Whole cell of pure Clostridium butyricum CBT-1 from anaerobic bioreactor effectively hydrolyzes agro-food waste into biohydrogen

Recycling organic waste and converting them into renewable energy are a promising route for environment protection and effective biochemical reactions suitable for industrial hydrogen synthesis. This study targeted to isolate a pure anaerobic culture with potential to hydrolyze different biomass and production of biohydrogen. For this, a sample of full-scale anaerobic digester, fed with a multicomponent solid, was inoculated on Reinforced Clostridial Medium (RCM) in strict anaerobic conditions. An anaerobic Clostridium butyricum CBT-1 strain was isolated, identified from morphological and 16S rRNA sequence. The CBT-1 culture expressed amylase, cellulase and peroxidases activities. The strain exhibited visual decolorization of both Azure B and crystal violet dyes. In batch fermentation experiment, the CBT-1 produced highest of 3.06, 2.67 and 2.46 mol/mol H2 yield from glucose, starch and cellulose respectively, whereas, the CBT-1 showed low 0.43 mol H2/mol of substrate from untreated rice straw due to lignin in compact structure and comparatively high H2 yield of 1.91 and 2.01 mol H2/mol of substrate rice straw hydrolysate and kitchen food waste (KFWS) respectively. The cumulative volumetric yield of H2 was 358.15, 300.8 and 294.5NmL/gSub from glucose, starch and cellulose respectively. Similarly, the cumulative H2 volume was 76.7, 184.4, 237.2 NmL/gVS from untreated rice straw, rice straw hydrolysate and kitchen food waste. This study emphasizes the prospects to find similar robust anaerobic culture for hydrolyzing complex biomass. Such strains could be used as standard co-inoculum for biohydrogen obtaining and as the biocatalyst for commercial scale applications.


