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2014年7月27日星期日

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

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Indian J Microbiol (July–Sept 2014) 54(3):262–267

DOI 10.1007/s12088-014-0467-7

ORIGINAL ARTICLE

Ecobiotechnological Strategy to Enhance Efﬁciency

of Bioconversion of Wastes into Hydrogen and Methane

Prasun Kumar • Dinesh Chander Pant •

Sanjeet Mehariya • Rishi Sharma • Arun Kansal •

Vipin C. Kalia

Received: 25 March 2014 / Accepted: 16 April 2014 / Published online: 29 April 2014

Association of Microbiologists of India 2014

Abstract Vegetable wastes (VW) and food wastes (FW) extent of 1.2- and 3.5-fold with FW and VW, respectively.

are generated in large quantities by municipal markets, The effective H2 yields were 17 and 85 l/kg TS fed,

restaurants and hotels. Waste slurries (250 ml) in 300 ml whereas effective CH4 yields were 61.7 and 63.3 l/kg TS

BOD bottles, containing 3, 5 and 7 % total solids (TS) fed, from VW and FW, respectively. This ecobiotechno-

were hydrolyzed with bacterial mixtures composed of: logical strategy can help to improve the conversion efﬁ-

Bacillus, Acinetobacter, Exiguobacterium, Pseudomonas, ciency of biowastes to biofuels.

Stenotrophomonas and Sphingobacterium species. Each of

these bacteria had high activities for the hydrolytic Keywords Anaerobic digestion Biowaste

enzymes: amylase, protease and lipase. Hydrolysate of Biomethanation Hydrolysis Mixed bacterial culture

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- Introduction

and 2.8-fold enhancement in H2 yield, respectively. Direct

biomethanation of hydrolysates of VW and FW resulted in Pollution Control Boards and Health Departments are

3.0- and 1.15-fold improvement in CH4 yield, respectively. constantly worried about the generation of huge quantities

A positive effect of hydrolysis was also observed with of wastes and the rapidly declining reservoirs of fossil

biomethanation of efﬂuent of H2 production stage, to the fuels. Uncontrolled fermentation and burning of these

wastes and fuels release obnoxious gases [ 1]. Among the

various proposals being explored to solve these problems,

Prasun Kumar and Dinesh Chander Pant have Contributed equally to anaerobic digestion (AD) appears to be the most lucrative.

this study. AD is a metabolically efﬁcient process, but is economically

Electronic supplementary material The online version of this very weak. In order to enhance the economic value of the

article (doi:10.1007/s12088-014-0467-7) contains supplementary process, suggestions have been made to derive value added

material, which is available to authorized users. products by diverting the intermediates of the waste solu-

bilizing step to hydrogen (H ), methane (CH ), bioplastic,

P. Kumar (&) S. Mehariya R. Sharma V. C. Kalia 2 4

Microbial Biotechnology and Genomics, CSIR -Institute of enzymes, etc. [2, 3]. AD is a multi-step process, which

Genomics and Integrative Biology (IGIB), Delhi University involves different bacteria with a wide range of metabolic

Campus, Mall Road, New Delhi 110007, India activities. Although, organic matter of the biowastes can be

e-mail: prasun.mcr@gmail.com digested up to 95 % into carbon dioxide and CH4 [4],

P. Kumar however, the whole process is limited by the hydrolytic

Department of Biotechnology, University of Pune, Pune 411007, step. The hydrolysis of organic matters is inﬂuenced by its

India composition, the most difﬁcult to digest are the lignocel-

lulosic biowaste [5]. Another issue which demands atten-

D. C. Pant A. Kansal

TERI University, 10, Industrial Area, Vasant Kunj, tion is the fact that although H2 is an intermediate of the

New Delhi 110070, India AD process, however, in nature, it results in CH4 as the

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Indian J Microbiol (July–Sept 2014) 54(3):262–267 263

