated SCB contained 30% carbon, 13.71 % oxygen, and 0.5% nitrogen as dominant compositions, according to EDX results. About 30 min of the acid hydrolysis pretreatment in SCB, the EDX spectra showed a higher percentage of carbon than oxygen, and sulfur was predominant relative to their binding energies. The relatively high metal content was commonly found in SCB, and the value that it is composed of 5.6 wt% Hydrogen, 45.5 wt% Carbon, 0.3 wt% Nitrogen and 45.2 wt% Oxygen which were consistent with other reports sun (Sun and Cheng 2005). The sugarcane bagasse waste also contains some elements, such as 3.87 wt% Calcium, 3.89 wt% Aluminum, 1.32 wt% Magnesium, 27.0 wt% Silicon and 0.97 wt% Sodium (J\u0026uacute;stiz-smith et al. 2008). Carbon, Oxygen, Nitrogen, Sulfur, and Silicon are the most prominent elements in the SCB and these elements have weight percentages of 41.39%, 36.28%, 2.30%, 10.81% and 9.91% at 60 min respectively. This reduction may be associated with lignin removal by heat pretreatment since silica is complex with lignin moieties. About 90 min acid hydrolysis pretreatment, the percentages of carbon increased, while oxygen and nitrogen also increased. The weight percentage of carbon (51.7% and 69.42%), oxygen (36.06% and 37.26%) and nitrogen (3.12% and 3.25%) were higher at 90 and 120 min by acid hydrolysis treatment of SCB. Acid hydrolysis pretreatment sulphur is present in the sample because sulfuric acid can be used in the hydrolysis activity. This interpretation is consistent with earlier reports (Cheng and Liu 2012). However, the N element was not identified in EDAX analyses after pretreatment at HT20+AT60min in integrated treatment, here confirming nitrogen has been removed in the SCB. Also, the amount of the element was changed in the case of after-pretreatment. The SCB sample showed an overall increase in oxygen, carbon, nitrogen and phosphorus, which is explained by the fact that with acid hydrolysis treatment, there might be a chance of oxygen bonding on the adsorbent surface. Nitrogen peaks were found to be significantly higher in EDX analysis with the sulfuric acid treatment experiments. The sulphur peaks could only be observed using sulfuric acid in this experiment lasting 120 minutes. After integrated treatment, showed a lower percentage of carbon and oxygen, in this study was found possible metal reduction for HT25+AT120 min \u003cstrong\u003e(Figure 7 c)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8.6) Effect of pretreatments on hydrogen fermentation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA batch experiment was conducted with dairy whey and Sugarcane bagasse to evaluate two heat and acid hydrolysis pretreatment and an untreated sample (as the control). The cumulative H\u003csub\u003e2\u003c/sub\u003e production achieved 96.2 mL/H\u003csub\u003e2\u003c/sub\u003e/L for heat treatments at 75mins by using pure culture in \u003cem\u003eS.bongori\u003c/em\u003e respectively. An evaluation of selective pretreatment for enriching H\u003csub\u003e2\u003c/sub\u003e production from dairy wastewater using mixed cultures was conducted by Mohan (Mohan 2008). Pretreatment with 2-Bromoethanesulfonic acid (0.22g/l for 24 h) possible higher H\u003csub\u003e2\u003c/sub\u003e yields along with high substrate degradation efficiency. Compared to untreated grass, acid and alkaline pretreatments increased hydrogen production, with acid pretreatment outperforming alkaline pretreatments regarding hydrogen yield from grass (Cui and Shen 2012). Bio-H\u003csub\u003e2\u003c/sub\u003e production from heat treated (FVW) fruit and vegetable waste through dark fermentation the maximum HPR and biomass removal efficiency was obtained 63.0 mL/g VS and 372.6 mL/L/d (Pascualone et al. 2019). Heat treatment is most suitable for hydrogen production as a positive effect on the substrates \u003cstrong\u003eFigure 8\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eThe effects of the Acid hydrolysis treatment on biohydrogen production were linked to the changes of the physicochemical characteristics of the liquid hydrolysis of pretreated \u0026nbsp;SCB and Solid hydrolysis of pretreated SCB. Fig. 5 illustrates the variations of cumulative H\u003csub\u003e2\u003c/sub\u003e production with time interval for different pretreatment methods. The average amount of hydrogen produced from from acid hydrolysis pretreated sugarcane bagasse of 189.6\u0026plusmn;4.3 mL/H\u003csub\u003e2\u003c/sub\u003e/L at 90mins by using a co-culture. Five different pretreatment techniques were evaluated, including autoclaving, acid pretreatment, alkali pretreatment, aeration, and fungal pretreatment, which used pretreated food waste for biohydrogen production\u0026nbsp;(Khan et al. 2018; Bhurat et al. 2023). The results showed that the cumulative H\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eyields at the various interval times of 120mins declined to 121 mL/H\u003csub\u003e2\u003c/sub\u003e/L in acid liquid hydrolysis pretreated sample by using co-culture, respectively, indicating that the too high starting times were all favorable for bio-H\u003csub\u003e2\u003c/sub\u003e production.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe dark fermentation by bacterial monocultures and consortium using Dairy whey (DW) and Sugarcane bagasse (SCB) feedstocks was performed and the findings revealed that pretreatment of the feed stock improves hydrogen yield and its production. Through pretreatment methods, microorganisms can easily digest and convert hydrogen and methane, simplifying the structural compounds and dissolving them into monomers. Compared to other methods, this one is more straightforward and less energy-intensive, and the end-products produced are non-toxic and non-polluting.