Ecobiotechnological Strategy to Enhance Efficiency of Bioconversion of Wastes into Hydrogen and Methane

Abstract

Vegetable wastes (VW) and food wastes (FW) are generated in large quantities by municipal markets, restaurants and hotels. Waste slurries (250 ml) in 300 ml BOD bottles, containing 3, 5 and 7 % total solids (TS) were hydrolyzed with bacterial mixtures composed of: Bacillus, Acinetobacter, Exiguobacterium, Pseudomonas, Stenotrophomonas and Sphingobacterium species. Each of these bacteria had high activities for the hydrolytic enzymes: amylase, protease and lipase. Hydrolysate of biowaste slurries were subjected to defined mixture of H₂ producers and culture enriched for methanogens. The impact of hydrolysis of VW and FW was observed as 2.6- and 2.8-fold enhancement in H₂ yield, respectively. Direct biomethanation of hydrolysates of VW and FW resulted in 3.0- and 1.15-fold improvement in CH₄ yield, respectively. A positive effect of hydrolysis was also observed with biomethanation of effluent of H₂ production stage, to the extent of 1.2- and 3.5-fold with FW and VW, respectively. The effective H₂ yields were 17 and 85 l/kg TS fed, whereas effective CH₄ yields were 61.7 and 63.3 l/kg TS fed, from VW and FW, respectively. This ecobiotechnological strategy can help to improve the conversion efficiency of biowastes to biofuels.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-014-0467-7) contains supplementary material, which is available to authorized users.

Introduction

Pollution Control Boards and Health Departments are constantly worried about the generation of huge quantities of wastes and the rapidly declining reservoirs of fossil fuels. Uncontrolled fermentation and burning of these wastes and fuels release obnoxious gases [1]. Among the various proposals being explored to solve these problems, anaerobic digestion (AD) appears to be the most lucrative. AD is a metabolically efficient process, but is economically very weak. In order to enhance the economic value of the process, suggestions have been made to derive value added products by diverting the intermediates of the waste solubilizing step to hydrogen (H₂), methane (CH₄), bioplastic, enzymes, etc. [2, 3]. AD is a multi-step process, which involves different bacteria with a wide range of metabolic activities. Although, organic matter of the biowastes can be digested up to 95 % into carbon dioxide and CH₄ [4], however, the whole process is limited by the hydrolytic step. The hydrolysis of organic matters is influenced by its composition, the most difficult to digest are the lignocellulosic biowaste [5]. Another issue which demands attention is the fact that although H₂ is an intermediate of the AD process, however, in nature, it results in CH₄ as the final byproduct with little or no H₂ evolution [6]. It is because of the fact that thermodynamically, H₂ production process is not stable and the equilibrium shifts to CH₄ production. This leads to a scenario of interspecies H₂ transfer reactions, where H₂ consumers out number the H₂ producers [7]. Another primary reason for low or no evolution of H₂ during AD is the feedback inhibition of H₂ process by high partial pressure of H₂. Studies to investigate H₂ and CH₄ potential of different biowastes have been evaluated under different physiological conditions [8–13]. It is difficult to produce H₂ from biowaste, since it is invariably accompanied by inherent microflora, which out number the H₂-producing bacteria [8, 14]. Sterilization of biowaste to get rid of contaminating bacteria is a costly proposal. Attempts to produce H₂ from un-sterile wastes have been successful to some extent [6, 15–17]. The need is to look for a robust set of organisms, which can survive under harsh conditions and produce H₂ and CH₄. The ecobiotechnological strategy is based on the concept of using a mixture of bacteria, which have been well defined to carry out the desired metabolic activity. Under a given set of physiological conditions prevailing in a fermenting biowaste, at least one of these well defined bacteria will be able to survive and carry out the process successfully [14, 18].

It has been realized that in all energy generation processes, the major limiting factor is the feed. Biowastes are an obvious choice because of their availability in large quantities and “consistent” supplies on daily basis. Most biowastes are composed of complex organic materials. The very first step in their utilization is the solubilization of macromolecules into simpler and easily metabolizable substrates [15, 19]. Biowastes originating from vegetable markets and food and fruit processing industries, which are rich in fats, carbohydrate and proteins. These macromolecules can be metabolized by bacteria possessing enzymes such as lipases, amylases and proteases [19]. The question is thus, Can an improvement in the hydrolytic process lead to enhancement of the digestion process? In this study, we have used an ecobiotechnological strategy to use well defined bacterial cultures for hydrolysis of unsterile wastes and subject the hydrolysate to another set of H₂-producers and enriched culture of methanogens, independently and in a sequential manner.