ﬁnal byproduct with little or no H2 evolution [6]. It is activities for the following enzymes: amylase, lipase and

because of the fact that thermodynamically, H2 production protease by method described previously [ 19, 20]. Fifty

process is not stable and the equilibrium shifts to CH4 bacteria with high hydrolytic activities were evaluated for

production. This leads to a scenario of interspecies H2 their performance at pH range 5.0–9.0. A set of 11 bacterial

transfer reactions, where H2 consumers out number the H2 strains were selected and identiﬁed through 16S rRNA gene

producers [7]. Another primary reason for low or no evo- [20]. These bacteria were employed for preparing 11 mixed

lution of H2 during AD is the feedback inhibition of H2 hydrolytic bacterial cultures (BC1–BC11) (Table S1),

process by high partial pressure of H . Studies to investi- designed on the basis of Plackett–Burman method [21]

2

gate H2 and CH4 potential of different biowastes have been (Tables S2). Similarly, for H2 production another set of

evaluated under different physiological conditions [8– 13]. mixed microbial culture (MMC4), previously screened on

It is difﬁcult to produce H2 from biowaste, since it is glucose was used [21]. MMC4 was composed of the fol-

invariably accompanied by inherent microﬂora, which out lowing 6 strains: Enterobacter aerogenes EGU16, Proteus

number the H2-producing bacteria [8, 14]. Sterilization of mirabilis EGU21, Bacillus cereus EGU43, B. thuringiensis

biowaste to get rid of contaminating bacteria is a costly EGU45, B. pumilus HPC 464, Bacillus sp. HPC459, which

proposal. Attempts to produce H2 from un-sterile wastes were previously established to be effective as mixed H2

have been successful to some extent [6, 15– 17]. The need producers [21]. Each mixed culture was prepared by com-

is to look for a robust set of organisms, which can survive bining 6 different microbes in equal proportions amounting

under harsh conditions and produce H and CH . The to a ﬁnal cell protein concentration of 10 lg/ml [19].

2 4

ecobiotechnological strategy is based on the concept of Enrichment of methanogens was done by incubating 3 %

using a mixture of bacteria, which have been well deﬁned total solids (TS) cattle dung slurry at 37 C for 20 days [22].

to carry out the desired metabolic activity. Under a given

set of physiological conditions prevailing in a fermenting Total Solids and Organic Solids

biowaste, at least one of these well deﬁned bacteria will be

able to survive and carry out the process successfully Samples of vegetable waste (VW) and kitchen food waste

[14, 18]. (FW) have been analysed for parameters like TS, and

It has been realized that in all energy generation pro- volatile solids, which were estimated by heating a sample

cesses, the major limiting factor is the feed. Biowastes are at 110 C for 24 h and at 600 C for 4 h, respectively [22].

an obvious choice because of their availability in large

quantities and ‘‘consistent’’ supplies on daily basis. Most Hydrolysis of Biowastes

biowastes are composed of complex organic materials. The

very ﬁrst step in their utilization is the solubilization of The biowaste slurries (250 ml) were hydrolysed with 11

macromolecules into simpler and easily metabolizable mixed microbial cultures namely BC1–BC11. The hydro-

substrates [15, 19]. Biowastes originating from vegetable lysis of waste was carried out for 5 days at 37 C.

markets and food and fruit processing industries, which are Hydrolysis was monitored through the production of vol-

rich in fats, carbohydrate and proteins. These macromole- atile fatty acids [19].

cules can be metabolized by bacteria possessing enzymes

such as lipases, amylases and proteases [19]. The question Hydrogen Production

is thus, Can an improvement in the hydrolytic process lead

to enhancement of the digestion process? In this study, we Biowaste feed (250 ml) at 3, 5 and 7 % TS was inoculated

have used an ecobiotechnological strategy to use well with MMC4 at the rate of 10 lg cell protein/ml of slurry.

deﬁned bacterial cultures for hydrolysis of unsterile wastes pH of the slurry was adjusted to 7.0 prior to incubation and

and subject the hydrolysate to another set of H2-producers the bottles were made air tight using glass stoppers. pH was

and enriched culture of methanogens, independently and in adjusted to 7.0 using 2 N NaOH or 2 N HCl and ﬂushed

a sequential manner. with argon, on a daily basis. The evolved gases were col-

lected by the water displacement method. Gas collection

and analysis of the samples were carried out until H2

Materials and Methods evolution ceased [ 19, 20]. The values presented here are

based on three replicates.