Introduction
Energy is the basic human need for daily activities. Currently, the world relies on energy of petroleum-based fuels generated from fossil sources. The problems with petroleum fuels are releasing of carbon dioxide and increase of global warming. Further, the fossil sources are depleting quite rapidly, and the cost of petroleum-based fuels is increasing globally (Roy and Das 2016). To control the price hike and environmental pollution, alternative energy sources have been put on the target (Shah et al. 2017). Renewable energy from cheap carbon sources, robust product specific strains and new bioprocess technological are the possible options (Shah et al. 2018a;Shehbaz et al. 2018). The agricultural waste biomass (grasses, straw) and kitchen waste could be utilized (Wong et al. 2016). The kitchen waste contains mostly fruits and vegetables, which are an easily digestible substrate for the microorganisms (Rodríguez-Valderrama et al. 2020). However, grasses and waste straw need some pretreatment, either chemical, physical or biological for an easy fermentation process (Shah et al. 2018c). The reason of pretreatment is to remove the lignin, from waste straw, which acts as a barrier in fermentation process. Lignin removal speeds up fermentation and increases the yield from the substrate per gram (Ali et al. 2020). Ideally, this treatment should be nominal and mild to lower the cost and be effective for the depolymerization of complex biomass (Shah et al. 2018b). Thus, dilute chemical treatment (H 2 SO 4 acid, NaOH alkali) process can be chosen as an inexpensive pretreatment approach Shah and Tabassum 2018).
Nature is full of wonderful microorganisms (Shah and Raheem 2019;Shah et al. 2018c); it only needs to be explored for the screening of target specific bioproduct (Saleem et al. 2020;Shah et al. 2016). Microorganisms are sources for production of various value added metabolites. In fact, a microbial cell is functioning as a factory, which synthesized different kinds of useful secondary materials (Balraj et al. 2021). Bacterial species are predominant among other microorganisms with enormous potential for biomolecule production. Previously, we reported pure Bacillus sp. strains capable of conversion of municipal organic food waste into biohydrogen.
This study further emphasized to screen out more anaerobic fermentative bacteria, which can convert multiple waste biomasses into fuels. Anaerobic digestion and anaerobic granular sludge are reported with a wide range of archaea, bacteria and fungi producing hydrogen and methane gas. In the list of these anaerobic microorganisms, Bacillus sp. strains and Clostridium sp. strains are the significant culture inside anaerobic digestion and fermentation of waste biomass (Rodríguez-Valderrama et al. 2020). Other cultures like enterobacters, Aeromonas, Pseudomonas, Streptomyces and Rhodobacter sphaeroides inoculum are reported for biohydrogen production (Budiman et al. 2017). However, these cultures are either low for H 2 potential or non H 2 producing. Additionally, these microbes are either unable to grow even on simple carbon beside complex biomasses. That does not determine them ideal for H 2 production and high scale processing. Any strain capable of converting multiple substrates like starch, cellulose, straw and food waste simultaneously could be the best culture for commercial applications (Nam et al. 2016).
Clostridium butyricum is gram positive, spore forming and one of the highest anaerobic bacterium existing in anaerobic digestion processes. Clostridium butyricum is reported with capabilities to ferment various polysaccharides (Cai et al. 2013;Cai et al. 2011). It can produce a variety of products like acetic acids, butyric acids, alcohols like glycerol, butanol and gases (H 2 ). However, many Clostridium strains have been isolated, but only few of them are found effective in fermentation of complex polysaccharides carbohydrates (Cai et al. 2013;Cai et al. 2011). The enzymes responsible of hydrogen synthesis are metalloenzymes of FeFe-hydrogenases, which are generally present in most of the anaerobic bacteria. In the anaerobic cellular reactions and metabolism, the hydrogenases separate surplus reducing molecules with the help of protons from water as terminal electron acceptors, giving rise to the production of hydrogen as a byproduct. The production of hydrogen occurs together with reoxidation of NADH /NAD(P)H as flavin-based electron division. The enzyme FeFe-hydrogenases also play an important role in physiological reactions like H 2 sensing, H 2 uptake and CO 2 fixation (Morra 2022). This study focused on biohydrogen production from a pure anaerobic strain capable of hydrolyzing rice straw hydrolysate and kitchen food waste. To get this objective, a granular sludge was collected from an anaerobic reactor actively operational on straw and food waste biomasses. The sample of this anaerobic reactor was screened to isolate the most active and vital Clostridium strains that have the capacity to hydrolyze various biomass including rice straw hydrolysate and kitchen waste. Successfully, Clostridium butyricum CBT-1 strain is purified and demonstrated capabilities of waste biomass hydrolysis. This is our first effort toward the purification of Clostridium butyricum CBT-1 for biohydrogen production. It can be stretched to explore more industrial biochemicals of Clostridium butyricum strains. The study emphasizes for improvement in batch fermentation process and development of standardized co-inoculum of pure anaerobic Clostridium strains capable of biohydrogen gas production. The experiments were designed as shown in graphical design ( Fig. 1) below.

Preparations of materials
Most of the chemicals tested in this study were from Sigma-Aldrich (Germany). An overnight dried rice straw was grounded by grinding machine to a fine powder of <0.5-cm size. The rice straw was measured as 40.0% C, 0.95% N and 4.1% H. The VS and TS composition was 85.3% and 81.5% of the rice straw, respectively, representing a high organic matter. The kitchen organic food waste was collected from the University Cafeteria. The unwanted materials like plastic bags, papers, glass and mud were removed. A homogeneous mixture is prepared, and the kitchen organic waste samples are stored at 4 °C. Rice straw hydrolysate (RSH) was prepared with 1% NaOH at 121 °C for 15-min autoclaving. After alkali treatment, the rice straw was subjected to another pretreatment step; this time with 1% H 2 SO 4 at 121 °C for 15-min autoclaving (Shah et al. 2018c). The RSH was filtered, washed with water and dried at room temperature. The untreated rice straw and rice straw hydrolysate samples were bound with gold palladium on black carbon tape. These samples of untreated rice straw and rice straw hydrolysate were photographed by a scanning electron microscope (SEM). SEM images were taken with a magnification resolution of 1000 μm to see the disrupted fibrous structure of untreated rice straw and rice straw hydrolysate after alkali and acid treatment (Qu et al. 2017).

