2.1 Material Preparation
Most of the chemicals tested in this study were of 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 is stored at 4oC. Rice straw hydrolysate (RSH) was prepared with 1% NaOH at 121ᵒC for 15 min autoclaving. After alkali treatment, the rice straw was subjected to another pretreatment step, this time with 1% H2SO4 at 121ᵒC for 15 min autoclaving. The RSH was filtered, washed with water and dried at room temperature (Minu et al. 2012). 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 scanning electron microscope (SEM). SEM images were taken with magnification resolution of 1000 um to see the disrupted fibrous structure of untreated rice straw and rice straw hydrolysate after alkali and acid treatment (Qu et al. 2017).
2.2 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 hr. A 100 mL of Reinforced Clostridial Medium (RCM) compose 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 minutes. The RCM bottles were inoculated with 20 mL volume from each granular sludge sample after heat treatment. The RCM bottles were flushed with nitrogen gas for 4 minutes and incubated in the thermal anaerobic chamber at 37°C temperature. After 72hr of growth, fresh sterile RCM broth of 100 mL volume was inoculated with 5 mL from each pregrown RCM bottles. The RCM bottles were incubated again in the thermal anaerobic chamber at 37°C temperature. This time, after 72 hr, the grown culture in RCM bottles was serially diluted in normal saline and 100 uL of culture inoculum from each dilution was spreaded in to sterile RCM agar plates. RCM plates were incubated in strict anaerobic conditions (placed inside an anaerobic chamber equipped with 10% H2, 5% CO2 and 85% N2 gas mixture) at temperature of 37°C. After incubation time for 48 hr culture growth was checked. From the growth culture, a pure single colony was streaked in to fresh nutrient agar plate. RCM media was used for long-term culture growth and spore generation. The pure culture cells was stored at refrigerator temperature (4°C) in sterile semisolid RCM broth tubes and glycerol tubes separately.
2.3 Colony Morphology and Enzyme Qualitative Assay
Spores were picked from the RCM agar plate and activated at 60°C for 15 minutes. Slide smear was prepared. Gram staining was performed to check the morphology of the pure culture under 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 hr. 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 was 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 minutes followed by washing with 1 M NaCl. The zone of hydrolysis on both starch and CMC plates were recorded (Shah et al. 2016b).
2.4 Growth and Degradation Potential of Dyes
CBT-1 pure culture was grown in RCM broth at 37°C, 120 rpm for 24 hr. A 100 µL of freshly grown CBT-1 cells volume from RCM broth was added into sterilized 50 mL bottles of mineral salt media (MSM). The MSM was supplemented with 0.5g/L Azure B and 0.5g/L crystal violet dye in separate bottles as defined previously (Picart et al. 2016, Ravi et al. 2017). Uninoculated media bottles of Azure B dye and Crystal violet dye were used as control media samples. The bottles were kept in shaking incubator conditions of 37ᵒC and 150 rpm for seven days. Daily 2 mL sample was taken to check absorbance at 620nm under UV-visible spectrophotometer for growth observation. Similarly, 5 mL sample before start of experiment and 5 mL samples after completion of seven days experiment were taken. The samples were centrifuged at 10000 rmp for 10 minutes for each sample bottle. The absorbance of supernatant was measured at 651nm for Azure B dye and 592nm for crystal violet dye decolorization, respectively (Picart et al. 2016). The amount of decolorization for both dyes was calculated using the following equation as:
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Where Xi = initial absorbance at first day
Xf = final absorbance at last day
2.5 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 hr. A 2 mL of cells volume from LB broth was centrifuged at 10000rpm for 15 minutes and the supernatant was discarded from the cells pellet. The cells pellet was treated with 300 µL TE Buffer (Tris HCl 10 mM, EDTA 1mM, 1 M NaCl, pH 8.0). A 100 µL of 10% SDS was added to the tubes. The cells pellet was heated at 80°C for 30 minutes. Proteinase K buffer in 1M Tris HCl of 200 µL was added and kept in water bath at 50°C for 60 minutes. A 50µL of 20g RNase was added at room temperature for 60 minutes. Then 200 µL of 6M-NaCl solution and chilled absolute ethanol was added and centrifuged at 10,000rpm for 10 minutes. The supernatant was transferred to other eppendorfs and chilled 1000 µL phenol-chloroform-iso amylalcohol was added again centrifuged at 10000rpm for 10 minutes. The upper most supernatant was wash with 70% chilled ethanol. The cells pellet is dissolved in 100 µL TE buffer. A 1% agarose gel was prepared and the DNA was loaded in 0.5x solution of TBE. The agarose was heated in microwave oven for 1 minute and cooled up to 45°C. A 0.3µg/mL ethidium bromide (Roche, Germany) was added for staining. Electrophoresis was carried out for 1 hour at 80 V. The DNA bands were visualized in UV Transilluminator (UVItec, EEC) and digital photograph was taken. The extracted DNA was then kept at -20 ºC for PCR reaction. The 16S rRNA region was amplified with forward primer FD1 (5/CCGAATTCGTCGACAACAGAGTTTGATCCTGGCTCAG3/) and reverse primer RD1 primer (5/CCCGGGATCCAAGCTTAAGGAGGTGATCCAGCC3/). Taq DNA polymerase (Fermentas, USA) of 1.5 µL, 1.5µL of 50ng genomic DNA, 15µL 10x PCR Super mix, 1.5µL from (25ng/µL) forward primer and reverse primer were added. PCR buffer water of 11.5µL were added to PCR tubes and gently vortexed for 5 seconds. PCR conditions were set as such : initial denaturation of one time at 94°C for 2 min, 94°C for 1 minute in each cycle, extension at 72°C for 1 minute and annealing at 55°C for 1 minute is processed for 30 PCR cycles. The PCR product was cleaned with QIAquick PCR Purification Kit (Qiagen, MD, USA). The amplified PCR sample was sequenced. The raw sequence of PCR was filtered through the sequence analysis package (DNA-Star). Fasta sequence of 16S rRNA was searched through NCBI Basic Local Alignment Search Tool (BLAST) for genetically similar species strains. Phylogenetic tree was constructed using MEGA7 software(Shah et al. 2019).