Preparation of Hydrolytic, H2 Producers

and Methanogens Methane Production

We isolated 1,000 bacteria from soils, river sediments and Biowaste feed (250 ml) at 3, 5 and 7 % TS was inoculated

cattle dung. These were screened for those having high with methanogens 10 % (v/v). pH of the slurry was

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264 Indian J Microbiol (July–Sept 2014) 54(3):262–267

adjusted to 7.0. The reactor bottle was ﬂushed with argon Hydrogen Evolution

to make the conditions anaeronbic. Biogas production was

monitored daily for 15 days and it was observed that bio- H2 evolution was observed from vegetable waste slurry

gas production stopped by 10 days except in controls [22]. (VWS) and food waste slurry (FWS) using deﬁned mixed

The values presented here are based on three replicates. microbial culture of H2-producers (MMC4) [21]. Unhy-

drolysed waste (control) was observed to generate

160–390 ml of biogas from 250 ml of VWS. Here, H2

Analytical method constituted 39.7–44.4 % of the total biogas, amounting to

a net observed volume of 65–155 ml/250 ml slurry. The

Gas Analysis effective H2 yield was in the range of 6–9 l/kg TS fed.

Pretreatment of VW with hydrolytic bacterial cultures

The composition of the biogas produced during fermenta- was very effective. Of the 11 mixed bacterial cultures,

tion processes was determined using gas chromatograph BC6, BC7, BC8 and BC10 were found to be effective in

(Nucon GC5765) equipped with Porapak-Q and molecular improving H2 yield (Table 1). At 3 % TS VWS, biogas

sieve columns using thermal conductivity detector [19, 21]. evolution increased up to 285 ml with BC7. It was

accompanied by a substantial enhancement in H2 evo-

Volatile Fatty Acid Estimation lution up to 130 ml, i.e., a 2-fold increase over control.

At 5 % TS VWS, maximum H2 evolution was observed

VFA analysis was carried out from 1.0 ml sample taken in with BC7. Although H2 component of the biogas did not

1.5 ml vials. 2–3 drops of ortho-phosphoric acid (25 % change much, however, BC7 resulted in 2.6-fold increase

v/v) were added to each vial for sample preservation. VFA in H2 yield. Further increase in the concentration of TS

concentrations were determined using gas chromatograph in the VWS to 7 % led to increase in the net evolution

(GC 6890 N) equipped with ﬂame ionization detector. A of biogas (up to 530 ml), however, it was not accom-

capillary column, DBWAXetr (30 m 9 53 lm 9 1 lm panied by a proportional increase in H2 evolution. H2

ID) was used for analysis. The oven, injector and detector evolution was almost similar to that recorded with con-

temperatures were 140, 220 and 230 C, respectively. trol. 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

Results also be remarked that high TS also inﬂuence the meta-

bolic process of H2 evolution since the H2 component of

In natural conditions, the biowaste containing biomacromol- the biogas was also reduced compared to those observed

ecules like carbohydrates, fats and proteins can be degraded at 3 and 5 % TS slurries. It may be reasonable to con-

by bacteria producing hydrolytic enzymes. Screening of 1,000 clude that BC7 is effective in hydrolyzing the VW

bacteria allowed us to select 50 having high activities for resulting in 2.0- to 2.6-fold enhancement in H2 yield

amylase, lipase and protease. Further evaluation of the (Table 1).

enzymatic activities at a wider pH range enabled us to select In contrast to VW, the fermentation process was more

11 having at least one of these enzymatic activities in the pH effective with FW, which may be due to easily digestible

range 5.0–9.0. Finally eleven bacteria so selected were iden- components of the waste. Here, 250 ml of FWS without

tiﬁed as: Bacillus aryabhattai MBG46 (KJ563237); Acine- any hydrolysis resulted in the net evolution of 685 ml

tobacter sp. MBG50 (KJ563241) and A. haemolyticus biogas at 3 % TS. It contained 225 ml H2, equivalent to