\u0026nbsp;Individual pretreatment can be paired with other pretreatment techniques to increase hydrogen production. The experiment successfully assessed the relative effectiveness of the various pretreatment techniques applied in this investigation.\u0026nbsp;In comparison with the control experiment, all pretreatment methods improved hydrogen and biogas production.\u0026nbsp;Maximum cumulative hydrogen production of 153.4 \u0026plusmn; 2.0\u0026nbsp;mL H\u003csub\u003e2\u003c/sub\u003e/L were achieved using the heat-treated DW, which is 20% higher than that of the untreated biomass.\u0026nbsp;The optimum acid hydrolysis pretreatment process of SCB substrate with 2 M H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e in an autoclave condition at 90 min for 121\u0026deg;C, showed the maximum specific hydrogen production of\u0026nbsp;189.6\u0026plusmn;4.3\u0026nbsp;mL H\u003csub\u003e2\u003c/sub\u003e/L. The results clearly revealed that acid hydrolysis pretreatment significantly promoted hydrogen production by SCB compared with other pretreatments.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe sincerely thank GRI-DTBU, the Head of the Biology Department, for supplying the required materials. We acknowledge that the Grammarly and Ithenticate software were provided by the GRI General Library.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo;Contribution:\u0026nbsp;\u003c/strong\u003eChelladurai mumtha (conceptualization, data curation, methodology, formal analysis, writing \u0026ndash; original draft), Pambayan Ulagan Mahalingam ( correcting the whole manuscript)\u003cstrong\u003e.\u003c/strong\u003e Each writer has contributed significantly, directly, and intellectually to the work and has given permission for it to be published\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e No governmental, private, or nonprofit entities provided funding for this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e: This published paper contains all of the data created or analysed during this investigation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e: Neither humans nor animals were used in any of the authors\u0026apos; studies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e: Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003e The authors declare no competing interests\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAkhtar N, Gupta K, Goyal D, Goyal A (2016) Recent Advances in Pretreatment Technologies for Efficient Hydrolysis of Lignocellulosic Biomass. 35:489\u0026ndash;511. https://doi.org/10.1002/ep\u003c/li\u003e\n \u003cli\u003eBadiei M, Jahim JM, Anuar N, et al (2012) Microbial community analysis of mixed anaerobic microflora in suspended sludge of ASBR producing hydrogen from palm oil mill effluent. 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INC\u003c/li\u003e\n \u003cli\u003e(2019) Natural gas : A transition fuel for sustainable energy system transformation ? 1075\u0026ndash;1094. https://doi.org/10.1002/ese3.380\u003c/li\u003e\n\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":"Heat treatment, Acid hydrolysis treatment, FTIR, XRD SEM and EDAX","lastPublishedDoi":"10.21203/rs.3.rs-3943615/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3943615/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHeat, ultrasonication, acid hydrolysis, and integrated treatment were the various pretreatment techniques employed by different substrates. For the two distinct biomass, integrated pretreatment techniques were used, including heat with acid hydrolysis in sugarcane bagasse and heat with ultrasonication in dairy whey (DW). A batch experiment with microorganisms was conducted to produce biohydrogen from dairy whey and sugarcane bagasse using three different pretreatment techniques. The heat-treated DW had a maximum cumulative hydrogen production of 153.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0 mL H\u003csub\u003e2\u003c/sub\u003e/L, which is 20% more than that of the untreated biomass. After pretreatment, FTIR, XRD, SEM, and EDAX were used to analyse the physicochemical changes in DW and SCB. Untreated and treated waste biomass were analyzed using FTIR spectroscopy to quantify their functional groups. According to EDX results, untreated SCB contains 30% Carbon, 13.71% Oxygen, and 0.50% Nitrogen. SCB was treated with acid using a hydrolysis time of 90mins at 121\u0026deg;C and H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e concentration 2 M the highest cumulative H\u003csub\u003e2\u003c/sub\u003e production of 189.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3 mL H\u003csub\u003e2\u003c/sub\u003e/L was obtained at 37℃ in co-culture. In the future, it may be possible to produce biomass biohydrogen that is both efficient and sustainable based on the findings of this study.\u003c/p\u003e","manuscriptTitle":"Enhancing the biological hydrogen production from different biomass through individual pretreatment method","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-18 15:53:36","doi":"10.21203/rs.3.rs-3943615/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":"09dad4b9-164e-4110-9677-75cee08152c9","owner":[],"postedDate":"July 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-01T08:46:21+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-18 15:53:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3943615","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3943615","identity":"rs-3943615","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}