Introduction

Hydrogen having high energy yield of 142 KJ/g is a crucial alternative source of energy carrier replacing fossil fuels. As it is pollution free, thus known as a clean energy source which is eco-friendly (Nath and Das, 2004; Rittmann and Herwig, 2012). Hydrogen is liberated during different processes like electrolysis of water, through natural gases, steam reforming of hydrocarbons and during biological processes.Hydrogen producing enzymes are present in a microbe which produces the bio-hydrogen. Bio-hydrogen productionfrom a variety of renewable resources utilized as a substrate by different approaches including direct bio-photolysis, indirect bio-photolysis, photo-fermentation and dark-fermentation (Chen et al. 2008; Homann 2003; Lo et al. 2010; Manish and Banerjee 2008; Redwood et al. 2012). Among these, dark-fermentation is a carbon neutral process for the production of bio-hydrogen in which carbohydrate rich substrates are a breakdown by facultative and obligate anaerobic bacteria. Many efforts have been directed to the development of efficient technologies to obtain renewable energy from lignocellulosic biomass, by recycling a large range of agricultural wastes which are non-food parts of crops: such as stems, leaves, flowers, etc. and the industrial waste products such as woodchips, skin and pulp from fruit pressing, wastes from oil refineries from seeds etc. These agricultural wastes are used for energy production. Anaerobic fermentation from bacteria by methanogenesis, that has already found wide application for the reduction and stabilization of agricultural wastes. Many microorganisms utilizes agricultural wastes as an organic source of carbohydrate and convert into hydrogen Bacteria which produce hydrogen include species of Enterobacter, Bacillus and Clostridium via pyruvate metabolism which is an intermediate, formed during the degradation of substrates (Martinez-Porqueras et al. 2013). Hydrogen evolution rate is higher in the dark-fermentation process in contrast to other processes.Theoretically, four moles of hydrogen can be produced per mole of glucose, assuming that all pyruvate is converted to acetate but in reality, it is not possible. The pH is an important key factor in bio-hydrogen production as it affects hydrogenase activity, metabolic pathways shifts the volatile acid production and microbial communities in bio-hydrogen production (Shridevi and Preetha 2015). Microbial growth during fermentation process was inhibited at lower pH leads to lower hydrogen production (Liu et al. 2011). This is because; the lower pH can change the fermentation process for bio-hydrogen production to butyric acid type, propionic acid type and ethanol type of fermentation. Generally, acidogens are less sensitive to pH change than the acetogens and methanogens. The pH drop from 6 to 4.5 was considered to be the ideal pH range for the anaerobic bacteria and for inhibition of methanogenic activity (Mohan et al. 2007). Bacterial families belonging to mesophilic bacteria includes Entero bacteriaceae and Clostridiaceae as well as the family Thermoanaerobacterales, which have attracted much attention in recent years, as it exhibits low hydrogen to glucose yields of 1-2 mol/mol because of the thermodynamic limitation associated with NADH utilized as an electron carrier   (Chou et al. 2008; Lee et al. 2011; Rittmann and Herwig 2012). Thermophilic H₂ production has been shown to be clearly advantageous over mesophilic H₂ production with respect to the yields of hydrogen to the substrate. Hydrogen yield has been shown to be high 1.46 mol/molformesophilic bio-hydrogen producing microorganisms as compared with thermophilic bacteria, 2.20 mol/mol (Verhaart et al. 2010). Ease of accessibility, cost-effectiveness, high carbohydrate content and biodegradability are taken into consideration while choosing any substrate for bio-hydrogen production (Das and Veziroglu 2008). India is at second position in world whereas, Chhattisgarh ranks fifth in India for maximum rice production. This indicates generation of large amount of agricultural wastes.

So, rice bran which is the by-product of rice can be suitable substrate for production of bio-hydrogen. Rice bran constitutes cellulose which is attached to sugars and hemicellulose which is attached to lignin. Rice bran is the by-product generated in rice bran oil industry. Various bacterial strains are capable of degrading complex carbohydrates but bio-hydrogen generation mainly depends on type and concentration of substrate, its pH, and hydraulic retention time, the substrate to inoculums ratio, metal ion concentration, etc. So considering all these parameters, the objectives fixed for the current piece of research are isolation and identification of maximum bio-hydrogen producing bacteria from rice industry waste and at the same time optimization of temperature and pH on bacterial growth.

Materials and methods

RB (Rice bran) and DORB (De-oiled Rice bran) samples were collected from Paras gold oil refinery industry, Urla, Chhattisgarh, India. Collected samples were kept in airtight container at room temperature.