MBG52 (KJ563243); Exiguobacterium sp. MBG53 32.8 % of the total biogas (Table 1). The effective H2

(KJ563244) and E. indicum MBG54 (KJ563245); Pseudo- yield was 30 l/kg TS fed. Hydrolysis with different bac-

monas mendocina strains MBG51 (KJ563242), MBG57 terial cultures resulted in gain in H2 yields, ranging up to

(KJ563248), MBG58 (KJ563249) and P. pseudoalcaligenes 85 l/kg TS fed with BC6. It was accompanied by a higher

MBG45 (KJ563236); Stenotrophomonas koreensis MBG44 H2 component of 62.1 %. Hydrolysis of FW resulted in

(KJ563235) and Sphingobacterium daejeonense MBG47 2.8-fold enhancement in H2 yields. Further increase in TS

(KJ563238) (Table S1). Of the 11 mixed bacterial cultures: of the slurry did not prove helpful in improving the H2

BC6, BC7, BC8 and BC10 were found to be effective for production process. H2 yields of 28–38 l/kg TS fed

hydrolyzing VW as indicated by the total volatile fatty acid from 5 % TS pretreated with BC1, BC6 and BC8 and

composition, over a period of 5 days of incubation (Tables 24–36 l/kg TS fed from 7 % TS slurries were higher than

S3). On the other hand, mixed bacterial culture designated as their respective controls. BC6 and BC8 were the most

BC1, BC6, BC8 and BC9 were found to be effective for effective mixtures of hydrolytic bacteria, which enhanced

hydrolyzing FW under similar incubation period. H2 yield. (Table 2).

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Indian J Microbiol (July–Sept 2014) 54(3):262–267 265

Table 1 Hydrogen producing abilities of mixed H -producersa from prehydrolyzed biowastes

2

Mixed bacterial Biogas H2 Biogas H2 Biogas H2

culture volume (ml) b volume (ml) volume (ml)

Vol (ml) % Yield 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

a Deﬁned mixed microbial culture of H -producers (MMC4)

2

b H2 production in l/kg Total solids fed

c Total solids

d No mixed bacterial culture added

Table 2 Comparison of methane yields from prehydrolyzed biow- routes-Direct and Indirect (preceeded by H2 production).

astes by direct and indirect biomethanation The impact of hydrolysis by mixed bacterial cultures on

Mixed Direct biomethanationa Indirect biomethanation biomethanation through both the routes was distinctly

bacterial observed.

3 % 5 % 7 % 3 % 5 % 7 %

culture

TSb TS TS TS TS TS

Direct Biomethanation

Vegetable waste

Controlc 20.0 26.5 36.4 8.0 13.4 8.6 Biomethanation of VWS (250 ml) was observed to vary

BC6 17.5 29.3 25.0 7.3 15.2 11.4 from 20 to 36.4 l/kg TS fed. It constituted around 61 % of

BC7 61.7 31.0 15.7 28.3 16.6 10.0 the total biogas produced over a period of 15 days. In

BC8 26.7 42.2 31.4 12.7 13.5 10.7 contrast, hydrolysis of VW by 11 different mixed bacterial

BC10 17.5 36.4 38.6 6.0 14.7 11.4 culture (BC1–BC11) having high hydrolytic enzyme

Food waste activities proved effective in improving the biomethanation

Control 55.0 26.0 20.3 26.3 16.7 10.7 process. The four BCs: BC6, BC7, BC8 and BC10 were

BC1 50.0 37.4 32.1 30.0 19.4 12.1 chosen for further studies as the VFA content of these

BC6 63.3 54.5 24.3 31.7 17.5 13.6 hydrolysates were quite high and consistent. BC7 proved to

BC8 46.7 45.2 42.1 21.4 19.2 12.8 be the most efﬁcient with a ﬁnal CH4 yield of 61.7 l/kg TS

BC9 48.3 52.3 37.9 31.5 18.5 12.1 fed at 3 % TS VWS. The net enhancement in CH4 yield

a was 3-fold. Although CH4 yields were higher at 5 and 7 %

CH4 production in l/kg Total solids fed

b TS VWS compared to control, however, BC7 treatment

Total solids

c resulted in lower CH4 yields at 5 and 7 % TS VWS,

No mixed bacterial culture added

compared to 3 % TS VWS. Hence, we may conclude that

Methane Evolution hydrolysis of VWS with BC7 is effective at 3 % TS VWS

in comparison to untreated VWS.

