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Improvement in biohythane production using organic solid waste and distillery effluent
Waste Management xxx (2017) xxx–xxx
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Improvement in biohythane production using organic solid waste and distillery effluent Preeti Mishra, G. Balachandar, Debabrata Das ⇑ Department of Biotechnology, Indian Institute of Technology, Kharagpur, West Bengal, India
a r t i c l e
i n f o
Article history: Received 19 January 2017 Revised 11 April 2017 Accepted 21 April 2017 Available online xxxx Keywords: Biohythane Distillery effluent Groundnut deoiled cake Mustard deoiled cake Distillers’ dried grain with solubles Algal biomass
a b s t r a c t Biohythane is a two-stage anaerobic fermentation process consisting of biohydrogen production followed by biomethanation. This serves as an environment friendly and economically sustainable approach for the improved valorization of organic wastes. The characteristics of organic wastes depend on their respective sources. The choice of an appropriate combination of complementary organic wastes can vastly improve the bioenergy generation besides achieving the significant cost reduction. The present study assess the suitability and economic viability of using the groundnut deoiled cake (GDOC), mustard deoiled cake (MDOC), distillers’ dried grain with solubles (DDGS) and algal biomass (AB) as a co-substrate for the biohythane process. Results showed that maximum gaseous energy of 23.93, 16.63, 23.44 and 16.21 kcal/L were produced using GDOC, MDOC, DDGS and AB in the two stage biohythane production, respectively. Both GDOC and DDGS were found to be better co-substrates as compared to MDOC and AB. The maximum cumulative hydrogen and methane production of 150 and 64 mmol/L were achieved using GDOC. 98% reduction in substrate input cost (SIC) was achieved using the co-supplementation procedure. Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction The burning issue of fossil energy depletion and severe environmental problems viz., global warming and greenhouse gas effect has forced the world’s scientific community to develop different ways to harness the bioenergy from diversified sources (Ellabban et al., 2014; Ghimire et al., 2015). Hydrogen and methane are the two main gaseous energy carriers which are widely used in the chemical industry. Hydrogen is regarded as the cleanest fuel with zero carbon emission and highest energy content (143 kJ/g) (Das and Veziroglu, 2001; Sharma and Ghoshal, 2015). Methane falls after hydrogen with an energy content 55 kJ/g. It is widely used as transportation fuel in the form of liquefied natural gas because of its higher calorific value and lower CO2 emissions as compared
Abbreviations: A/B ratio, acetate to butyrate ratio; AB, algal biomass; AMC, acidogenic mixed consortium; APHA, American Public Health Association; BOD, biological oxygen demand; CPCB, central pollution control board; COD,, chemical oxygen demand; DDGS, distillers’ dried grain with solubles; DE, distillery effluent; GC, gas chromatograph; GDOC, groundnut deoiled cake; FID, flame ionization detector; MDOC, mustard deoiled cake; MMC, methanogenic mixed consortium; VFA, volatile fatty acid; TCD, thermal conductivity detector. ⇑ Corresponding author. E-mail address: [email protected] (D. Das).
to oil and coal. In addition, unlike other fuels, methane combustion does not produce any NOx (nitrous oxide) and SOx (sulphur dioxide) which are the major contributors for air pollution (Gaffney and Marley, 2009). So, both hydrogen and methane have independently attracted broad commercial interest and are highly valued. Recently, hythane, a mixture of hydrogen (10–25% by volume) and methane, is garnering growing attention due to its versatile advantages. It acts as an ideal transition fuel for the change to hydrogen because it provides significant reduction in the NOx emission as compared to the natural gas. Unlike hydrogen, it is relatively inexpensive and easy to store (Fulton et al., 2010; Liu et al., 2013). The conversion of waste into hythane can mitigate the dual problem of environmental pollution and energy crisis. Chemical production of hythane requires costly substrate, is an energy intensive process, and releases hazardous end products which are detrimental to the environment. On the contrary, use of industrial waste for the biochemical conversion into hythane has a potential to use these end products as a substrate for subsequent methanogenesis (Arizzi et al., 2016; Kothari et al., 2012). Dark fermentation is the most efficient biological process for commercial hydrogen production. However, it produces metabolic end-products such as acetate, butyrate, propionate, ethanol and lactic acid which remain unused (Das and Veziroglu, 2001). Therefore, gaseous energy
http://dx.doi.org/10.1016/j.wasman.2017.04.040 0956-053X/Ó 2017 Elsevier Ltd. All rights reserved.
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recovery can be increased up to 65–70% by using the spent media for subsequent methane production (Schievano et al., 2014). Different organic wastes viz., potato wastes (Zhu et al., 2008), ethanol stillage (Luo et al., 2011), food waste (Lee et al., 2010), wheat straw hydrolysate (Kongjan et al., 2011) and garbage slurry and paper waste (Uneo et al., 2007) have been used for the two stage biohythane production in a Mesophilic conditions. Khongkliang et al. (2015) have reported a high hydrogen and methane yield of 81.5 and 310.5 L/kg COD, respectively using thermophilic consortium. Among different waste substrates, organic wastewater including distillery effluent has high organic load which is detrimental for the environment (Lin et al., 2012). According to Central Pollution Control Board (CPCB, 2010), India, distilleries are ranked among the top 17 heavily polluting industries of the country. Annually, 40 billion litres of effluent (spent wash) is disposed from distilleries. The distillery wastewater, owing to its huge organic load, is an ideal substrate for the biohydrogen production. It comprises of high COD (60,000–120,000 mg/L) and BOD (45,000– 60,000 mg/L) (Pant et al., 2007; Krishnamoorthy et al., 2017). It contains very high amounts of potassium, calcium, chloride and sulphate. However, the distillery effluent lacks most of the essential micro nutrients such as Fe, Mg, P, Cu, and Zn (Mohana et al., 2009; Basu, 1975). They serve as cofactors of the different enzymes involved in the fermentation pathway. Therefore, the supplementation of such micronutrients is essential for the improvement of the biohydrogen yield (Karadag et al., 2010; Mishra and Das, 2014). Reports show that the supplementation of pure nitrogen and mineral salts to the distillery effluent results in the increased cost of the biohydrogen production process (Da Silva et al., 2014; Saharan et al., 2011). This problem can be solved by supplementing inexpensive nutrient sources. Distillers’ grain with solubles (DDGS) is a rich source of proteins and minerals (Li et al., 2002). DDGS is made by blending distiller’s dried grains (DDG) with solubles. The water and solids remaining after distillation of ethanol are called whole stillage. Whole stillage is comprised primarily of water, fiber, protein, and fat. This mixture is centrifuged to separate coarse solids from liquid. The liquid, called thin stillage, goes through an evaporator to remove additional moisture resulting in condensed distiller’s solubles (syrup) which contains approximately 20–40% (w/w) proteins and 30% dry matter. The coarse solids are called distiller’s dried grains (wet cake) and contain 35% (w/v) dry matter and 60% (w/w) protein (Ratanapariyanuch et al., 2016). The sources of DDGS are mainly wheat, maize and corn. DDGS has been used for different purposes viz., specific protein extraction (Gupta et al., 2016; Chatzifragkou et al., 2016), production of liquid hydrocarbons (Taneeru and Steele, 2015), bio-oil production (Lei et al., 2011), chemical hydrogen production (Tavasoli et al., 2009) and photo fermentative hydrogen production (Sargsyan et al., 2016). However, the use of DDGS as a supplement for fermentative biohydrogen production has not been explored. DDGS, DOC are rich source of nitrogen and minerals and used as another co-substrate owing to its high production rate of 253 million tons oil seeds per year (Barnwal and Sharma, 2005). After extraction of the oil, the de-oiled cakes can be used for the biohydrogen production. DOC has been used for bioenergy generation in the form of bio oil (Volli and Singh, 2012), biodiesel (SánchezArreola et al., 2015), biogas (Barik and Murugan, 2015) and biohydrogen (Kumar et al., 2015) production. Further, algal biomass was considered as another co-substrate for biohythane production because of its high protein and mineral content. Besides being rich nutrient source, it has several other advantages viz., higher productivities, use of non-arable land and harvest solar energy. The low lignin content (<2%) of algae like Coleochaete and some of the filamentous fresh water algal species surpasses the energy intensive de-lignification step enabling faster
substrate utilization during fermentation (Ververis et al., 2007; Montingelli et al., 2015; Maurya et al., 2016). Excess algal growth (algal bloom) in natural water bodies causes eutrophication which leads to the death of the aquatic plants and animals. Cleaning of algal bloom needs additional man power and energy. Utilization of such wastewater grown algal biomass for biohythane production can save energy and cost of the process (Chiu et al., 2015). These substrates have been used as major or sole nutrient source for biohydrogen production. The study on the effect of fortifying such solid organic wastes (rich in nitrogen and minerals) with a liquid waste containing high carbohydrate load such as the distillery effluent has not yet been fully investigated. In the present study, algal biomass (AB), distillers dried grains with solubles (DDGS), mustard deoiled cake (MDOC) and groundnut deoiled cake (GDOC) as a co-substrate were considered for the improvement of the biohythane production. Emphasis has been laid in reducing the substrate cost besides achieving waste remediation in a two stage biohythane production process.