Isolation of bacteria and characterization

Bacteria were isolated from the RB and DORB samples. 1g of the RB and DORB samples was added to 10 ml of distilled water. Serial dilution (10^-­¹ to 10^-9) was performed on a nutrient agar plate (peptone 5g/l; beef extract 3g/l; NaCl 5g/l; agar 15g/l; pH 6.8) by spread plate method and incubated for 24hrs at 37±2°C (Thakur et al.2012). Isolated colonies were purified using streak plate method and maintained in nutrient agar slants as stock. Fermentation medium (peptone 10g/l; glucose 5g/l; NaCl 15g/l; phenol red 0.018g/l and pH -7.3) was prepared and then transferred into a test tube having Durham's tube for detection of gas production. Inoculation of culture in fermentative media and incubated at 37±2°C for 48hr. Phenol red was used as an indicator which turns the medium from pinkish red to yellow. The screened isolates were maintained on nutrient agar slants at 37±2°C and stored at 4°C. Colony characterization of the isolated bacteria from different season by their number, shape, elevation, margin, and appearance was performed (Prescott and Harley 2002). Staining tests for the screening of bacteria by gram staining, acid-fast staining, endospore staining, and motility test were performed. Amylase test, urease, IMViC test (indole test, methyl red test, Voges-Proskauer test, citric acid utilization test) and catalase tests were performed for their biochemical characterization (Prescott and Harley 2002).

Effect of pH and temperature on bacterial cell growth

Temperature is a very important parameter in bio hydrogen production and also influences cell growth. The pH is an important key factor in bio-hydrogen production as it affects hydrogenase activity, metabolic pathways shifts the volatile acid production and microbial communities in bio-hydrogen production. Thus, the effect of pH on bacterial growth and bio-hydrogen production capacity ranges from 4.5, 5, 5.5, 6, 6.5, and 7. The effect of temperature on bacterial growth was investigated at different temperatures ranging from 25°C, 30°C, 35°C, 40°C and 45°C and for bio-hydrogen producing capacity from 30°C, 35°C, 40°C, 45°C and 50°C. 

Batch fermentation hydrogen production

Batch fermentation studies were carried out in 250ml Erlenmeyer flasks by the process of liquid displacement method of all isolated culture (Fig.1). In this method, two 250 ml conical flask was capped by rubber cork connected with plastic pipe and an outlet in one flask which was in the measuring cylinder to measure the production of bio-hydrogen in a millilitre. The first flask contained substrate along with bacterial culture and second flask contained 10 % KOH, for the absorption of carbon dioxide(CO₂), which was again connected to a measuring cylinder for collection of KOH (Kotay and Das 2006). Bio-hydrogen and CO₂released from headspace of fermentor from first flask containing 1:10 (w/v) of substrate to inoculums ratio through 10 % KOH where CO₂ absorbed and collected in the measuring cylinder to measure the production of bio-hydrogen in millilitre (Zanchetta et al. 2007). The initial pH value for hydrogen production was maintained at 6 compared to neutral pH under mesophilic condition (37±2°C) (Mohan et al. 2008).

Figure 1: Experimental setup (Batch fermentation process) for bio-hydrogen production.

This setup was placed on hot magnetic stirrer maintained at 37±2°C. Data obtained from secondary screening for bio-hydrogen production and specific hydrogen production rate (SHPR) was compared and analysed using Excel Software.

Results and Discussion

Isolation of bacteria

Bacteria were isolated from the RB and DORB samples. 1g of the RB and DORB samples was added to 10 ml of distilled water. Serial dilutions (10^-­¹ to 10^-9)was performed on nutrient agar plate by spread plate method and incubated for 24hrs at 37±2°C(Thakur et al.2012).After incubation for 24 hrs, mixed culture of the bacteria was found on the plates of dilution at 10^-3, 10^-4 and 10^-5. Pure culture was performed by streak plate method and pure cultures were obtained by plating repeatedly. From these only six bacterial isolates were selected which showed best results for bio-hydrogen production and goes for a further test. Out of these cultures, RB II showed maximum bio-hydrogen producing capacity of 510±3.9 ml H₂/L with SHPR 0.70±0.33ml H₂/g substrate/h.

Microscopic and biochemical analysis

Bacterial species were isolated from the RB sample and from DORB sample and their microscopic study was performed (table 1).

Table 1: Microscopic and Biochemical test performed in bacteria isolated from RB and DORB

All bacterial cultures are gram-negative, acid-fast negative, endospores negative. Zhang et al. (2007) identified the spore-forming rod-shaped during their studies on enhanced bio-hydrogen production from corn stalk wastes with pre-treatment by acidification by mixed anaerobic cultures. Basically, the bacteria can degrade organic and inorganic compounds by converting them into various intermediate compounds by the action of various enzymes. They derive their nutritional requirements from the compounds present in the ent waste. All isolates of RB sample were indole negative, citrate positive, catalase positive. All but one isolate showed MR negative and two showed VP positive results.