Isolation process, reinforced clostridial medium (RCM)
Anaerobic granular sludge sample was collected in a bucket from an anaerobic reactor fed with organic municipal waste and straw residue after proper mixing to get a homogenized sample. From this bucket, 100 mL samples in triplicates were taken and sealed tightly in 250-mL anaerobic bottles. The 250-mL bottles were kept at 80 °C for 3 h (Alibardi et al. 2012). A 100 mL of reinforced clostridial medium (RCM) composed of meat extract 0.1g/100 mL, starch 0.1g/100 mL, yeast extract 0.3g/100 mL, glucose 0.5g/100 mL, peptones 0.1g/100 mL, sodium acetate 0.3g/100 mL, sodium chloride 0.5g/100 mL, L-cysteinium chloride 0.3g/100 mL and agar 0.5g/100 mL was sterilized in autoclaved at 121 °C for 15 min. The RCM bottles were inoculated with 20-mL volume from each granular sludge sample after heat treatment. The RCM bottles of 250 mL were flushed with a pure 99.99% nitrogen gas flow rate of 0.6 L/min for 4 min, added with filtered L-cysteine-HCL (0.1%) and filtered 0.04% resazurin using a sterile syringe and incubated in the thermal anaerobic chamber at 37 °C temperature. After 72 h of growth, fresh sterile RCM broth of 100-mL volume was inoculated with 5 mL from each pregrown RCM bottle. The RCM bottles were incubated again in the thermal anaerobic chamber at 37 °C temperature. This time, after 72 h, the grown culture in RCM bottles was serially diluted in normal saline, and 100 μL of culture inoculum from each dilution was spread into sterile RCM agar plates. RCM plates were incubated in strict anaerobic conditions placed inside an anaerobic chamber (Bactron, Model No. Baclite-2, Sheldon manufacturer) fitted with gloves, equipped with 5% H 2 , 5% CO 2 and 90% N 2 gas mixture) at a temperature of 37 °C. For anaerobiosis, the media was added with filtered L-cysteine-HCL (0.1%) and filtered 0.04% resazurin using a sterile syringe after autoclaving media. In the anaerobic chamber, catalyst cartridge of palladium pellets removes traces of oxygen from the chamber to maintain anaerobiosis. After incubation time for 48 h culture growth was checked. From the growth culture, a pure single colony was streaked into fresh nutrient agar plate. RCM media was used for long-term culture preservation. The pure culture cells were stored at refrigerator temperature (4 °C) in sterile semisolid RCM broth tubes and glycerol tubes separately.

Colony morphology and enzyme qualitative assay
The pure culture cells of freshly grown CBT-1 were picked from the RCM agar plate and were gently heated to fix with the slide. Slide smear was prepared. Gram staining was performed to check the morphology of the pure culture under a microscope. Also, on fresh RCM and nutrient agar plates media, the macroscopic morphology like color, shape and texture of the colony was observed. Further identification was done by performing biochemical tests using procedure of Bergey's Manual of Systematic Bacteriology. The pure culture was subjected to qualitative enzymatic study for cellulase and amylase using standard plate method. Pure culture was grown in RCM broth at 37 °C, 120 rpm for 24 h. A 100 μL of cells volume from RCM broth was diluted in 0.9% NaCL. A 3-μL sample of diluted cell suspension was freckled onto media plates in triplicates. For cellulase 5 g/L carboxymethylcellulose (CMC), and for amylase 20 g/L starch were added respectively. The agar plates of CMC and starch were incubated at 37 °C for 72 h. The enzyme activity was checked by flooding gram iodine solution for amylase test and 0.1% Congo red solution for cellulase test. The plates were left on room temperature for 30 min followed by washing with 1 M NaCl. The zone of hydrolysis on both starch and CMC plates was recorded (Shah et al. 2016).

Decolorization potential of dyes
CBT-1 pure culture was grown in RCM broth at 37 °C, 120 rpm for 24 h. A 100 μL of freshly grown CBT-1 cells volume from RCM broth was added into sterilized 50 mL of mineral salt media (MSM) in 250-mL bottles. The MSM was added with sterilized (separately autoclaved 121 °C for 10 min) glucose solution of 2 g/100 mL and 0.5g/L Azure B and 0.5g/L crystal violet dye in separate bottles. Uninoculated media bottles of Azure B dye and crystal violet dye added with sterilized (separately autoclaved 121 °C for 10 min) glucose solution of 2g/100 mL were used as control media samples. The media bottles were flushed with a pure 99.99% nitrogen gas flow rate of 0.6L/min for 4 min. The bottles were supplemented with filtered L-cysteine-HCL (0.1%) and filtered 0.04% resazurin using sterile syringe and incubated in the thermal anaerobic chamber (Bactron, Model No. Baclite-2, Sheldon manufacturer) at 37 °C temperature for 7 days. A visible change in the dye decolorization was monitored.