2. Materials and methods 2.1. Microorganism and culture conditions 2.1.1. Cultivation of acidogenic mixed consortium Acidogenic mixed consortium (AMC) was developed from the anaerobic sludge collected from IFB Agro Industries Ltd., Kolkata (Mishra et al., 2015). The AMC was maintained in a broth media containing glucose (1% w/v), tryptone (1% w/v) and trace amount of FeSO4 at 37 °C and pH 6.5 with regular subculturing. 2.1.2. Cultivation of methanogenic mixed consortium Methanogenic mixed consortium (MMC) was developed from anaerobic sludge collected from IFB Agro Industries Ltd., Kolkata. It was used as an inoculum for the second stage methane production. The consortium was maintained in the media (per Litre) containing acetic acid 2.0 g; butyric acid 1.0 g; NH4Cl 0.84 g; K2HPO4 0.234 g; KH2PO4 0.136 g; NaCl 0.6 g; MgCl26H2O 0.084 g; CaCl22H2O 0.006 g; FeCl3 0.05 g; Na2SO4 0.5 g; trace elements solution 10 mL (Per Litre: nitrilotriacetic acid 1.5 g; MgCl26H2O 2.476 g; Mn(CH3COO)24H2O 0.655 g; NaCl 1.0 g;CoCl26H2O 0.106 g; CaCl22H2O 0.10 g; ZnCl2 0.18 g; CuCl22H2O 0.007 g; AlCl3 0.0056 g; H3BO3 0.01 g; Na2MoO42H2O 0.01 mg; NiCl26H2O 0.025 mg; Na2SeO35H2O 0.30 mg); and vitamin solution 10 mL (Per Litre: folic acid 2.0 mg; pyridoxine - HCl 10.0 mg; riboflavin 5.0 mg; nicotinic acid 5.0 mg; D-Ca-pantothenate 5.0 mg; vitamin B12 0.10 mg; paminobenzoic acid 5.0 mg). The consortium was maintained in its active form by sub-culturing at 35 °C and initial pH of 7.8 after every 15 d of interval. 2.2. Collection and characterization of different organic solid wastes/ wastewater Rice grain based distillery effluent, collected from IFB agro, West Bengal, India, was used in the study. The distillery effluent was characterized in our previous work (Mishra and Das, 2014). The AB was collected from a fresh water pond, Jakpur, West Bengal, India in the month of March. Leaves and other such materials were manually separated and the residual clay/soil particles were removed by washing. The powdered algal biomass was used as a co-substrate for the hydrogen production. DDGS was collected from IFB agro, West Bengal. The GDOC and MDOC were collected from a local market in Kharagpur, West Bengal, India. The physico-chemical characteristics were determined using standard protocols APHA (1995) (Table 1).
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P. Mishra et al. / Waste Management xxx (2017) xxx–xxx Table 1 Characterization of different organic solid wastes. Parameters
DDGS
GDOC
MDOC
AB
Crude protein Total carbohydrate Reducing sugar COD TOC Ammonia Phosphate C H O N S C/N molar ratio C/H molar ratio Empirical formula
42.60 ± 0.13 23.30 ± 1.9 17.50 ± 0.09 27.70 ± 2.2 57.1 ± 1.1 0.39 ± 0.02 0.60 ± 0.01 44.36 6.85 41.63 6.89 0.27 7.51 0.54 C7.5H13.81O5.29NS0.02
45.0 ± 0.40 20.00 ± 0.06 7.20 ± 0.01 30.0 ± 0.90 61.0 ± 0.84 0.45 ± 0.05 0.25 ± 0.05 45.39 6.93 40.51 6.00 1.17 8.82 0.55 C8.82H16.05O5.91NS0.08
36.00 ± 0.21 13.00 ± 0.03 4.00 ± 0.02 22.0 ± 1.20 50.0 ± 0.30 0.32 ± 0.03 0.15 ± 0.01 45.37 2.33 44.66 6.73 0.91 7.86 1.63 C7.86H4.81O5.81NS0.06
21.2 ± 1.30 23.9 ± 1.0 5.4 ± 0.59 28.5 ± 0.35 47.1 ± 0.45 – – 38.50 6.20 50.67 4.07 0.56 11.03 0.52 C11.03H21.17O10.89NS0.06
*All value are given in % w/w except molar values and empirical formula.
2.3. Experimental conditions 2.3.1. First stage: Biohydrogen production 10% (v/v) overnight grown AMC was used as an inoculum throughout the study. Each of the chosen co-substrates was used as sole substrate and their individual hydrogen production potentials were studied. The experiments were conducted in a 100 mL serum bottle with a working volume of 80 mL. The pH of the media was adjusted prior to the experiment. Media was maintained at the initial pH of 7.0 and then sparged with nitrogen to maintain anaerobic environment. Media was inoculated with AMC anaerobically. Experiments were conducted at 37 °C for 24 h at 180 rpm. After 24 h, total gas produced in the serum bottle was allowed to pass through 40% (w/v) KOH solution for the selective absorption of CO2 (Khanna et al., 2011). The remaining gas was collected by liquid displacement method in a gas collector filled with 10% (w/v) saline and 0.1 M K2Cr2O7 at normal temperature and atmospheric pressure. All experimental runs were performed in triplicates. Further, suitability of these organic wastes as a co-substrate with the distillery effluent was analysed. Concentration of the cosubstrates was varied from 5–40 g/L to determine the optimum value. The setup was monitored for the hydrogen production up to 24 h. A scale up study was done in a controlled fermenter (New Brunswick Scientific, NJ, USA) to study the hydrogen production and substrate degradation profiles at the optimum concentration of the co-substrates. All experiments were performed in a batch mode with a working volume of 2 L. Experiments were repeated for three times with all co-substrates.
column packed with Porapak Q (80/100) and a thermal conductivity detector (TCD). The operational temperatures of the injection port, the oven and the detector were 80 °C, 150 °C and 200 °C, respectively. Nitrogen was used as the carrier gas with a flow rate of 20 mL/min. The flame ionization detector (FID) was used for the estimation of the volatile fatty acids and ethanol. The FID operational temperatures of the injection port, the detector, and programmed column were 220 °C, 240 °C, and 130–175 °C, respectively. For the flame generation, a mixture of hydrogen and air was used at a flow rate of 30 mL/min (Khanna et al., 2011). Total carbohydrate content was estimated spectrophotometrically (UV–VIS Spectrophotometer Perkin–Elmer, k = 490 nm) using the phenol–sulphuric acid assay (Ginkel et al., 2001). The COD was estimated based on the APHA standard protocols using a COD measurement instrument (DRB200 & DR2800 Portable Spectrophotometer HACHÒ, USA). The pH was monitored using a desktop pH meter (pH510, Cyberscan, Singapore). The biomass was measured at 600 nm using UV/Vis spectrophotometer (Lambda 25, PerkinElmer LLC, MA, USA) and confirmed using dry cell weights. Standard protocol for ammonia estimation was done according to APHA (1995). Concentration of metal ions was measured by using an atomic absorption spectrometer (Perkin–Elmer). The elemental composition of the biomass was analysed using CHNS analyzer (Vario MACRO cube CHNS, Elementar, USA). The calorific value of the samples were determined by using an Automatic Oxygen Bomb Calorimeter (6100, Parr Instruments, USA).
