Introduction

The energy sector has assumed significant importance in India due to the ever increasing energy needs of the growing population. Burning of fossil fuel which produces extensive greenhouse gas and toxic gases, such as CO₂, SO₂, NO_x and other pollutants, causing global warming and acid rain (Meng et al. 2006). Nowadays increasing fossil fuels reserves utilization, for combustion has serious negative effects on environment. For these reasons, many researchers have been working on the biomass energy sources (Kapdan and Kargi 2006). Biomass is most abundant renewable resources which are formed by fixing carbon dioxide in the atmosphere during the process plant photosynthesis, so it is carbon neutral. Biomass research is receiving increasing attention because conversion of waste to energy but low efficiency of utilizing is one of the drawbacks (Meng et al. 2006).

Hydrogen gas is a clean energy source with high energy content if 122 kJ g-1 (Huibo et al. 2010). It is a versatile energy carrier with the potential for extensive use in power generation and in many other applications (Nath and Das 2004). Processes for production of hydrogen are numerous such as bio-photolysis, electrolysis, fermentation and hybrid method or two stage fermentation processes. Hydrogen production through the utilization of microorganism is called bio-hydrogen (Hallenbeck and Benemann 2002). Water hyacinth can use as raw material for bio-hydrogen production because it contains high energy, cheap and have high protein contents (Wang and Wu 2004; Sagar & Kumari 2013). Water hyacinth is monocotyledonous plant from family Pontederiaceae. It is a freshwater aquatic plant used as ornamental plant and in medicinal use also as a toxic compounds removal from the polluted pond. It can proliferate rapidly and can spread over large area of water which may cause practical problems for marine transportation and fishing but it be used as substrate for the production of bio-fuel. It composed from cellulose, hemicelluloses and lignin in which cellulose-20%, lignin-10% and hemicellulose-33%. Thus less amount of lignin, leads to the cellulose and hemicellulose more easily converted to fermentable sugar for utilizing biomass for bio-hydrogen production (Sagar and Kumari 2013). Although, it is a good source of energy and this can be used for bio-hydrogen production. Among chemical pre-treatments, several acids like HCl, H₂SO₄, HNO₃, H₃PO₄, are used for the bio-hydrogen production. Biomass degradation rate increased in concentrated acid hydrolysis (Jung et al. 2013; Wang et al. 2012). However, there is a need of high alkaline dosages for neutralization of salt formation before discharge because it is highly corrosive. During acid pre-treatment, biomass hydrolysis occurs due to delignification from cellulose and hemicellulose by breakage of glycosidic bonds (Zheng et al. 2014). Hydrogen can be produced from the different form of sugar monomers (C₅ and C₆) after degradation of lignocellulosic substrate through bacteria (Lai et al. 2014). For pilot scale bio-hydrogen production to overcome of energy crisis in the world, water hyacinth is ideal source energy.

Materials and methods

Sample collection and processing

In the present work water hyacinth the aquatic plant was used as substrate. Fresh water hyacinth plants were collected from Aamanaka, Raipur (C.G.) in a clean polybag. Collected water hyacinth was washed to remove adhering dirt. The leaves, stems and roots were separated carefully; the roots were discarded because they absorb heavy metal pollutants from water bodies (Das and Lenz 2000). Leaves and petioles were washed with tap water and sun dried. The dried leaves and petioles were grinded into powdered form. Prepared powder was used as feedstock in all fermentation experiments (Fig.1). It was stored in air tight container at room temperature.

Experimental setup was carried out in batch fermentation setup, in 250 ml conical flasks having substrate medium at pH of 6.0 and placed on hot plate magnetic stirrer temperature adjusted at temperature 37°C. This flask was connected to another flask containing 10% KOH for the absorption of carbon dioxide produced as a byproduct of the fermentation process (Thakur et al. 2012). KOH containing flask was connected to another measuring cylinder for collection of KOH by liquid displacement method to measure the production of bio-hydrogen (Zanchetta et al. 2007).

Figure 1: Sample collection and processing of water hyacinth (a) plant in pond (b) water after cleaning in lab (c) dried and cut sample (d) powdered sample (e) substrate prepared in distilled water and (f) batch fermentation setup for bio-hydrogen production.

Effect of age of inoculums on bio-hydrogen production

For the study the effect of age of inoculums on bio-hydrogen production 6 gm substrate powder was taken in 100 ml of distill water by adjusting pH 6.0 and then autoclaved.  Sample was taken for sugar test before fermentation. Bacterial culture of Klebsiella oxytoca ATCC 13182 was taken from laboratory of School of Studies in biotechnology, Pt. Ravishankar Shukla University, Raipur (C.G.). Culture was maintained on Nutrient Agar Media composed of peptone, 5 g/l; beef extract, 3 g/l; NaCl, 5 g/l and agar, 15 g/l and pH adjusted at 6.8. NAM slants were prepared for bacterial culture, grown at 37°C for 24 hours and then maintained in 4°C in freeze. Culture was maintained at nutrient broth media composed of peptone, 5 g/l; beef extract, 3 g/l; NaCl, 5 g/l and pH was maintained at 6.8 for experiment (Thakur et al. 2014). After incubation the suspension was taken as the inoculums for fermentation experiments. The Klebsiella oxytoca ATCC 13182 was incubated for different time duration (18 h, 20 h and 24 h) for optimization of age of inoculums (Thakur et al. 2014). Batch fermentation setup were carried out in conical flask (250 ml Erlenmeyer flasks) containing substrate media which was adjusted at pH 6.0 and temperature 37°C then placed on the hot plate magnetic stirrer for bio-hydrogen production (Thakur et al. 2012). This flask was connected with pipe to conical flask containing KOH and then again pipe was connected to measuring cylinder for collection of KOH by displacement for the production of bio-hydrogen (Zanchetta et al. 2007).