2.6 Batch Fermentation for Biohydrogen Potential (BFBP) from Glucose, Starch and Cellulose
Glucose, starch and cellulose 15 g/L was added into sodium phosphate buffer (SPB) of pH 6.5 supplemented with K2HPO4 (2.5 g/L), (2.5 g/L) NaHCO3 solution, 2 mL of vitamin solution in 250 mL Pyrex bottles. The volume of media was kept to 100 mL equally in all Pyrex bottle. 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 0.3nm optical density (600 nm) was equally added into 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 N2 gas flushing for 4 minutes 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 using water displacement process. A 25% acidified (pH < 3) NaCL solution of 0.5 Liter flask was prepared to record the volume of daily gas released from the headspace of each Pyrex bottle. The amount of water move in graduated cylinder is correspondingly equal to the amount of gas released from the headspace of each Pyrex bottle. The hydrogen, carbon dioxide composition were measured by Gas chromatography (micro-GC Varian 490GC).
2.7 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 bioH2 production from complex substrates. The CBT-1 was freshly grown in 100 mL Pyrex bottle of LB medium at 37°C, for 48hr in a thermostatic incubator. Sodium phosphate buffer (SPB) of pH 6.0 supplemented with K2HPO4 (2.5 g/L), (2.5 g/L) NaHCO3 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 0.3nm optical density of CBT-1 was inoculated in the rice straw hydrlysate, untreated rice straw and kitchen food waste Pyrex bottles. The control Pyrex bottles were left uninoculated. Each Pyrex bottle was flushed for 5 min with N2 gas. 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 2.6 section.
2.8 Kinetic Calculations
The kinetic for hydrogen production rate and yield was measured as did earlier in our study (Shah et al. 2016a).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 (H2), volume of headspace, concentration of H2 at time t and t-1. Whereas, concentration (X) of total H2 volume at time t and the specific H2 concentration at time t and t-1. The daily H2 volume raw data was normalized at standard temperature and pressure (STP). The cumulative H2 volume of each substrate in mL/gVS and mol/mol yield was mathematically calculated using the equation Eq-2 from the daily (H2) volume and concentrations.
\(H2Vol.t=XH2,GH2V,t+HSV.(XH2,t - XH2,t - 1)\) (Eq-2)
Total volume of H2 in mL = H2 Vol, t,
Concentration of H2 = XH2
Gas volume of H2 each time = G H2V, t
Headspace volume = HSV
The H2 yield was calculated as shown in the equation Eq-3.
\(H2{\text{ }}(Y)=\frac{{Cumulative{\text{ }}H2}}{{Weight{\text{ }}of{\text{ }}substrate}}\) (Eq-3)
Where H2 yield (Y) = measured as mL/gVS as substrate load was based on VS to each Pyrex bottle in BHFP.
The H2 yield (Y) mol H2/g glucose was measured by equation Eq. 4.
\(H2(y)=\frac{{Cumulative{\text{ }}H2}}{{\frac{{22.4}}{{\frac{{Weight{\text{ }}of{\text{ }}glu\cos e}}{{180}}}}}}\) (Eq-4)
where ,
1 mole of ideal gas at standard temperature and pressure (STP) = 22.4 L/mol volume
1 mole of glucose = 180 g/mol
The estimated kinetic of total hydrogen potential was measure by modified Gompertz Model (Eq. 5) 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.
\(Y\left( t \right)=P \times {\text{ }}exp{\text{ }}\left\{ { - {\text{ }}exp{\text{ }}\left[ \begin{gathered} \underline {{{R_*}^{e}}} \hfill \\ p\left( {\Delta - t} \right) \hfill \\ \end{gathered} \right]{\text{ }}\left( {L - t} \right)+1} \right\}\) (Eq. 5)
Where Y (t) is total yield of hydrogen (mL) in total time of incubation (t), P is the hydrogen production (mL), R is highest rate of production (mL/d), and L is the lag phase time in days (d), e is equal to 2.718282.
2.9 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 gram of rice straw and kitchen food waste, 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 this Eq. 6:
TS (%) = \(\frac{{WSD - WD}}{{DS - WD}}\) (Eq. 6)
Where, WSD = Weight of dried residue + dish, WD = Weight of dish, DS = Dish + substrate.
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 minutes. 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. 7:
VS (%) = \(\frac{{WDR - WA}}{{DS - WA}}\) (Eq. 7)
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 VFA analysis. For carbohydrates detection, a standard solution of (H20 and methanol) were run as 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 (H3PO4). 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. 2016a).