3. Results and discussion 2.3.2. Second stage: Biomethane production Spent media after the hydrogen production was centrifuged (10,000 rpm for 10 min). The supernatant was collected and pH was maintained at 7.8 by using 2 M NaOH. The media was sparged with nitrogen gas to ensure an anaerobic condition and cysteine HCl (1 g/L) was added as a reducing agent to maintain the anaerobic state of the media. Methanogenic mixed consortium (absorbance 0.8–1.0) was added to the media in a ratio of 3:1 (media: inoculum). The process was monitored up to 12 d. The gas sample collected was allowed to pass through 40% (w/v) KOH solution for the selective absorption of CO2. 2.4. Analytical methods The hydrogen produced during batch fermentation was analysed by the gas chromatograph (GC Agilent Technology 7890A, U.S.A). The gas chromatograph was equipped with a stainless steel
3.1. Suitability of different wastes for hydrogen production Different substrates viz., GDOC, MDOC, DDGS and AB were used in a range of 0–100 g/L to study the suitable substrate concentration for biohydrogen production. GDOC showed highest cumulative hydrogen production followed by DDGS. Hydrogen production was observed to be decreasing beyond 50, 60 and 90 g/L for GDOC, DDGS and MDOC, respectively. For algal biomass no such effect was observed up to 100 g/L. This could be attributed to the fact that AMC used in this study produced maximum hydrogen at an optimum glucose concentration of 10 g/L and an inhibitory effect on the hydrogen production was observed beyond 18 g/L (Mishra and Das, 2015). Comparatively lower carbohydrate content was found in MDOC (Table 1) as compared to GDOC and DDGS. So, maximum hydrogen production was obtained at 90 g/L which is equivalent to 11.7 g carbohydrate/L (Fig. 1). The reducing
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Fig. 1. Cumulative hydrogen production using groundnut deoiled cake (GDOC), mustard deoiled cake (MDOC), Distillers’ dried grains with solubles (DDGS), and algal biomass (AB).
sugar content in the algal biomass was very low (5.4 ± 0.59% w/w), therefore, no substrate inhibition was observed even beyond 100 g/L. Highest cumulative hydrogen production of 99.8 ± 2.56 and 79.5 ± 1.62 mmol/L were obtained using GDOC and DDGS which make them a promising substrate for hydrogen production. 3.2. Suitability as a co-substrate with distillery effluent for hydrogen production Rice grain based distillery effluent was used in this study which has a very high organic load (Total COD: 59.0 ± 2.0 g/L; soluble COD: 38.0 ± 3.0 g/L). However, it contains very less amount of micronutrients (Supplementary Table 1). Therefore, distillery effluent was used mainly as a carbon source and it was supplemented with different organic solid wastes mainly as a mineral supplements. As the GDOC and DDGS showed inhibitory effect beyond 50 and 60 g/L, a co-substrate concentration was kept below 50 g/L for this study. The distillery effluent was supplemented with varied concentration (0–40 g/L) of GDOC, MDOC, DDGS and AB. A maximum cumulative hydrogen production of 128.0 ± 3.8 and 123.7 ± 4.4 mmol/L and COD reduction of 38.6 ± 0.24% and 37.3 ± 0.87% were achieved at 20 and 25 g/L of GDOC and DDGS, respectively. Further increase in their concentrations resulted in a slower production rate and reduced hydrogen productivity. MDOC showed cumulative hydrogen production and COD reduction of 95 ± 1.02 mmol/L and 33.2 ± 1.26%, respectively at 35 g/L. Further increase in the biomass concentration showed inhibitory effect on the hydrogen production process. However, in case of AB, an increase in the cumulative hydrogen production was observed with increasing substrate concentration. Supplementation of DDGS and GDOC has shown 2.15 and 2.23 folds increase in the cumulative hydrogen production as compared to the nonsupplemented distillery effluent (Fig. 2). Hydrogen production and substrate reduction profile was studied at the optimum concentration of GDOC (20.0 g/L), MDOC (35.0 g/L), DDGS (25.0 g/L) and AB (40.0 g/L) at a scaled up volume of 2 L (Fig. 3). A maximum cumulative hydrogen production was obtained using GDOC (150.7 ± 0.9 mmol/L) which was found 38.1% and 29.9% higher as compared to the MDOC (109.1 ± 4.4 mmol/L) and AB (116.1 ± 4.2 mmol/L), respectively. Hydrogen production profile using DDGS was equivalent to that of GDOC. Maximum COD reduction of 49.3 ± 1.2, 41.7 ± 0.9 and 48.5 ± 0.8, 38.1 ± 1.1% were obtained with GDOC, MDOC, DDGS
Fig. 2. (a) Cumulative hydrogen production and (b) COD reduction using raw DE supplemented with different concentrations of groundnut deoiled cake (GDOC), mustard deoiled cake (MDOC), Distillers’ dried grains with solubles (DDGS), and algal biomass (AB).
and AB, respectively. Hydrogen yield of 5.29 mol H2/ kg CODreduced was obtained using 20 g/L of GDOC with DE. Specific hydrogen yield of 11.36, 11.09, 8.87 and 5.54 mol H2/g VSS was obtained using GDOC, DDGS, MDOC and AB, respectively. The consortium used in this study has a microbial population dominated with Clostridium spp. which is reported to contain FeFe hydrogenase, a key enzyme for hydrogen production (Sinha et al., 2016; Mishra et al., 2015). Elemental analysis of these substrates showed higher Fe content in GDOC and DDGS as compared to MDOC and AB (Table 2). DDGS and GDOC are also rich in Mg which acts as a co-factor for several glycolytic enzymes and chelates with adenine nucleotides for cellular phosphate transfer reactions (Walker, 1994; Fujio and Furuya, 1985). Higher Fe and Mg content inGDOC and DDGS could be one major cause for the higher hydrogen production as compared to MDOC and AB. Distillery effluent supplemented with the AB showed lower cumulative hydrogen and specific hydrogen yield as compared to other cosubstrates. This could be due to the lower nitrogen and protein content in the AB (Table 1). Microscopic study of the wastewater grown algal consortium showed spiral arrangement of chloroplast which indicates presence of Spirogyra spp. (Fig. 4). Spirogyra is reported to contain lower nitrogen and protein content compared to the other algal species viz., Chlamydomonas, Anabaena, Microcystis, etc. (Raheem et al., 2015). The volatile fatty acid analysis (Fig. 5) showed acetate to butyrate (A/B) ratio of 0.68, 0.65, 0.52 and 0.48 in the spent media of
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Fig. 3. Hydrogen production and COD profiles by AMC using raw DE supplemented with (a) Groundnut deoiled cake (GDOC), (b) mustard deoiled cake (MDOC), (c) Distillers’ dried grains with solubles (DDGS, and (d) algal biomass (AB) at optimum substrate concentration.
Table 2 Elemental analysis of metal ions present in different organic solid wastes. Micro-elements
GDOC
MDOC
DDGS
AB
Mg Fe Mn Cu K Ni Zn
262.00 167.10 8.40 4.50 192.30 0.80 6.7
268 74.28 7.9 3.5 192.1 NA 8.5
393.70 135.60 11.90 2.80 96.20 – 7.6
186.80 75.40 42.10 6.00 191.90 – 10.8
process whose end product is alcohol. Although, several studies have reported the improvement of hydrogen production by using different pre-treatment methods including acid, alkali, sonication and thermal pre-treatment, however, it increases the cost of the process (Dong et al., 2016). To make the process cost effective, substrates were used without pre-treatment in the present study. Effect of pretreated co-substrates on the biohydrogen production can be considered for the future study. 3.3. Biomethanation using spent media
*All values are given in mg/100 g.
GDOC, DDGS, MDOC and AB, respectively. 1 mol of glucose produces 4 mol of hydrogen when it follows acetate production pathway. However, only 2 mol of hydrogen is produced from 1 mol glucose in the butyrate production pathway. Therefore, higher acetate to butyrate ratio corresponds to higher hydrogen production in the mesophilic hydrogen producers (Turon et al., 2015). Higher acetate to butyrate (A/B) ratio in the spent media of GDOC and DDGS also supports the fact that higher hydrogen was produced in the former as compared to MDOC and AB. A higher ethanol concentration (>1600 ppm) was observed in the spent media of all the co-substrates. This could be attributed to the distillation
A cumulative methane production of 64.1 ± 0.78, 43.1 ± 0.47, 63.8 ± 2.04 and 38.9 ± 1.42 mmol/L were obtained using spent media of GDOC, MDOC, DDGS and AB, respectively (Fig. 6). An ammonia concentration of 0.08–0.10 g NH3-N/L was found in the spent media used for methanogenesis which is below to the reported inhibitory range (Schnürer and Nordberg, 2008; Prochazka et al., 2012). This also supports the fact that spent media can be an ideal substrate for biomethanogenesis. The methanogenic consortium used in this study was enriched and maintained in the acetate containing media for more than 6–8 months which might have resulted in the dominance of acetoclastic methanogens in the consortium. This could be one reason for higher acetate (>90%) and butyrate (>85%) conversion as compared to ethanol (50%) as shown in Fig. 5.