Polymerase chain reaction (PCR) of 16S rRNA gene
A pure colony was picked from RCM agar plate and was grown in sterile Luria Broth (LB Oxiod pH 0.6) at 37 °C, 120 rpm for 24 h. The DNA was extracted and kept at −20 °C for PCR reaction. The 16S rRNA region was amplified with forward primer FD1 (5 / CCG AAT TCG TCG ACA ACA GAG TTT GAT CCT GGC TCA G3 / ) and reverse primer RD1 primer (5 / CCC GGG ATC CAA GCT TAA GGA GGT GAT CCA GCC3 / ). After sequencing, sequence of 16S rRNA was searched through NCBI Basic Local Alignment Search Tool (BLAST) for genetically similar species strains, and Phylogenetic tree was constructed as described previously ).

Batch fermentation for biohydrogen potential (BFBP) from glucose, starch and cellulose
Glucose, starch and cellulose 15 g/L were added into sodium phosphate buffer (SPB) of pH 6.5 supplemented with K 2 HPO 4 (2.5 g/L), (2.5 g/L) NaHCO 3 solution, 2 mL of vitamin solution in 250-mL Pyrex bottles. The volume of media was kept to 100 mL equally in all Pyrex bottles. The pH was balanced at 6.5 for each Pyrex bottle. Then, media was autoclaved for 15 min at 121 °C. Pure colony of CBT-1 was anaerobically grown in sterilized LB broth at 37 °C overnight. The media Pyrex bottles containing 15 g/L glucose, starch and cellulose separately were inoculated. A starting value of CBT-1 with 0.3-nm optical density (600 nm) was equally added to all triplicates bottles. Control (uninoculated) media Pyrex bottles in triplicates containing 15 g/L glucose, starch and cellulose without culture inoculation were run in parallel at the same conditions. All media Pyrex bottles were closed using a silicon plug. Anaerobic conditions were adjusted by N 2 gas flushing at 0.6L/ min for 4 min in all experimental bottles. The BFBP experimental Pyrex bottles were incubated at 37 °C in a thermostatic chamber at static condition for 14 days. The daily volume of biohydrogen produced was measured with a sterile needle syringe using water displacement process. A 25% acidified (pH < 3) NaCL solution of 0.5-L flask was prepared to record the volume of daily gas released from the headspace of each Pyrex bottle. The amount of water movement in graduated cylinder is correspondingly equal to the amount of gas released from the headspace of each Pyrex bottle. The hydrogen, carbon dioxide compositions were measured by gas chromatography (micro-GC Varian 490GC).

BFBP from organic food waste and rice straw hydrolysate
After simple substrates (cellulose, glucose and starch) BFBP confirmation experiments, the culture of CBT-1 was assessed for capability of bioH 2 production from complex substrates. The CBT-1 was freshly grown in a 100-mL Pyrex bottle of LB medium at 37 °C, for 48 h in a thermostatic incubator. Sodium phosphate buffer (SPB) of pH 6.0 supplemented with K 2 HPO 4 (2.5 g/L), (2.5 g/L) NaHCO 3 solution, 2 mL of vitamin solution and 15 g/L rice straw hydrlysate, 15 g/L untreated rice straw and 15 g/L VS of kitchen food waste were prepared for media. A total of 100-mL media volume was kept in each 250-mL Pyrex bottle. Control Pyrex bottles added with untreated rice straw, rice straw hydrolysate and kitchen food waste were run in parallel. All Pyrex bottles were autoclaved for 15 min at 121 °C. A 0.3-nm optical density of CBT-1 was inoculated in the rice straw hydrolysate, untreated rice straw and kitchen food waste Pyrex bottles. The control Pyrex bottles were left uninoculated. Each Pyrex bottle was flushed for 4 min with N 2 gas at 0.6L/min flow rate. All the samples were managed in triplicate. The batch fermentation was run for 14 days at static condition by incubating in 37 °C thermostatic chamber. The daily volume of biohydrogen produced was measured using water displacement process as described above in Section 2.6.