Materials and Methods

Preparation of Hydrolytic, H₂ Producers and Methanogens

We isolated 1,000 bacteria from soils, river sediments and cattle dung. These were screened for those having high activities for the following enzymes: amylase, lipase and protease by method described previously [19, 20]. Fifty bacteria with high hydrolytic activities were evaluated for their performance at pH range 5.0–9.0. A set of 11 bacterial strains were selected and identified through 16S rRNA gene [20]. These bacteria were employed for preparing 11 mixed hydrolytic bacterial cultures (BC1–BC11) (Table S1), designed on the basis of Plackett–Burman method [21] (Tables S2). Similarly, for H₂ production another set of mixed microbial culture (MMC4), previously screened on glucose was used [21]. MMC4 was composed of the following 6 strains: Enterobacter aerogenes EGU16, Proteus mirabilis EGU21, Bacillus cereus EGU43, B. thuringiensis EGU45, B. pumilus HPC 464, Bacillus sp. HPC459, which were previously established to be effective as mixed H₂ producers [21]. Each mixed culture was prepared by combining 6 different microbes in equal proportions amounting to a final cell protein concentration of 10 µg/ml [19]. Enrichment of methanogens was done by incubating 3 % total solids (TS) cattle dung slurry at 37 °C for 20 days [22].

Total Solids and Organic Solids

Samples of vegetable waste (VW) and kitchen food waste (FW) have been analysed for parameters like TS, and volatile solids, which were estimated by heating a sample at 110 °C for 24 h and at 600 °C for 4 h, respectively [22].

Hydrolysis of Biowastes

The biowaste slurries (250 ml) were hydrolysed with 11 mixed microbial cultures namely BC1–BC11. The hydrolysis of waste was carried out for 5 days at 37 °C. Hydrolysis was monitored through the production of volatile fatty acids [19].

Hydrogen Production

Biowaste feed (250 ml) at 3, 5 and 7 % TS was inoculated with MMC4 at the rate of 10 µg cell protein/ml of slurry. pH of the slurry was adjusted to 7.0 prior to incubation and the bottles were made air tight using glass stoppers. pH was adjusted to 7.0 using 2 N NaOH or 2 N HCl and flushed with argon, on a daily basis. The evolved gases were collected by the water displacement method. Gas collection and analysis of the samples were carried out until H₂ evolution ceased [19, 20]. The values presented here are based on three replicates.

Methane Production

Biowaste feed (250 ml) at 3, 5 and 7 % TS was inoculated with methanogens 10 % (v/v). pH of the slurry was adjusted to 7.0. The reactor bottle was flushed with argon to make the conditions anaeronbic. Biogas production was monitored daily for 15 days and it was observed that biogas production stopped by 10 days except in controls [22]. The values presented here are based on three replicates.

Analytical method

Gas Analysis

The composition of the biogas produced during fermentation processes was determined using gas chromatograph (Nucon GC5765) equipped with Porapak-Q and molecular sieve columns using thermal conductivity detector [19, 21].

Volatile Fatty Acid Estimation

VFA analysis was carried out from 1.0 ml sample taken in 1.5 ml vials. 2–3 drops of ortho-phosphoric acid (25 % v/v) were added to each vial for sample preservation. VFA concentrations were determined using gas chromatograph (GC 6890 N) equipped with flame ionization detector. A capillary column, DBWAXetr (30 m × 53 μm × 1 μm ID) was used for analysis. The oven, injector and detector temperatures were 140, 220 and 230 °C, respectively.