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Fig. 4. Microscopic image of collected algal biomass: (a) 10 magnification and (b) 40 magnification.
Hydrogen and methane yields are compared with the published reports on two stage biohythane production using wastes (Table 5). Hydrogen yield is found higher as compared to the reported literature (Luo et al., 2011; Kisielewska et al., 2014). The current study was done in a mesophilic condition, which is lesser energy intensive and easy to operate, but comparatively higher hythane yield can be obtained by using a thermophilic consortium (Kongjan and Angelidaki, 2013). Although, hydrogen yield was found higher to most of the reported work, however methane yield was found comparatively lower (Khongkliang et al., 2015; Cheng et al., 2016). This could be due to the use of acidogenic mixed consortium (AMC) acclimatized with the distillery effluent (Mishra et al., 2015). So, a similar acclimatization study for the methanogenic consortium could be helpful for the improvement of methane yield. Future study can also focus on the improvement of the ethanol conversion efficiency.
3.4. Cost and energy analysis Fig. 5. End metabolites concentration in the spent media of dark fermentative hydrogen production.
Fig. 6. Cumulative methane production and substrate reduction using spent of dark fermentation.
Replacement of tryptone with cheaper organic solid waste biomass resulted in a drastic reduction of substrate input cost (SIC) (Table 3). The cumulative Hydrogen and methane production using distillery effluent supplemented with 10 g/L tryptone was156.0 and 69.64 mmol/L, which was comparable to both GDOC (150.7 and 64.1 mmol/L) and DDGS (144.2 and 63.8 mmol/L). However, the SIC for GDOC and DDGS were reduced to 98.3 and 97.9%, respectively as compared to controlled distillery effluent supplemented with tryptone. In case of MDOC, the cost reduction was 97.7% with cumulative hydrogen and methane production of 109.1 and 43.13 mmol/L, respectively. AB was collected from a local pond and dried under sunlight. Therefore, apparently zero input cost can be considered for AB. Apart from zero input cost, utilization of wastewater grown algal biomass also helps in bioremediation by minimizing eutrophication. Hence, it can infer that utilization of such alternative solid waste products as a cosubstrate for the hydrogen production can result in a drastic reduction in the substrate cost. Total gaseous energy of 23.93 and 23.44 kcal/L were produced using distillery effluent supplemented with GDOC and DDGS, respectively (Table 4) which was 2.6 times higher compared to the control (9.08 kcal/L). GDOC and DDGS showed 54.6 and 51.4% higher gaseous energy production as compared to tryptone (15.48 kcal/L) with significant reduction in the substrate cost reduction. This makes GDOC and DDGS as a potential cosubstrate with distillery effluent for biohythane production.
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P. Mishra et al. / Waste Management xxx (2017) xxx–xxx Table 3 Substrate input cost (SIC) analysis of the process.
a b c d
Substrate
Cost (INR/MT)d
Substrates used (g/L of DE)
Cost of substrate used/L (Rs)
H2 produced (mmol/L)
Cost reduction compared to tryptone as supplement. (%)
GDOC MDOC DDGS AB Tryptone
25,500a 20,000a 25,000b 0 3,000,000c
20.0 35.0 25.0 40.0 10.0
0.51 0.70 0.62 0.00 30.00
150 109 144 116 156
98.3 97.7 97.9 100.0
http://www.agriwatch.com. http://dir.indiamart.com/impcat/rice-ddgs.html. Sisco Research Laboratories Pvt. Ltd. (SRL). INR: Indian Rupees.
Table 4 Hydrogen and methane production using co-substrates with distillery effluent. Substrate
Control DE
GDOC MDOC DDGS AB Tryptone
Total gas produced (mmol/L)
Gaseous energy produced (kcal/L)
H2
CH4
H2
CH4
Total
55.13 150.70 109.10 144.20 116.10 156.00
24.95 64.06 43.13 63.84 38.88 69.64
3.77 10.31 7.46 9.86 7.94 10.67
5.30 13.62 9.17 13.57 8.27 14.81
9.08 23.93 16.63 23.44 16.21 15.48
Table 5 Comparative studies on the two stage hydrogen and methane production using different organic wastes. Substrate
H2 production phase
CH4 production phase
Reference
Inoculum
Conditions
Yield
Inoculum
Conditions
Yield
Garbage and paper wastes Potato waste OFMSW Cheese whey Food waste Water hyacinth leave Organic biowaste Ethanol stillage Desugared molasses Food waste Whey permeate POME Starchy wastewater Food waste with sewage sludge
ASC AS AS IM AS AS AS ADM TC IM AS AS TC AS
CSTR, C CSTR, C CSTR, C CSTR, C CSTR, SC SB, B CSTR, SC CSTR, C UASB, C CSTR, C UASB, C ASBR, C CSTR, B CSTR,B
282 L/kg COD 30 L/kg TS 205 L/kg VS 92 L/kg COD 270 L/kg COD 51.7 L/kg VS 52 L/kg VS 48 L/kg VS 132 L/kg VS 60 L/kg VS 93.8 L/kg COD 210 L/kg COD 81.5 L/kg COD 174.6 L/kg VS
AS AS AS AS AS AS AS ADM TC AS AS AS TC AS
PBR CSTR, C FBR, C CSTR, C A* SB, B CSTR, SC CSTR, C UASB, C CSTR, C UASB, C UASB, C CSTR, B CSTR, B
389 L/kg COD 183 L/kg TS 464 L/kg VS 136 L/kg COD 287 L/kg COD 143.4 L/kg VS 410 L/kg VS 344 L/kg VS 239 L/kg VS – 120 L/kg COD 315 L/kg COD 310 L/kg COD 264.1 L/kg VS
Ueno et al. (2007) Zhu et al. (2008) Chu et al. (2008) Venetsaneas et al. (2009) Lee et al. (2010) Cheng et al. (2010) Cavinato et al. (2011) Luo et al. (2011) Kongjan and Angelidaki (2013) Micolucci et al. (2014) Kisielewska et al. (2014) Mamimin et al. (2015) Khongkliang et al. (2015) Cheng et al. (2016)
DE with GDOC
AMC
CSTR, B
118.5 L/kg COD
MMC
CSTR, B
158.1 L/kg COD
Present study
A*, Biogas sparging type reactor; PBR, packed-bed reactor; FBR, Fluid bed reactor; CSTR, Continuously stirred tank reactor; C, Continuous mode of operation; B, Batch mode of operation; SC, Semi continuous mode of operation; ASBR, Anaerobic sequencing batch reactor; AS, Anaerobic digester sludge; ADM, Anaerobic digested manure; ASC, Activated sludge compost; TC, Thermophilic consortium; COD, Chemical oxygen demand; IM, Indigenous microflora; OFMSW, Organic fraction of municipal solid waste; POME, Palm oil mill effluent; TS, Total solid; VS, Volatile solid, VSS, Volatile suspended solids; DE, Distillery effluent; DDGS, Distillers’ dried grains with soluble.
4. Conclusion
Acknowledgement
Suitability of GDOC, MDOC, DDGS and AB as a supplement to the distillery effluent for biohythane production was explored. GDOC was found as the most suitable co-substrate with the distillery effluent followed by DDGS. GDOC and DDGS showed 2.73 and 2.61-fold improvement in the cumulative hydrogen production and 2.57 and 2.56-fold improvement in the cumulative methane production as compared to the non-supplemented distillery effluent (control). A significant reduction in the substrate input cost (SIC) can be attained by replacing tryptone with cheaper organic co-substrates like wastewater grown algal biomass with comparable hydrogen and methane productionbesides decreasing organic load on the environment. DGGE analysis of AMC consortia is in progress to find out the hydrogen producing microorganisms.
The authors gratefully acknowledge Ministry of New and Renewable Energy (MNRE) and Department of Biotechnology (DBT), Government of India for providing the financial support. Authors are also thankful to the IIT Kharagpur for providing the research facilities and IFB agro, Kolkata for providing distillery effluent.
Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.wasman.2017.04. 040.