Kinetic calculations
The kinetic for hydrogen production rate and yield was measured as done earlier in our study (Shah et al. 2016). The volume of gas produced in the control Pyrex bottle was subtracted from the gas volume in inoculated Pyrex bottle to calculate actual gas yield. The Microsoft Excel program was used to calculate volume of hydrogen (H 2 ), volume of headspace, concentration of H 2 at time t and t−1, whereas, concentration (X) of total H 2 volume at time t and the specific H 2 concentration at time t and t−1. The daily H 2 volume raw data was normalized at standard temperature and pressure (STP). The cumulative H 2 volume of each substrate in mL/gVS and mol/mol yield was mathematically calculated using Eq. (1) from the daily (H 2 ) volume and concentrations. where, The estimated kinetic of total hydrogen potential was measured by modified Gompertz Model (Eq. 4) by statistical software (IBM SPSS Statistic 23) through nonlinear regression model for each sample separately and the values of P, R and L were calculated. where is total yield of hydrogen (mL) in total time of incubation (t) P is the hydrogen production (mL) R is the highest rate of production (mL/d) and L is the lag phase time in days (d) e is equal to 2.718282. (1)

Analytical methods
Rice straw and kitchen food waste was analyzed for carbohydrate composition. Total solids (TS), volatile solids (VS), ash, moisture, carbon content, nitrogen, lignin, glucan, xylose, galactose, mannose and arabinose were measured as described in the standard laboratory analytical procedure (LAP) (Sluiter et al. 2013). The TS measurement was calculated by taking 1 g of rice straw and kitchen food waste, and oven dried at 105 °C overnight in a crucible. The weight of oven dried rice straw and kitchen food waste was measured again, and TS value was calculated using Eq. (5): where The VS of rice straw and kitchen food waste was calculated by burning the oven dried rice straw and kitchen food waste samples at 550 °C for 30 min. The samples of rice straw and kitchen food waste were cooled down in a desiccator at room temperature. The difference in measured weight was found using Eq. (6): where WDR weight of dried residue + dish WA weight of ash DS dish + substrate.
Rice straw sample was acid treated using the National Renewable Energy Laboratory (NREL)'s analytical method. Carbohydrate monomers (glucan, xylose, galactose, mannose and arabinose) were measured by HPLC (Shimadzu, SPD-MZ0A). Samples before fermentation and post-fermentation experiments of glucose, cellulose, starch, rice straw, rice straw hydrolysate and kitchen food waste were collected for volatile fatty acid (VFA) analysis. For carbohydrate detection, a standard solution of H 2 O and methanol was run as a carrier at a speed reaction of 0.6 mL/min and 80 °C. The filtered samples before and after completion of batch fermentation assay were treated with phosphoric acid (H 3 PO 4 ). The samples were run in HPLC (C18 column, mobile phase 1:1) parallel to standard concentrations of ethanol, methanol, n-butyric, propionic, acetic and valaric acid (Shah et al. 2016). A two-way analysis of variance (ANOVA) was conducted for the biohydrogen production yield in triplicates for

Selection of targeted strain
A sample from anaerobic reactor was grown on RCM to enrich only anaerobic culture. Out of four different culture isolates, only one colony was picked that showed qualitative expression for peroxidases, amylase and cellulase potential. The DNA was extracted from pure colony of this isolate named as CBT-1. Primers FD1 and RD1 were used to amplify the 16S rRNA gene by PCR. From PCR sequence and NCBI Blast result, 16S rRNA sequence of the pure CBT-1 culture showed 99.1% homology with Clostridium butyricum strains. The sequence of CBT-1 has been assigned with accession number OM698377 in NCBI Gene submission program. The similarity pattern of this strain was made through MEGA 7.0 by neighbor joining method as shown in phylogenetic tree (Fig. 2). The CBT-1 strain demonstrated its homology with other anaerobic Clostridium strains as well.