Effect of volume of inoculums on bio-hydrogen production

Optimized 20 hours old culture was taken as inoculums and the volume of inoculums was optimized with 10%, 20%, 30% and 40% inoculums. Cultures are maintained at nutrient broth medium (peptone 5 g/l; beef extract 3 g/l; NaCl 5 g/l; pH 6.8). Batch fermentative process was conducted in 200 ml volume in 250 ml flask with initial pH 6.0 and initial temperature at 35°C.

Effect of acid pretreatment on bio-hydrogen production

Dissolved 2 gm/l of the dried sample in distilled water and add 0.5%, 1%, 1.5% and 2% of concentrated sulfuric acid (H₂SO₄) and autoclaved. After cooled the mixture was filtered at room temperature with whatman filter paper and adjusted the pH of substrate medium at 6.0. The filtrate was taken and autoclaved, sample was taken in a collection tube for sugar test. Set up for fermentation experiment. Total sugar (anthrone test) and reducing sugar test (DNS test) was performed. Sugar in the presence of sulfuric acid dehydrate to furfural or hydro methyl furfural, which further react with anthrone to yield a bluish green color complex that has an absorbance maximum at 620 nm (Scott and Melvin 1953).

It is used to measure the quantity of reducing sugars produced by the saccharification process and measured at 540 nm, OD value by making a standard calibration curve. Higher OD values, the quantity of reducing sugars produced were also high.

Carbohydrate conversion efficiency (CCE): (Amount of substrate utilized)/ (amount of substrate supplied) × 100

Specific hydrogen production rate (SHPR): (amount of hydrogen production (ml))/ (mass of substrate used × time duration)

Results and Discussion

Effect of age of inoculums on bio-hydrogen production

The anaerobic, mesophilic, pure culture of Klebsiella oxytoca ATCC 13182 is hydrogen producing bacterial strain. The age and volume of inoculums was optimized for best result.  The Klebsiella oxytoca ATCC 13182 was incubated for different time duration (18h, 20h and 24h) for optimization of age of inoculums. Inoculums of 20h olds culture gave maximum hydrogen production of 33.67±8.17 ml, SHPR was 0.35±0.08 ml/g/l and CCE was observed 62.04±1.07 % with decrease in pH after fermentation from 6.0 to 5.36. 24h old culture gave lowest production of 16.67±4.4 ml with SHPR 0.17±0.05 ml/g/l, CCE 67.67±2.6 and pH 5.6 (Fig. 2 and 3). Similarly, Muanruksha et al. (2016) also produce bio-hydrogen and estimate age of inoculums and volume of inoculums respectively. 

Figure 2: Effect of age of inoculums on bio-hydrogen production in for different time duration.

Figure 3: SHPR after different age of inoculums on bio-hydrogen production.

Effect of volume of inoculums on bio-hydrogen production

Optimized 20 hours old culture was taken for fermentation and the volume of inoculums was optimized with 10%, 20%, 30% and 40% inoculums. 30% inoculums (60 ml inoculums) showing maximum bio-hydrogen production 31.5±3.5 ml with SHPR 0.45±0.00 ml/g/l having CCE 61.1±2.8% and pH after fermentation is 5.6. 10% inoculums volume (20ml inoculums) gave the minimum bio-hydrogen production 15±1.73 ml with SHPR 0.45±0.00 ml/g/l having CCE 61.1±2.8% and pH after fermentation decreases to 5.7 (Fig.4 and 5).

Figure 4: Effect of volume of inoculums on bio-hydrogen production.

Figure 5: SHRP of effect of inoculums volume on bio-hydrogen.

Figure 6: Effect of concentrated acid (H₂SO₄) on bio-hydrogen production.

Effect of acid pretreatment (H₂SO₄) on bio-hydrogen production

Water hyacinth is a lignocellulosic feedstock which contains cellulose, hemicelluloses and small amount of lignin. Lignin is a cementing agent which acts as polymer around hemicelluloses micro fibrils and cellulose molecules making together and protecting from chemical degradation. For penetration of lignin which cannot be converted into sugars acid pretreatment was required. At different concentrations (0.5%, 1.0%, 1.5% and 2%) of sulfuric acid (H₂SO₄) was used for pretreatment method. 1% H₂SO₄ pretreated substrate maximum bio-hydrogen production of 125.0±5.0ml with SHPR 1.30±0.64ml/g/l and CCE 88.11±0.86% and pH after fermentation was observed 4.4. H₂SO₄ 2% pretreated feedstock gave minimum production of 75.0±5.0 ml with SHPR 0.78±0.12 ml/g/l and CCE 80.81.7±0.7% and pH after fermentation pH slightly decreases from 6 to 5.25 (Fig. 6 and 7).

Figure 7: SHPR of effect of conc. H₂SO₄ on bio-hydrogen production.

H₂SO₄ was selected because dilute H₂SO₄ can effectively converted hemicellulose to xylose rather than other acid (Mosier et al. 2005). Khan et al. (2015), 1.5% H₂SO₄ pre-treated de-oiled rice bran hydrosylate gave maximum of 97.5±2.5 ml bio-hydrogen production.

Conclusion

Biological hydrogen production from water hyacinth is much suitable feedstock for bio-hydrogen production. The decomposition rate of water hyacinth with the hydrogen production rate at first increase then decrease with time. The pH of the sludge was decreases after fermentation and CCE increases as duration of fermentation increases. The maximum hydrogen production is 125.0±5.0 ml with SHPR 1.30±0.64 ml/g/l and CCE 88.11±0.86% and pH after fermentation 4.4 was observed. Among all processes, anaerobic dark fermentation is less expensive and produces hydrogen at faster rate.

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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