Please cite this article in press as: Mishra, P., et al. Improvement in biohythane production using organic solid waste and distillery effluent. Waste Management (2017), http://dx.doi.org/10.1016/j.wasman.2017.04.040
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Improvement in biohythane production using organic solid waste and distillery effluent Preeti Mishra, G. Balachandar, Debabrata Das ⇑ Department of Biotechnology, Indian Institute of Technology, Kharagpur, West Bengal, India
a r t i c l e
i n f o
Article history: Received 19 January 2017 Revised 11 April 2017 Accepted 21 April 2017 Available online xxxx Keywords: Biohythane Distillery effluent Groundnut deoiled cake Mustard deoiled cake Distillers’ dried grain with solubles Algal biomass
a b s t r a c t Biohythane is a two-stage anaerobic fermentation process consisting of biohydrogen production followed by biomethanation. This serves as an environment friendly and economically sustainable approach for the improved valorization of organic wastes. The characteristics of organic wastes depend on their respective sources. The choice of an appropriate combination of complementary organic wastes can vastly improve the bioenergy generation besides achieving the significant cost reduction. The present study assess the suitability and economic viability of using the groundnut deoiled cake (GDOC), mustard deoiled cake (MDOC), distillers’ dried grain with solubles (DDGS) and algal biomass (AB) as a co-substrate for the biohythane process. Results showed that maximum gaseous energy of 23.93, 16.63, 23.44 and 16.21 kcal/L were produced using GDOC, MDOC, DDGS and AB in the two stage biohythane production, respectively. Both GDOC and DDGS were found to be better co-substrates as compared to MDOC and AB. The maximum cumulative hydrogen and methane production of 150 and 64 mmol/L were achieved using GDOC. 98% reduction in substrate input cost (SIC) was achieved using the co-supplementation procedure. Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction The burning issue of fossil energy depletion and severe environmental problems viz., global warming and greenhouse gas effect has forced the world’s scientific community to develop different ways to harness the bioenergy from diversified sources (Ellabban et al., 2014; Ghimire et al., 2015). Hydrogen and methane are the two main gaseous energy carriers which are widely used in the chemical industry. Hydrogen is regarded as the cleanest fuel with zero carbon emission and highest energy content (143 kJ/g) (Das and Veziroglu, 2001; Sharma and Ghoshal, 2015). Methane falls after hydrogen with an energy content 55 kJ/g. It is widely used as transportation fuel in the form of liquefied natural gas because of its higher calorific value and lower CO2 emissions as compared
Abbreviations: A/B ratio, acetate to butyrate ratio; AB, algal biomass; AMC, acidogenic mixed consortium; APHA, American Public Health Association; BOD, biological oxygen demand; CPCB, central pollution control board; COD,, chemical oxygen demand; DDGS, distillers’ dried grain with solubles; DE, distillery effluent; GC, gas chromatograph; GDOC, groundnut deoiled cake; FID, flame ionization detector; MDOC, mustard deoiled cake; MMC, methanogenic mixed consortium; VFA, volatile fatty acid; TCD, thermal conductivity detector. ⇑ Corresponding author. E-mail address: [email protected] (D. Das).
to oil and coal. In addition, unlike other fuels, methane combustion does not produce any NOx (nitrous oxide) and SOx (sulphur dioxide) which are the major contributors for air pollution (Gaffney and Marley, 2009). So, both hydrogen and methane have independently attracted broad commercial interest and are highly valued. Recently, hythane, a mixture of hydrogen (10–25% by volume) and methane, is garnering growing attention due to its versatile advantages. It acts as an ideal transition fuel for the change to hydrogen because it provides significant reduction in the NOx emission as compared to the natural gas. Unlike hydrogen, it is relatively inexpensive and easy to store (Fulton et al., 2010; Liu et al., 2013). The conversion of waste into hythane can mitigate the dual problem of environmental pollution and energy crisis. Chemical production of hythane requires costly substrate, is an energy intensive process, and releases hazardous end products which are detrimental to the environment. On the contrary, use of industrial waste for the biochemical conversion into hythane has a potential to use these end products as a substrate for subsequent methanogenesis (Arizzi et al., 2016; Kothari et al., 2012). Dark fermentation is the most efficient biological process for commercial hydrogen production. However, it produces metabolic end-products such as acetate, butyrate, propionate, ethanol and lactic acid which remain unused (Das and Veziroglu, 2001). Therefore, gaseous energy
http://dx.doi.org/10.1016/j.wasman.2017.04.040 0956-053X/Ó 2017 Elsevier Ltd. All rights reserved.
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recovery can be increased up to 65–70% by using the spent media for subsequent methane production (Schievano et al., 2014). Different organic wastes viz., potato wastes (Zhu et al., 2008), ethanol stillage (Luo et al., 2011), food waste (Lee et al., 2010), wheat straw hydrolysate (Kongjan et al., 2011) and garbage slurry and paper waste (Uneo et al., 2007) have been used for the two stage biohythane production in a Mesophilic conditions. Khongkliang et al. (2015) have reported a high hydrogen and methane yield of 81.5 and 310.5 L/kg COD, respectively using thermophilic consortium. Among different waste substrates, organic wastewater including distillery effluent has high organic load which is detrimental for the environment (Lin et al., 2012). According to Central Pollution Control Board (CPCB, 2010), India, distilleries are ranked among the top 17 heavily polluting industries of the country. Annually, 40 billion litres of effluent (spent wash) is disposed from distilleries. The distillery wastewater, owing to its huge organic load, is an ideal substrate for the biohydrogen production. It comprises of high COD (60,000–120,000 mg/L) and BOD (45,000– 60,000 mg/L) (Pant et al., 2007; Krishnamoorthy et al., 2017). It contains very high amounts of potassium, calcium, chloride and sulphate. However, the distillery effluent lacks most of the essential micro nutrients such as Fe, Mg, P, Cu, and Zn (Mohana et al., 2009; Basu, 1975). They serve as cofactors of the different enzymes involved in the fermentation pathway. Therefore, the supplementation of such micronutrients is essential for the improvement of the biohydrogen yield (Karadag et al., 2010; Mishra and Das, 2014). Reports show that the supplementation of pure nitrogen and mineral salts to the distillery effluent results in the increased cost of the biohydrogen production process (Da Silva et al., 2014; Saharan et al., 2011). This problem can be solved by supplementing inexpensive nutrient sources. Distillers’ grain with solubles (DDGS) is a rich source of proteins and minerals (Li et al., 2002). DDGS is made by blending distiller’s dried grains (DDG) with solubles. The water and solids remaining after distillation of ethanol are called whole stillage. Whole stillage is comprised primarily of water, fiber, protein, and fat. This mixture is centrifuged to separate coarse solids from liquid. The liquid, called thin stillage, goes through an evaporator to remove additional moisture resulting in condensed distiller’s solubles (syrup) which contains approximately 20–40% (w/w) proteins and 30% dry matter. The coarse solids are called distiller’s dried grains (wet cake) and contain 35% (w/v) dry matter and 60% (w/w) protein (Ratanapariyanuch et al., 2016). The sources of DDGS are mainly wheat, maize and corn. DDGS has been used for different purposes viz., specific protein extraction (Gupta et al., 2016; Chatzifragkou et al., 2016), production of liquid hydrocarbons (Taneeru and Steele, 2015), bio-oil production (Lei et al., 2011), chemical hydrogen production (Tavasoli et al., 2009) and photo fermentative hydrogen production (Sargsyan et al., 2016). However, the use of DDGS as a supplement for fermentative biohydrogen production has not been explored. DDGS, DOC are rich source of nitrogen and minerals and used as another co-substrate owing to its high production rate of 253 million tons oil seeds per year (Barnwal and Sharma, 2005). After extraction of the oil, the de-oiled cakes can be used for the biohydrogen production. DOC has been used for bioenergy generation in the form of bio oil (Volli and Singh, 2012), biodiesel (SánchezArreola et al., 2015), biogas (Barik and Murugan, 2015) and biohydrogen (Kumar et al., 2015) production. Further, algal biomass was considered as another co-substrate for biohythane production because of its high protein and mineral content. Besides being rich nutrient source, it has several other advantages viz., higher productivities, use of non-arable land and harvest solar energy. The low lignin content (<2%) of algae like Coleochaete and some of the filamentous fresh water algal species surpasses the energy intensive de-lignification step enabling faster
substrate utilization during fermentation (Ververis et al., 2007; Montingelli et al., 2015; Maurya et al., 2016). Excess algal growth (algal bloom) in natural water bodies causes eutrophication which leads to the death of the aquatic plants and animals. Cleaning of algal bloom needs additional man power and energy. Utilization of such wastewater grown algal biomass for biohythane production can save energy and cost of the process (Chiu et al., 2015). These substrates have been used as major or sole nutrient source for biohydrogen production. The study on the effect of fortifying such solid organic wastes (rich in nitrogen and minerals) with a liquid waste containing high carbohydrate load such as the distillery effluent has not yet been fully investigated. In the present study, algal biomass (AB), distillers dried grains with solubles (DDGS), mustard deoiled cake (MDOC) and groundnut deoiled cake (GDOC) as a co-substrate were considered for the improvement of the biohythane production. Emphasis has been laid in reducing the substrate cost besides achieving waste remediation in a two stage biohythane production process.