Effect of pH and temperature on bacterial growth

Environmental conditions are the major parameters to be controlled in bacterial growth for bio-hydrogen production. In this study, the effect of pH ranges from 5 to 8 and temperature ranges from 25°C to 40°C on bacterial growth was observed. At pH 6.0, bacterial growths were observed maximum while at pH 8, bacterial growths were observed minimum (fig. 2). Bacterial strains had the capacity to degrade complex carbohydrates. Studies concluded that pH play an important role in hydrogen production, and the optimum pH for enhanced yield of hydrogen recorded was in the wide range of pH (5.0-9.0) for various kinds of hydrogen producing microbes by Mu et al. (2014).This may be due to the suppression of methanogenic activity under acidic conditions. At higher or lower than this pH accumulation of acids causes a sharp drop of culture pH and subsequent inhibition of bacterial hydrogen production (Haridoss 2016). Pachapur et al. (2016) noted that impact of H₂ production with increasing pH up to optimum (7.0) had a comparative impact of increase in pH through crude glycerol. Ren et al. (2007) observed maximum hydrogen yield of 1.77mmol/mmol of glucose was achieved at pH 6.0in batch fermentation processes showed a similar result. While on the study of the effect of temperature on bacterial growth, 35°C temperature was the best for all selected bacteria whereas at 40°C bacterial growth was found minimum (fig. 3). Chittibabu et al. (2006) observed efficiency of bacterial growth decreased when temperature was increased from 38°C to 45°C because of denaturation of the key enzymes or inactivation in the metabolic pathway at high temperature. Maximum bacterial growth was observed at pH 6.0 and suitable temperature 35°C.

Figure 2: Bacterial growth of BHP bacteria isolated from RB and DORB at different pH (5-8 pH).

Figure 3: Bacterial growth of BHP bacteria isolated from RB and DORB at different temperature (25-40°C).

Bio-hydrogen producing capacity at different pH and temperature

Hydrogen production is a growth associated product where mostly gas was produced during the exponential phase carried out in 250ml batch fermentation process by liquid displacement method.Temperature is one of the most important physical factors affecting microorganism and at optimum temperature enhances bio-hydrogen production. The maximum bio-hydrogen production was found at pH 6.0 1351.6±15.89 ml H₂/L with maximum SHPR 0.187±0.002 ml H₂/g substrate/h (Fig. 4). Bio-hydrogen producing capacity was maximum at 35°Cand 30°C having 1291.6ml H₂/L with maximum SHPR 0.179±0.0001ml H₂/g substrate/h and 1358.3±8.3ml H₂/L with maximum SHPR 0.188±0.05 ml H₂/g substrate/h respectively (Fig. 5). At 35°C, showed maximum production due to the accumulation of VFA’s over time, it causes the death of the bacteria hence decline in hydrogen production was observed with more time interval. Shukla et al. (2015) also reported bio-hydrogen production fromrice bran and rice mill effluent by using Clostridium acetobutylicum NCIM 2877 at 35°C and pH 6.0 and found production was 68.7±0.9 ml with 85±1.0% substrate utilization. Chaoudhary et al. (2015) also observed maximum bio-hydrogen production of 112 ml at 48 hours of hydraulic retention time, 92 ml production at 35°C and 180 ml at initial pH 8 from isolated.

Cell growth and hydrogen production were decreased when the temperature was increased from 38°C to 45°C and there was no hydrogen production at 45°C.

Figure 4(a): Bio-hydrogen productions in ml H₂/L±SE of batch fermentation at temperature 37°C±2°C,

Figure 4(b): SHPR in ml/g substrate/h

Figure 5(a):Bio-hydrogen productions in mlH₂/l±SE of batch fermentation at pH 6.0

Figure 5 (b): SHPR in ml/g substrate/h.

Conclusion

A hydrogen producing strain was isolated from municipal sewage sludge and RB II showed maximum hydrogen production of 1358.3±8.3ml H₂/L with maximum SHPR 0.188±0.05 ml H₂/g substrate/h. Maximum bio-hydrogen production at ambient pH 6.0 and temperature 35°C will be isolated and utilized for enhanced bio-hydrogen production was optimized. Further biological and chemical pretreatments will improve bio-hydrogen yields. The dark fermentation process can renovate unvalued organic waste into bio-hydrogen rich gas. This approach also reduces the efficient production costs which are ultimately beneficial from economic point of view.

Conflict of interest

Authors had no conflict of interest.

Acknowledgement

Authors are thankful to the Department of Science and Technology, New Delhi for financial support through DIST-FIST scheme to support to School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur (C.G.).Authors are also grateful to the RGNF, UGC (University Grants Commission), New Delhi, circular number F1-17.1/2015-16/RGNF-2015-17-SC-CHH-2144/(SA-III/Website) for financial support.

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