Results

In natural conditions, the biowaste containing biomacromolecules like carbohydrates, fats and proteins can be degraded by bacteria producing hydrolytic enzymes. Screening of 1,000 bacteria allowed us to select 50 having high activities for amylase, lipase and protease. Further evaluation of the enzymatic activities at a wider pH range enabled us to select 11 having at least one of these enzymatic activities in the pH range 5.0–9.0. Finally eleven bacteria so selected were identified as: Bacillus aryabhattai MBG46 (KJ563237); Acinetobacter sp. MBG50 (KJ563241) and A. haemolyticus MBG52 (KJ563243); Exiguobacterium sp. MBG53 (KJ563244) and E. indicum MBG54 (KJ563245); Pseudomonas mendocina strains MBG51 (KJ563242), MBG57 (KJ563248), MBG58 (KJ563249) and P. pseudoalcaligenes MBG45 (KJ563236); Stenotrophomonas koreensis MBG44 (KJ563235) and Sphingobacterium daejeonense MBG47 (KJ563238) (Table S1). Of the 11 mixed bacterial cultures: BC6, BC7, BC8 and BC10 were found to be effective for hydrolyzing VW as indicated by the total volatile fatty acid composition, over a period of 5 days of incubation (Tables S3). On the other hand, mixed bacterial culture designated as BC1, BC6, BC8 and BC9 were found to be effective for hydrolyzing FW under similar incubation period.

Hydrogen Evolution

H₂ evolution was observed from vegetable waste slurry (VWS) and food waste slurry (FWS) using defined mixed microbial culture of H₂-producers (MMC4) [21]. Unhydrolysed waste (control) was observed to generate 160–390 ml of biogas from 250 ml of VWS. Here, H₂ constituted 39.7–44.4 % of the total biogas, amounting to a net observed volume of 65–155 ml/250 ml slurry. The effective H₂ yield was in the range of 6–9 l/kg TS fed. Pretreatment of VW with hydrolytic bacterial cultures was very effective. Of the 11 mixed bacterial cultures, BC6, BC7, BC8 and BC10 were found to be effective in improving H₂ yield (Table 1). At 3 % TS VWS, biogas evolution increased up to 285 ml with BC7. It was accompanied by a substantial enhancement in H₂ evolution up to 130 ml, i.e., a 2-fold increase over control. At 5 % TS VWS, maximum H₂ evolution was observed with BC7. Although H₂ component of the biogas did not change much, however, BC7 resulted in 2.6-fold increase in H₂ yield. Further increase in the concentration of TS in the VWS to 7 % led to increase in the net evolution of biogas (up to 530 ml), however, it was not accompanied by a proportional increase in H₂ evolution. H₂ evolution was almost similar to that recorded with control. In fact, it has been reported previously that H₂ evolution process is negatively influenced by the increase in carbohydrate concentration in the slurry [23]. It may also be remarked that high TS also influence the metabolic process of H₂ evolution since the H₂ component of the biogas was also reduced compared to those observed at 3 and 5 % TS slurries. It may be reasonable to conclude that BC7 is effective in hydrolyzing the VW resulting in 2.0- to 2.6-fold enhancement in H₂ yield (Table 1).

Table 1.

Hydrogen producing abilities of mixed H₂-producers^a from prehydrolyzed biowastes

Mixed bacterial culture	Biogas volume (ml)	H2			Biogas volume (ml)	H2			Biogas volume (ml)	H2
		Vol (ml)	%	Yieldb		Vol (ml)	%	Yield		Vol (ml)	%	Yield
					Vegetable Waste
	3 % TSc				5 % TS				7 % TS
Controld	160	65	40.6	9	180	80	44.4	6	390	155	39.7	9
BC6	180	70	38.9	10	270	85	31.5	7	465	125	26.9	7
BC7	285	130	45.6	17	475	210	44.2	17	530	150	28.3	9
BC8	260	115	44.2	16	380	165	43.4	13	350	75	21.4	4
BC10	250	90	36.0	12	250	100	40.0	8	240	105	43.7	6
					Food Waste
	3 % TS				5 % TS				7 % TS
Control	685	225	32.8	30	635	205	32.3	16	620	295	47.6	17
BC1	535	230	43.0	30	735	345	46.9	28	650	265	40.8	15
BC6	1,030	640	62.1	85	800	395	49.4	32	885	420	47.4	24
BC8	485	250	51.5	33	875	470	53.7	38	945	625	66.1	36
BC9	635	290	45.7	39	525	225	42.8	18	515	190	36.9	11

^aDefined mixed microbial culture of H₂-producers (MMC4)