2. Materials and methods 2.1. Microorganism and culture conditions 2.1.1. Cultivation of acidogenic mixed consortium Acidogenic mixed consortium (AMC) was developed from the anaerobic sludge collected from IFB Agro Industries Ltd., Kolkata (Mishra et al., 2015). The AMC was maintained in a broth media containing glucose (1% w/v), tryptone (1% w/v) and trace amount of FeSO4 at 37 °C and pH 6.5 with regular subculturing. 2.1.2. Cultivation of methanogenic mixed consortium Methanogenic mixed consortium (MMC) was developed from anaerobic sludge collected from IFB Agro Industries Ltd., Kolkata. It was used as an inoculum for the second stage methane production. The consortium was maintained in the media (per Litre) containing acetic acid 2.0 g; butyric acid 1.0 g; NH4Cl 0.84 g; K2HPO4 0.234 g; KH2PO4 0.136 g; NaCl 0.6 g; MgCl26H2O 0.084 g; CaCl22H2O 0.006 g; FeCl3 0.05 g; Na2SO4 0.5 g; trace elements solution 10 mL (Per Litre: nitrilotriacetic acid 1.5 g; MgCl26H2O 2.476 g; Mn(CH3COO)24H2O 0.655 g; NaCl 1.0 g;CoCl26H2O 0.106 g; CaCl22H2O 0.10 g; ZnCl2 0.18 g; CuCl22H2O 0.007 g; AlCl3 0.0056 g; H3BO3 0.01 g; Na2MoO42H2O 0.01 mg; NiCl26H2O 0.025 mg; Na2SeO35H2O 0.30 mg); and vitamin solution 10 mL (Per Litre: folic acid 2.0 mg; pyridoxine - HCl 10.0 mg; riboflavin 5.0 mg; nicotinic acid 5.0 mg; D-Ca-pantothenate 5.0 mg; vitamin B12 0.10 mg; paminobenzoic acid 5.0 mg). The consortium was maintained in its active form by sub-culturing at 35 °C and initial pH of 7.8 after every 15 d of interval. 2.2. Collection and characterization of different organic solid wastes/ wastewater Rice grain based distillery effluent, collected from IFB agro, West Bengal, India, was used in the study. The distillery effluent was characterized in our previous work (Mishra and Das, 2014). The AB was collected from a fresh water pond, Jakpur, West Bengal, India in the month of March. Leaves and other such materials were manually separated and the residual clay/soil particles were removed by washing. The powdered algal biomass was used as a co-substrate for the hydrogen production. DDGS was collected from IFB agro, West Bengal. The GDOC and MDOC were collected from a local market in Kharagpur, West Bengal, India. The physico-chemical characteristics were determined using standard protocols APHA (1995) (Table 1).
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P. Mishra et al. / Waste Management xxx (2017) xxx–xxx Table 1 Characterization of different organic solid wastes. Parameters
DDGS
GDOC
MDOC
AB
Crude protein Total carbohydrate Reducing sugar COD TOC Ammonia Phosphate C H O N S C/N molar ratio C/H molar ratio Empirical formula
42.60 ± 0.13 23.30 ± 1.9 17.50 ± 0.09 27.70 ± 2.2 57.1 ± 1.1 0.39 ± 0.02 0.60 ± 0.01 44.36 6.85 41.63 6.89 0.27 7.51 0.54 C7.5H13.81O5.29NS0.02
45.0 ± 0.40 20.00 ± 0.06 7.20 ± 0.01 30.0 ± 0.90 61.0 ± 0.84 0.45 ± 0.05 0.25 ± 0.05 45.39 6.93 40.51 6.00 1.17 8.82 0.55 C8.82H16.05O5.91NS0.08
36.00 ± 0.21 13.00 ± 0.03 4.00 ± 0.02 22.0 ± 1.20 50.0 ± 0.30 0.32 ± 0.03 0.15 ± 0.01 45.37 2.33 44.66 6.73 0.91 7.86 1.63 C7.86H4.81O5.81NS0.06
21.2 ± 1.30 23.9 ± 1.0 5.4 ± 0.59 28.5 ± 0.35 47.1 ± 0.45 – – 38.50 6.20 50.67 4.07 0.56 11.03 0.52 C11.03H21.17O10.89NS0.06
*All value are given in % w/w except molar values and empirical formula.
2.3. Experimental conditions 2.3.1. First stage: Biohydrogen production 10% (v/v) overnight grown AMC was used as an inoculum throughout the study. Each of the chosen co-substrates was used as sole substrate and their individual hydrogen production potentials were studied. The experiments were conducted in a 100 mL serum bottle with a working volume of 80 mL. The pH of the media was adjusted prior to the experiment. Media was maintained at the initial pH of 7.0 and then sparged with nitrogen to maintain anaerobic environment. Media was inoculated with AMC anaerobically. Experiments were conducted at 37 °C for 24 h at 180 rpm. After 24 h, total gas produced in the serum bottle was allowed to pass through 40% (w/v) KOH solution for the selective absorption of CO2 (Khanna et al., 2011). The remaining gas was collected by liquid displacement method in a gas collector filled with 10% (w/v) saline and 0.1 M K2Cr2O7 at normal temperature and atmospheric pressure. All experimental runs were performed in triplicates. Further, suitability of these organic wastes as a co-substrate with the distillery effluent was analysed. Concentration of the cosubstrates was varied from 5–40 g/L to determine the optimum value. The setup was monitored for the hydrogen production up to 24 h. A scale up study was done in a controlled fermenter (New Brunswick Scientific, NJ, USA) to study the hydrogen production and substrate degradation profiles at the optimum concentration of the co-substrates. All experiments were performed in a batch mode with a working volume of 2 L. Experiments were repeated for three times with all co-substrates.
column packed with Porapak Q (80/100) and a thermal conductivity detector (TCD). The operational temperatures of the injection port, the oven and the detector were 80 °C, 150 °C and 200 °C, respectively. Nitrogen was used as the carrier gas with a flow rate of 20 mL/min. The flame ionization detector (FID) was used for the estimation of the volatile fatty acids and ethanol. The FID operational temperatures of the injection port, the detector, and programmed column were 220 °C, 240 °C, and 130–175 °C, respectively. For the flame generation, a mixture of hydrogen and air was used at a flow rate of 30 mL/min (Khanna et al., 2011). Total carbohydrate content was estimated spectrophotometrically (UV–VIS Spectrophotometer Perkin–Elmer, k = 490 nm) using the phenol–sulphuric acid assay (Ginkel et al., 2001). The COD was estimated based on the APHA standard protocols using a COD measurement instrument (DRB200 & DR2800 Portable Spectrophotometer HACHÒ, USA). The pH was monitored using a desktop pH meter (pH510, Cyberscan, Singapore). The biomass was measured at 600 nm using UV/Vis spectrophotometer (Lambda 25, PerkinElmer LLC, MA, USA) and confirmed using dry cell weights. Standard protocol for ammonia estimation was done according to APHA (1995). Concentration of metal ions was measured by using an atomic absorption spectrometer (Perkin–Elmer). The elemental composition of the biomass was analysed using CHNS analyzer (Vario MACRO cube CHNS, Elementar, USA). The calorific value of the samples were determined by using an Automatic Oxygen Bomb Calorimeter (6100, Parr Instruments, USA).