Characterization of the strain
A total of four different pure microbial colonies were picked from the RCM agar plates based on morphology differences. These colonies were screened for peroxidases, amylases and cellulases activities by qualitative hydrolysis of Azure B dye, crystal violet dye, starch and cellulose as sole carbon sources respectively. Out of four cell colonies, only the CBT-1 isolate showed expression of hydrolytic enzymes and decolorization potential of Azure B and crystal violet dyes. In preliminary screening, it was detected that the CBT-1 strain is a gram-positive, rod-shape and anaerobic culture. Colony of the CBT-1 was convex, lobate and off white on LB agar medium at 37 °C for 48 h. In the gram staining slide smear, its cells appeared as straight rods occurring in single or pairs (data not show). In Fig. 3, the CBT-1 showed zone of hydrolysis on both CMC and starch plate, which indicated positive for its enzyme activities (amylases and cellulases). In starch supplemented media, a zone around colonies showed starch hydrolyzing activity. Similarly, in the CMC medium plate, a zone around colonies showed existence of cellulolytic activity. The ability of the CBT-1 strain to grow on starch and CMC as a carbon source and formation of zone around colonies showed that they could metabolize it into products. Also, they can hydrolyze other biomass containing cellulase and starch composition. The CBT-1 decolorization of dyes in liquid media supplemented with glucose showed that the culture was capable of decolorization of Azure B dye and of crystal violet dye in 7 days of incubation time (data not show). The decolorization of Azure B dye using pure culture of Bacillus sp. MZS10 and the mechanism of cellular growth on the Azure B dye are studied well (Li et al. 2014). These pure cultures of Bacillus sp. strains were producing peroxidases (ligninases and laccase). The investigation of dye decolorization potential was for the validation of enzyme system and expression of ligninases, laccase and plate assay of CMC for cellulases and starch for amylase. This strategy helps in confirmation of lignocellulosic and kitchen food waste digestion for biohydrogen production. This strain was selected for further analysis.

Substrate composition
Rice straw and kitchen waste were analyzed for their compositional analysis. The rice straw was containing 20.2% lignin, total organic carbon 51.5%, total nitrogen 0.55%, total solid 92.5% and volatile solid 85%, whereas, the kitchen waste  Table 1. The compositions of rice straw are consistent with earlier described results ). Further, the rice straw was treated with acid and NaOH. The effect of treatment was checked for the untreated rice straw, NaOH-treated rice straw and after both treatment of 1% NaOH and 1%H 2 SO 4 acid. The SEM images make it obvious that the untreated rice straw micrograph was a smooth, flat and very compact structure (Fig. 4a). After the NaOH hydrolysis, the compact structure of rice straw was degraded (Fig. 4b). The impact was even more severe after both NaOH and H 2 SO 4 acid treatment. This could be due the removal of basic barrier element lignin from the surface of rice straw along with hemicellulose component, shown in Fig. 4c. The SEM images of Fig. 4 clearly revealed breaks in the silicon waxy morphology of the rice straw. Therefore, the rice straw hydrolysate was containing less lignin then as rice straw. Similar changes in surface destruction of the lignocellulosic biomass due to pretreatment is also reported (Zeng et al. 2011).