^bH₂ production in l/kg Total solids fed

^cTotal solids

^dNo mixed bacterial culture added

In contrast to VW, the fermentation process was more effective with FW, which may be due to easily digestible components of the waste. Here, 250 ml of FWS without any hydrolysis resulted in the net evolution of 685 ml biogas at 3 % TS. It contained 225 ml H₂, equivalent to 32.8 % of the total biogas (Table 1). The effective H₂ yield was 30 l/kg TS fed. Hydrolysis with different bacterial cultures resulted in gain in H₂ yields, ranging up to 85 l/kg TS fed with BC6. It was accompanied by a higher H₂ component of 62.1 %. Hydrolysis of FW resulted in 2.8-fold enhancement in H₂ yields. Further increase in TS of the slurry did not prove helpful in improving the H₂ production process. H₂ yields of 28–38 l/kg TS fed from 5 % TS pretreated with BC1, BC6 and BC8 and 24–36 l/kg TS fed from 7 % TS slurries were higher than their respective controls. BC6 and BC8 were the most effective mixtures of hydrolytic bacteria, which enhanced H₂ yield. (Table 2).

Table 2.

Comparison of methane yields from prehydrolyzed biowastes by direct and indirect biomethanation

Mixed bacterial culture	Direct biomethanationa			Indirect biomethanation
	3 % TSb	5 % TS	7 % TS	3 % TS	5 % TS	7 % TS
Vegetable waste
Controlc	20.0	26.5	36.4	8.0	13.4	8.6
BC6	17.5	29.3	25.0	7.3	15.2	11.4
BC7	61.7	31.0	15.7	28.3	16.6	10.0
BC8	26.7	42.2	31.4	12.7	13.5	10.7
BC10	17.5	36.4	38.6	6.0	14.7	11.4
Food waste
Control	55.0	26.0	20.3	26.3	16.7	10.7
BC1	50.0	37.4	32.1	30.0	19.4	12.1
BC6	63.3	54.5	24.3	31.7	17.5	13.6
BC8	46.7	45.2	42.1	21.4	19.2	12.8
BC9	48.3	52.3	37.9	31.5	18.5	12.1

^aCH₄ production in l/kg Total solids fed

^b Total solids

^c No mixed bacterial culture added

Methane Evolution

Biomethanation has been a suitable post H₂ treatment process for effective utilization of biowastes. CH₄ evolution was observed on hydrolysed biowastes through two routes-Direct and Indirect (preceeded by H₂ production). The impact of hydrolysis by mixed bacterial cultures on biomethanation through both the routes was distinctly observed.

Direct Biomethanation

Biomethanation of VWS (250 ml) was observed to vary from 20 to 36.4 l/kg TS fed. It constituted around 61 % of the total biogas produced over a period of 15 days. In contrast, hydrolysis of VW by 11 different mixed bacterial culture (BC1–BC11) having high hydrolytic enzyme activities proved effective in improving the biomethanation process. The four BCs: BC6, BC7, BC8 and BC10 were chosen for further studies as the VFA content of these hydrolysates were quite high and consistent. BC7 proved to be the most efficient with a final CH₄ yield of 61.7 l/kg TS fed at 3 % TS VWS. The net enhancement in CH₄ yield was 3-fold. Although CH₄ yields were higher at 5 and 7 % TS VWS compared to control, however, BC7 treatment resulted in lower CH₄ yields at 5 and 7 % TS VWS, compared to 3 % TS VWS. Hence, we may conclude that hydrolysis of VWS with BC7 is effective at 3 % TS VWS in comparison to untreated VWS.

Direct biomethanation of untreated FWS was more effective in comparison to VWS. Here, the net CH₄ yield was 55 l/kg TS fed. Biomethanation was found to decline from FWS (control) at higher TS concentrations of 5 and 7 %, where the CH₄ yield were found to be 26.0 and 20.3 l/kg TS fed, respectively. Hydrolysis of FW with well defined mixed bacterial cultures was quite effective with BC6, which enabled us to improve the biomethanation process to yield 63.3 l CH₄/kg TS fed at 3 % TS FWS. Although, the CH₄ yields were higher than the control even at 5 % and 7 % TS FWS, however, these values were relatively lower than those obtained from 3 % TS FWS with BC6.

Indirect Biomethanation

The effect of pretreatment with hydrolytic bacteria was evident even with effluent emanating from H₂ production process. Untreated VWS, resulted in 8.0–13.4 l CH₄/kg TS fed at 3–7 % TS concentrations. In contrast, VWS subjected to hydrolysis by BC7 proved effective even in indirect biomethanation process, with a net gain of 1.16- to 3.53-fold. The best results were observed at 3 % TS VWS. Incidentally, the same combination was the most efficient even via direct biomethanation. On the other hand, FWS slurry was also digested most efficiently by BC6 in the cases of direct and indirect biomethanation. Via indirect biomethanation, a 1.2-fold enhanmcement in CH₄ yield was recorded in comparison to its respective control.