3. Results and discussion 2.3.2. Second stage: Biomethane production Spent media after the hydrogen production was centrifuged (10,000 rpm for 10 min). The supernatant was collected and pH was maintained at 7.8 by using 2 M NaOH. The media was sparged with nitrogen gas to ensure an anaerobic condition and cysteine HCl (1 g/L) was added as a reducing agent to maintain the anaerobic state of the media. Methanogenic mixed consortium (absorbance 0.8–1.0) was added to the media in a ratio of 3:1 (media: inoculum). The process was monitored up to 12 d. The gas sample collected was allowed to pass through 40% (w/v) KOH solution for the selective absorption of CO2. 2.4. Analytical methods The hydrogen produced during batch fermentation was analysed by the gas chromatograph (GC Agilent Technology 7890A, U.S.A). The gas chromatograph was equipped with a stainless steel
3.1. Suitability of different wastes for hydrogen production Different substrates viz., GDOC, MDOC, DDGS and AB were used in a range of 0–100 g/L to study the suitable substrate concentration for biohydrogen production. GDOC showed highest cumulative hydrogen production followed by DDGS. Hydrogen production was observed to be decreasing beyond 50, 60 and 90 g/L for GDOC, DDGS and MDOC, respectively. For algal biomass no such effect was observed up to 100 g/L. This could be attributed to the fact that AMC used in this study produced maximum hydrogen at an optimum glucose concentration of 10 g/L and an inhibitory effect on the hydrogen production was observed beyond 18 g/L (Mishra and Das, 2015). Comparatively lower carbohydrate content was found in MDOC (Table 1) as compared to GDOC and DDGS. So, maximum hydrogen production was obtained at 90 g/L which is equivalent to 11.7 g carbohydrate/L (Fig. 1). The reducing
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Fig. 1. Cumulative hydrogen production using groundnut deoiled cake (GDOC), mustard deoiled cake (MDOC), Distillers’ dried grains with solubles (DDGS), and algal biomass (AB).
sugar content in the algal biomass was very low (5.4 ± 0.59% w/w), therefore, no substrate inhibition was observed even beyond 100 g/L. Highest cumulative hydrogen production of 99.8 ± 2.56 and 79.5 ± 1.62 mmol/L were obtained using GDOC and DDGS which make them a promising substrate for hydrogen production. 3.2. Suitability as a co-substrate with distillery effluent for hydrogen production Rice grain based distillery effluent was used in this study which has a very high organic load (Total COD: 59.0 ± 2.0 g/L; soluble COD: 38.0 ± 3.0 g/L). However, it contains very less amount of micronutrients (Supplementary Table 1). Therefore, distillery effluent was used mainly as a carbon source and it was supplemented with different organic solid wastes mainly as a mineral supplements. As the GDOC and DDGS showed inhibitory effect beyond 50 and 60 g/L, a co-substrate concentration was kept below 50 g/L for this study. The distillery effluent was supplemented with varied concentration (0–40 g/L) of GDOC, MDOC, DDGS and AB. A maximum cumulative hydrogen production of 128.0 ± 3.8 and 123.7 ± 4.4 mmol/L and COD reduction of 38.6 ± 0.24% and 37.3 ± 0.87% were achieved at 20 and 25 g/L of GDOC and DDGS, respectively. Further increase in their concentrations resulted in a slower production rate and reduced hydrogen productivity. MDOC showed cumulative hydrogen production and COD reduction of 95 ± 1.02 mmol/L and 33.2 ± 1.26%, respectively at 35 g/L. Further increase in the biomass concentration showed inhibitory effect on the hydrogen production process. However, in case of AB, an increase in the cumulative hydrogen production was observed with increasing substrate concentration. Supplementation of DDGS and GDOC has shown 2.15 and 2.23 folds increase in the cumulative hydrogen production as compared to the nonsupplemented distillery effluent (Fig. 2). Hydrogen production and substrate reduction profile was studied at the optimum concentration of GDOC (20.0 g/L), MDOC (35.0 g/L), DDGS (25.0 g/L) and AB (40.0 g/L) at a scaled up volume of 2 L (Fig. 3). A maximum cumulative hydrogen production was obtained using GDOC (150.7 ± 0.9 mmol/L) which was found 38.1% and 29.9% higher as compared to the MDOC (109.1 ± 4.4 mmol/L) and AB (116.1 ± 4.2 mmol/L), respectively. Hydrogen production profile using DDGS was equivalent to that of GDOC. Maximum COD reduction of 49.3 ± 1.2, 41.7 ± 0.9 and 48.5 ± 0.8, 38.1 ± 1.1% were obtained with GDOC, MDOC, DDGS
Fig. 2. (a) Cumulative hydrogen production and (b) COD reduction using raw DE supplemented with different concentrations of groundnut deoiled cake (GDOC), mustard deoiled cake (MDOC), Distillers’ dried grains with solubles (DDGS), and algal biomass (AB).
and AB, respectively. Hydrogen yield of 5.29 mol H2/ kg CODreduced was obtained using 20 g/L of GDOC with DE. Specific hydrogen yield of 11.36, 11.09, 8.87 and 5.54 mol H2/g VSS was obtained using GDOC, DDGS, MDOC and AB, respectively. The consortium used in this study has a microbial population dominated with Clostridium spp. which is reported to contain FeFe hydrogenase, a key enzyme for hydrogen production (Sinha et al., 2016; Mishra et al., 2015). Elemental analysis of these substrates showed higher Fe content in GDOC and DDGS as compared to MDOC and AB (Table 2). DDGS and GDOC are also rich in Mg which acts as a co-factor for several glycolytic enzymes and chelates with adenine nucleotides for cellular phosphate transfer reactions (Walker, 1994; Fujio and Furuya, 1985). Higher Fe and Mg content inGDOC and DDGS could be one major cause for the higher hydrogen production as compared to MDOC and AB. Distillery effluent supplemented with the AB showed lower cumulative hydrogen and specific hydrogen yield as compared to other cosubstrates. This could be due to the lower nitrogen and protein content in the AB (Table 1). Microscopic study of the wastewater grown algal consortium showed spiral arrangement of chloroplast which indicates presence of Spirogyra spp. (Fig. 4). Spirogyra is reported to contain lower nitrogen and protein content compared to the other algal species viz., Chlamydomonas, Anabaena, Microcystis, etc. (Raheem et al., 2015). The volatile fatty acid analysis (Fig. 5) showed acetate to butyrate (A/B) ratio of 0.68, 0.65, 0.52 and 0.48 in the spent media of
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Fig. 3. Hydrogen production and COD profiles by AMC using raw DE supplemented with (a) Groundnut deoiled cake (GDOC), (b) mustard deoiled cake (MDOC), (c) Distillers’ dried grains with solubles (DDGS, and (d) algal biomass (AB) at optimum substrate concentration.
Table 2 Elemental analysis of metal ions present in different organic solid wastes. Micro-elements
GDOC
MDOC
DDGS
AB
Mg Fe Mn Cu K Ni Zn
262.00 167.10 8.40 4.50 192.30 0.80 6.7
268 74.28 7.9 3.5 192.1 NA 8.5
393.70 135.60 11.90 2.80 96.20 – 7.6
186.80 75.40 42.10 6.00 191.90 – 10.8
process whose end product is alcohol. Although, several studies have reported the improvement of hydrogen production by using different pre-treatment methods including acid, alkali, sonication and thermal pre-treatment, however, it increases the cost of the process (Dong et al., 2016). To make the process cost effective, substrates were used without pre-treatment in the present study. Effect of pretreated co-substrates on the biohydrogen production can be considered for the future study. 3.3. Biomethanation using spent media
*All values are given in mg/100 g.
GDOC, DDGS, MDOC and AB, respectively. 1 mol of glucose produces 4 mol of hydrogen when it follows acetate production pathway. However, only 2 mol of hydrogen is produced from 1 mol glucose in the butyrate production pathway. Therefore, higher acetate to butyrate ratio corresponds to higher hydrogen production in the mesophilic hydrogen producers (Turon et al., 2015). Higher acetate to butyrate (A/B) ratio in the spent media of GDOC and DDGS also supports the fact that higher hydrogen was produced in the former as compared to MDOC and AB. A higher ethanol concentration (>1600 ppm) was observed in the spent media of all the co-substrates. This could be attributed to the distillation
A cumulative methane production of 64.1 ± 0.78, 43.1 ± 0.47, 63.8 ± 2.04 and 38.9 ± 1.42 mmol/L were obtained using spent media of GDOC, MDOC, DDGS and AB, respectively (Fig. 6). An ammonia concentration of 0.08–0.10 g NH3-N/L was found in the spent media used for methanogenesis which is below to the reported inhibitory range (Schnürer and Nordberg, 2008; Prochazka et al., 2012). This also supports the fact that spent media can be an ideal substrate for biomethanogenesis. The methanogenic consortium used in this study was enriched and maintained in the acetate containing media for more than 6–8 months which might have resulted in the dominance of acetoclastic methanogens in the consortium. This could be one reason for higher acetate (>90%) and butyrate (>85%) conversion as compared to ethanol (50%) as shown in Fig. 5.