Daily and cumulative hydrogen yield
The Clostridium species consist of a huge number of extracellular enzymatic systems (cellulosomes) necessary for hydrolysis of complex biomass (Guo et al. 2015). Several bacterial species similar to Clostridium strains in metabolism are reported for utilization of pure carbohydrates (i.e., sucrose, glucose and galactose), polysaccharides enriched municipal waste and lignocellulosic biomass composed of hemicelluloses and celluloses (Amin et al. 2016;Nam et al. 2016). The efficient utilization of lignocellulosic biomass requires a pretreatment step to convert the substrate into sugar units for easy uptake and fast fermentation process (Malhotra and Suman 2021). This study first confirms the extracellular enzymes (peroxidase, cellulases and amylases) and then tested for biohydrogen production from pure carbohydrates. Rice straw hydrolysate was included, which could be an encouraging step toward the use of plant biomass for the synthesis of biohydrogen and other valuable products.
As per objective of the study, the culture of Clostridium butyricum CBT-1 was first evaluated for H 2 production from glucose, cellulose and starch. It was found that Fig. 3 In the above Fig. 3, the CBT-1 produced zone of hydrolysis for amylase on starch agar plate flooded with iodine solution (blue color), and zone of hydrolysis of cellulase positive on CMC plate (red color) is shown.
Zone of hydrolysis of CBT-1 on CMC medium Zone of hydrolysis of CBT-1 on starch medium NmLH 2 /substrate from starch and 294.5 NmLH 2 /substrate from cellulose respectively as shown in Fig. 5. The hydrogen yield of this study from simple carbohydrates is nearly the same from starch and cellulose of a previously reported H 2 yield from these carbohydrate substrates (Zagrodnik and Seifert 2020). Recombinant C. thermocellum DSM 1313 is reported for H 2 production from cellulose and hemicellulose substrates, which suggested that pure culture can use plant waste straw for value added products (Xiong et al. 2018). A co-culture of E. aerogenes and C. butyricum showed a 2mol H 2 /mol glucose production rate from starch waste and corn, and the most abundant species in anaerobic digestion of waste biomass reported for significant increase of biohydrogen are Clostridium sp. and Bacillus sp. (Nam et al. 2016), whereas, the highest rate H 2 yield of 3-3.8 mol H 2 / a b c ) and cellulose ( ) (b) respectively mol hexose has been observed from Pyrococcus furiosus, Thermotoga spp and Thermoanaerobacterium spp (Verhaart et al. 2010). Figure 6 illustrates the daily and cumulative H 2 production from untreated rice straw, rice straw hydrolysate and kitchen food waste. The daily highest H 2 volume was 11 NmL/gVS from untreated rice straw, 30.2 NmL/gVS from rice straw hydrolysate and 34.1 NmL/gVS from kitchen food waste. The maximum cumulative volume of H 2 produced was only 76.7 NmL/gVS from untreated rice straw, 184.4 NmL/gVS from rice straw hydrolysate and 237.2 NmL/gVS from kitchen food waste respectively.
No methane gas was found from the batch fermentation of all the substrates (glucose, starch, cellulose, rice straw and kitchen food waste fermentation experiments). The concentration of H 2 detected in GC was in the range of 25-40% for the tested substrates. The outcomes of the present study are comparable to previously reported cumulative H 2 yield from similar biomass and carbon carbohydrate saccharides (Shah et al. 2016;Xing et al. 2009).