Most of the biological wastes undergo AD process with no net evolution of H₂. It is primarily because of inter species H₂ transfer phenomenon. Since H₂ generation results in accounting for 35 % of the total energy present in the organic matter content of the feed, it becomes imperative to subject the effluent from H₂ stage to methanogens. Here, we can expect a maximum of 65 % of the energy as CH₄, with respect to CH₄ yield observed via direct biomethanation as 100 %. In VWS (3 % TS) and BC7 combination, we observed 63.3 l CH₄/kg TS fed via direct biomethanation. Via indirect biomethanation, we can expect a CH₄ yield of 41.1 l. Since we could observe VWS (3 % TS) and BC7 combination to generate 31.7 l CH₄, it is equivalent to 77 % of the expected value. On the other hand, with FWS (3 % TS) and BC6 combination, we could generate 61.7 l CH₄/kg TS fed via direct biomethanation and 28.3 l CH₄/kg TS fed via indirect biomethanation. Thus we could recover 70 % of the CH₄ yield expected via direct biomethanation. In both the cases, we could recover 70–75 % of the expected CH₄ yields.

Discussion

Bioprocesses involving single bacterial cultures are always at the risk of getting contaminated [14]. In order to run the process continuously there is a need to maintain conditions which are favorable to the bacteria in question and at the same time prevent others from growing. Invariably, it demands sterile feed material. In the case of fermentation of biowastes, it is difficult to sterilize the feed [19]. Hence, the presence of inherent bacteria continues to pose a threat, as they metabolize the organic matter into undesirable by-products. Ecobiotechnological approach relies on the use of robust bacteria with well defined activities. Mixed defined bacteria as inoculum enhances the chance of survival of at least one or two types of bacteria, which are sufficient to ensure consistency and reproducibility of the process. This approach has been exploited previously for producing polyhydroxyalkanoates [14, 18]. In the present work, the whole process is quite complex. For complete degradation of biowastes, coordinated activities of different set of bacteria are operative: (1) Hydrolytic bacteria (2) H₂ producers and 3) methanogens [1]. The major metabolic limitations are: (1) The hydrolytic process, and (2) H₂ transfer reaction [7]. For hydrolysis of organic matter, the need is to have well defined bacteria with high hydrolytic activities. And such bacteria are present in small numbers in natural populations. The other issue is the fact that H₂ produced by one set of bacteria is immediately quenched by methanogens, such that there is little or no net evolution of H₂ [15]. In the present study, bacteria with high relative enzyme efficiencies were mixed in equal proportions. Of the 11 such mixed bacterial cultures, BC7 and BC6 found to be effective in enhancing H₂ yield from vegetable waste and food waste to the extent of 1.9- and 2.8-fold, respectively, in comparison to control. Hydrolysate generated by BC6 and BC7 were effective in 1.15- and 3.1-fold improvement in CH₄ yield. In the case of hydrolysate initially subjected to H₂ producers and subsequently by methanogens, BC7 resulted in 3.53-fold and BC6 led to 1.2-fold enhancement in CH₄ yields. Thus under all conditions, hydrolytic bacterial mixture proved effective in enhancing the processes for generating bioenergy. Secondly, the split of H₂ stage and CH₄ stage allowed us to overcome the problem of H₂-energy transfer [8, 23]. These findings provide an evidence that hydrolysate of organic matter can be easily converted into bioproducts of high economic values. Combining these metabolic pathways may enable complete and efficient degradation along with sustainability.

Conclusion

Hydrolysis of biowastes with defined bacterial cultures helps to improve H₂ and CH₄ production from VW at 3 % by 1.9- and 3.1-fold, whereas with FW the corresponding enhancements were 2.83- and 1.15-fold, respectively. FW is a better feed for H₂ (5-fold) compared to VW. 3 % TS is the best concentration observed for H₂ and CH₄ generation with both VW and FW. The effective H₂ yields were 17 and 85 l/kg TS fed, where as effective CH₄ yields were 61.7 and 63.3 l/kg TS fed from VWS and FWS, respectively. Hydrolysis thus proved beneficial in achieving cost-effective conversion of waste to energy.