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Fig. 4. Microscopic image of collected algal biomass: (a) 10 magnification and (b) 40 magnification.
Hydrogen and methane yields are compared with the published reports on two stage biohythane production using wastes (Table 5). Hydrogen yield is found higher as compared to the reported literature (Luo et al., 2011; Kisielewska et al., 2014). The current study was done in a mesophilic condition, which is lesser energy intensive and easy to operate, but comparatively higher hythane yield can be obtained by using a thermophilic consortium (Kongjan and Angelidaki, 2013). Although, hydrogen yield was found higher to most of the reported work, however methane yield was found comparatively lower (Khongkliang et al., 2015; Cheng et al., 2016). This could be due to the use of acidogenic mixed consortium (AMC) acclimatized with the distillery effluent (Mishra et al., 2015). So, a similar acclimatization study for the methanogenic consortium could be helpful for the improvement of methane yield. Future study can also focus on the improvement of the ethanol conversion efficiency.
3.4. Cost and energy analysis Fig. 5. End metabolites concentration in the spent media of dark fermentative hydrogen production.
Fig. 6. Cumulative methane production and substrate reduction using spent of dark fermentation.
Replacement of tryptone with cheaper organic solid waste biomass resulted in a drastic reduction of substrate input cost (SIC) (Table 3). The cumulative Hydrogen and methane production using distillery effluent supplemented with 10 g/L tryptone was156.0 and 69.64 mmol/L, which was comparable to both GDOC (150.7 and 64.1 mmol/L) and DDGS (144.2 and 63.8 mmol/L). However, the SIC for GDOC and DDGS were reduced to 98.3 and 97.9%, respectively as compared to controlled distillery effluent supplemented with tryptone. In case of MDOC, the cost reduction was 97.7% with cumulative hydrogen and methane production of 109.1 and 43.13 mmol/L, respectively. AB was collected from a local pond and dried under sunlight. Therefore, apparently zero input cost can be considered for AB. Apart from zero input cost, utilization of wastewater grown algal biomass also helps in bioremediation by minimizing eutrophication. Hence, it can infer that utilization of such alternative solid waste products as a cosubstrate for the hydrogen production can result in a drastic reduction in the substrate cost. Total gaseous energy of 23.93 and 23.44 kcal/L were produced using distillery effluent supplemented with GDOC and DDGS, respectively (Table 4) which was 2.6 times higher compared to the control (9.08 kcal/L). GDOC and DDGS showed 54.6 and 51.4% higher gaseous energy production as compared to tryptone (15.48 kcal/L) with significant reduction in the substrate cost reduction. This makes GDOC and DDGS as a potential cosubstrate with distillery effluent for biohythane production.
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P. Mishra et al. / Waste Management xxx (2017) xxx–xxx Table 3 Substrate input cost (SIC) analysis of the process.
a b c d
Substrate
Cost (INR/MT)d
Substrates used (g/L of DE)
Cost of substrate used/L (Rs)
H2 produced (mmol/L)
Cost reduction compared to tryptone as supplement. (%)
GDOC MDOC DDGS AB Tryptone
25,500a 20,000a 25,000b 0 3,000,000c
20.0 35.0 25.0 40.0 10.0
0.51 0.70 0.62 0.00 30.00
150 109 144 116 156
98.3 97.7 97.9 100.0
http://www.agriwatch.com. http://dir.indiamart.com/impcat/rice-ddgs.html. Sisco Research Laboratories Pvt. Ltd. (SRL). INR: Indian Rupees.
Table 4 Hydrogen and methane production using co-substrates with distillery effluent. Substrate
Control DE
GDOC MDOC DDGS AB Tryptone
Total gas produced (mmol/L)
Gaseous energy produced (kcal/L)
H2
CH4
H2
CH4
Total
55.13 150.70 109.10 144.20 116.10 156.00
24.95 64.06 43.13 63.84 38.88 69.64
3.77 10.31 7.46 9.86 7.94 10.67
5.30 13.62 9.17 13.57 8.27 14.81
9.08 23.93 16.63 23.44 16.21 15.48
Table 5 Comparative studies on the two stage hydrogen and methane production using different organic wastes. Substrate
H2 production phase
CH4 production phase
Reference
Inoculum
Conditions
Yield
Inoculum
Conditions
Yield
Garbage and paper wastes Potato waste OFMSW Cheese whey Food waste Water hyacinth leave Organic biowaste Ethanol stillage Desugared molasses Food waste Whey permeate POME Starchy wastewater Food waste with sewage sludge
ASC AS AS IM AS AS AS ADM TC IM AS AS TC AS
CSTR, C CSTR, C CSTR, C CSTR, C CSTR, SC SB, B CSTR, SC CSTR, C UASB, C CSTR, C UASB, C ASBR, C CSTR, B CSTR,B
282 L/kg COD 30 L/kg TS 205 L/kg VS 92 L/kg COD 270 L/kg COD 51.7 L/kg VS 52 L/kg VS 48 L/kg VS 132 L/kg VS 60 L/kg VS 93.8 L/kg COD 210 L/kg COD 81.5 L/kg COD 174.6 L/kg VS
AS AS AS AS AS AS AS ADM TC AS AS AS TC AS
PBR CSTR, C FBR, C CSTR, C A* SB, B CSTR, SC CSTR, C UASB, C CSTR, C UASB, C UASB, C CSTR, B CSTR, B
389 L/kg COD 183 L/kg TS 464 L/kg VS 136 L/kg COD 287 L/kg COD 143.4 L/kg VS 410 L/kg VS 344 L/kg VS 239 L/kg VS – 120 L/kg COD 315 L/kg COD 310 L/kg COD 264.1 L/kg VS
Ueno et al. (2007) Zhu et al. (2008) Chu et al. (2008) Venetsaneas et al. (2009) Lee et al. (2010) Cheng et al. (2010) Cavinato et al. (2011) Luo et al. (2011) Kongjan and Angelidaki (2013) Micolucci et al. (2014) Kisielewska et al. (2014) Mamimin et al. (2015) Khongkliang et al. (2015) Cheng et al. (2016)
DE with GDOC
AMC
CSTR, B
118.5 L/kg COD
MMC
CSTR, B
158.1 L/kg COD
Present study
A*, Biogas sparging type reactor; PBR, packed-bed reactor; FBR, Fluid bed reactor; CSTR, Continuously stirred tank reactor; C, Continuous mode of operation; B, Batch mode of operation; SC, Semi continuous mode of operation; ASBR, Anaerobic sequencing batch reactor; AS, Anaerobic digester sludge; ADM, Anaerobic digested manure; ASC, Activated sludge compost; TC, Thermophilic consortium; COD, Chemical oxygen demand; IM, Indigenous microflora; OFMSW, Organic fraction of municipal solid waste; POME, Palm oil mill effluent; TS, Total solid; VS, Volatile solid, VSS, Volatile suspended solids; DE, Distillery effluent; DDGS, Distillers’ dried grains with soluble.
4. Conclusion
Acknowledgement
Suitability of GDOC, MDOC, DDGS and AB as a supplement to the distillery effluent for biohythane production was explored. GDOC was found as the most suitable co-substrate with the distillery effluent followed by DDGS. GDOC and DDGS showed 2.73 and 2.61-fold improvement in the cumulative hydrogen production and 2.57 and 2.56-fold improvement in the cumulative methane production as compared to the non-supplemented distillery effluent (control). A significant reduction in the substrate input cost (SIC) can be attained by replacing tryptone with cheaper organic co-substrates like wastewater grown algal biomass with comparable hydrogen and methane productionbesides decreasing organic load on the environment. DGGE analysis of AMC consortia is in progress to find out the hydrogen producing microorganisms.
The authors gratefully acknowledge Ministry of New and Renewable Energy (MNRE) and Department of Biotechnology (DBT), Government of India for providing the financial support. Authors are also thankful to the IIT Kharagpur for providing the research facilities and IFB agro, Kolkata for providing distillery effluent.
Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.wasman.2017.04. 040.
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