Advantages and comparison of pure culture for hydrogen production
In this study, the Clostridium butyricum CBT-1 strain hydrogen yield was compared to already similar Clostridium strains as shown in Table 2. It is noteworthy that the Clostridium species are able to metabolize different substrates and capable of relatively good amount of hydrogen yield in batch fermentation. The yield is different from each substrate, in Clostridium strains, which could be due to differences in experiment conditions, nature of strains and capabilities of the tested strains on the applied waste biomass (Saratale et al. 2015).
It was found that the highest H 2 yield exhibited 393.18 NmL/gVS from glucose, whereas, the cellulose and starch that comparatively produced similar H 2 yield exhibited 314.18 and 325.62 NmL/gVS respectively. The untreated rice straw, rice straw hydrolysate and kitchen food waste showed 78.4, 197.4 and 263.3 NmL/gVS H 2 yield respectively as shown in Table 3. In Fig. 7a and Table 4, a comparison of the experimental cumulative H 2 volume and theoretical cumulative volume from glucose in 14 days is shown. The experimental H 2 volume is relatively similar to the theoretically estimated H 2 volume of the glucose. Similar H 2 volume was observed for other substrates, too (data not show). The overall results of Clostridium butyricum CBT-1 strain are notable, because only few studies reported biohydrogen potential from a pure culture. However, the capability of H 2 potential from lignocellulosic biomass (rice straw and rice straw hydrolysate in this study) is more interesting. The H 2 yield from rice straw by Clostridium butyricum CBT-1 is higher than Ca(OH) 2 and acid-treated biomass straw as reported previously (Nasirian et al. 2011;Reilly et al. 2014). The chemical treatment is used to enhance the hydrogen yield from waste biomass (Reginatto and Antônio 2015). So, it is significant that the H 2 yield could be improved from pretreated agriculture straw with acid or alkali . Similarly, from untreated cornstalk waste, a 61.4 mL/g of cumulative H 2 yield was observed using Clostridium thermocellum as a single culture. Even, Clostridium thermocellum and Clostridium thermosaccharolyticum co-culture produced 75 mL of H 2 /g H 2 yield . It is noteworthy that most of the studies either used mix microorganism of anaerobic sludge as inoculum or expensive chemothermal pretreatments. H 2 production potential of Clostridium saccharolyticus from wheat straw hydrolysate treated at 130 °C for 30 min has been reported (Ivanova et al. 2009). In addition, the hydrogen yield of Clostridium butyricum CBT-1 is higher than in previously reported studies. The application and H 2 yield of Clostridium butyricum CBT-1 proved that pure active and biomass degrading culture can avoid the necessity of expensive chemical pretreatment of waste straw carbon sources. Clostridium butyricum 293.5,299.8,184.4 and 236.2% more hydrogen yield compared to untreated rice straw sample. In our previous study, pure ligninolytic culture Brevibacillus agri AN-3 showed 293.7% increase of hydrogen yield from wheat straw compared to untreated wheat straw sample (Shah et al. 2018a). The biohydrogen production is directly proportional to the concentration of VFA consumed during anaerobic digestion process. The level of VFA produced and consumed reflected the performance of substrate fermentation assay and elucidated the H 2 metabolic pathways (Luo et al. 2019). In this study, no VFA concentrations were detected at the completion of glucose, cellulose and starch fermentation assay, signifying the total consumption of VFA to products by the tested Clostridium butyricum CBT-1. However, in a sample of rice straw hydrolysate and kitchen food waste, a considerable level of VFA was detected. The higher concentration was of butyrate, iso-butyrate, acetate and propionate. The detection of butyrate and acetate VFA production in Clostridium butyricum CBT-1 proved it to be an ideal candidate for bioH 2 production. Similar results of VFA (butyrate and acetate) are reported in the H 2 producing microorganisms specifying similar H 2 catabolic pathways (Fang et al. 2006;Shin et al. 2004). The leftover VFA and slurry after anaerobic fermentation process is a rich source of beneficial nutrients for plants, crops and vegetables growth, which can be used as organic fertilizers to enhance their growth and yield (Hamid et al. 2021). To see a significant variance among each tested substrate in all the experiments for comparison of biohydrogen yield. A two-way ANOVA in triplicates values was run to check the statistical difference in biohydrogen yield. All the tested substrates untreated rice straw, rice straw hydrolysate, kitchen food waste, glucose, cellulose and starch were compared; the average result showed a uniform P values of 0.001, demonstrating that the responses were significant for most of the conditions. A perfect significant change between the substrates value can be seen in Fig. 7b and Table 4. The difference in biohydrogen yield was high in case of glucose as substrate followed by starch, cellulose, kitchen food waste Table 2 The Clostridium butyricum CBT-1 of this study is compared to previously reported study (Saratale et al. 2015). The yield is presented in molH 2 /mol  and rice straw hydrolysate. The lowest biohydrogen yield was obtained from the untreated rice straw (Fig. 7b). The overall results of this study highlight the importance of isolation of pure culture capable of different biomass hydrolysis and fermentation into value added metabolites. This strategy can be used to collect specific strains for pure bioproducts as well as a biocatalyst for hydrolysis of complex biomass at large scale.

Conclusion
In this study, Clostridium butyricum CBT-1 strain was isolated and purified that was capable of expressing amylase, cellulase and peroxidases enzymes. These enzyme activities were determined from a significant amount of decolorization of Azure B and crystal violet dyes. In addition, the strain showed considerably high H 2 potential from glucose, starch, cellulose, KFWS and rice straw hydrolysate. Especially, the Clostridium butyricum CBT-1 cumulative H 2 yield was significant from rice straw hydrolysate and kitchen food waste. It produced 180-240% more H 2 volume from rice straw hydrolysate and kitchen food waste compared to the untreated rice straw. These results indicate a promising line for enhancing H 2 yield of pure anaerobic culture from organic waste biomasses, although, culture condition optimization may also play an important role to uplift the yield of specific product from similar anaerobic culture.
Author contribution TAS did investigation, conceptualization, experiments, writing and manuscript preparation: LZ and AZ helped in writing and manuscript preparation; LZ provided supervision and editing; DL did results and sample analysis; WF and HX did sample analysis, equipment and resources management.
Funding This work was supported by the National Key R&D Program of China (Grant No. 2019YFD1100600).

Data availability
The authors confirm that the all data generated or analyzed during this study are included in this published article.

Declarations
Ethics approval and consent to participate Not applicable.

Competing interests
The authors declare no competing interests.