Biomethanation has been a suitable post H2 treatment Direct biomethanation of untreated FWS was more

process for effective utilization of biowastes. CH4 evolu- effective in comparison to VWS. Here, the net CH4 yield

tion was observed on hydrolysed biowastes through two was 55 l/kg TS fed. Biomethanation was found to decline

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266 Indian J Microbiol (July–Sept 2014) 54(3):262–267

from FWS (control) at higher TS concentrations of 5 and which are favorable to the bacteria in question and at the

7 %, where the CH4 yield were found to be 26.0 and 20.3 l/ same time prevent others from growing. Invariably, it

kg TS fed, respectively. Hydrolysis of FW with well demands sterile feed material. In the case of fermentation

deﬁned mixed bacterial cultures was quite effective with of biowastes, it is difﬁcult to sterilize the feed [19]. Hence,

BC6, which enabled us to improve the biomethanation the presence of inherent bacteria continues to pose a threat,

process to yield 63.3 l CH4/kg TS fed at 3 % TS FWS. as they metabolize the organic matter into undesirable by-

Although, the CH4 yields were higher than the control even products. Ecobiotechnological approach relies on the use of

at 5 % and 7 % TS FWS, however, these values were robust bacteria with well deﬁned activities. Mixed deﬁned

relatively lower than those obtained from 3 % TS FWS bacteria as inoculum enhances the chance of survival of at

with BC6. least one or two types of bacteria, which are sufﬁcient to

ensure consistency and reproducibility of the process. This

Indirect Biomethanation approach has been exploited previously for producing

polyhydroxyalkanoates [ 14, 18]. In the present work, the

The effect of pretreatment with hydrolytic bacteria was whole process is quite complex. For complete degradation

evident even with efﬂuent emanating from H2 production of biowastes, coordinated activities of different set of

process. Untreated VWS, resulted in 8.0–13.4 l CH4/kg TS bacteria are operative: (1) Hydrolytic bacteria (2) H2 pro-

fed at 3–7 % TS concentrations. In contrast, VWS sub- ducers and 3) methanogens [ 1]. The major metabolic lim-

jected to hydrolysis by BC7 proved effective even in itations are: (1) The hydrolytic process, and (2) H2 transfer

indirect biomethanation process, with a net gain of 1.16- to reaction [7]. For hydrolysis of organic matter, the need is to

3.53-fold. The best results were observed at 3 % TS VWS. have well deﬁned bacteria with high hydrolytic activities.

Incidentally, the same combination was the most efﬁcient And such bacteria are present in small numbers in natural

even via direct biomethanation. On the other hand, FWS populations. The other issue is the fact that H2 produced by

slurry was also digested most efﬁciently by BC6 in the one set of bacteria is immediately quenched by methano-

cases of direct and indirect biomethanation. Via indirect gens, such that there is little or no net evolution of H2 [15].

biomethanation, a 1.2-fold enhanmcement in CH4 yield In the present study, bacteria with high relative enzyme

was recorded in comparison to its respective control. efﬁciencies were mixed in equal proportions. Of the 11

Most of the biological wastes undergo AD process with such mixed bacterial cultures, BC7 and BC6 found to be

no net evolution of H2. It is primarily because of inter effective in enhancing H2 yield from vegetable waste and

species H2 transfer phenomenon. Since H2 generation food waste to the extent of 1.9- and 2.8-fold, respectively,

results in accounting for 35 % of the total energy present in in comparison to control. Hydrolysate generated by BC6

the organic matter content of the feed, it becomes imper- and BC7 were effective in 1.15- and 3.1-fold improvement

ative to subject the efﬂuent from H2 stage to methanogens. in CH4 yield. In the case of hydrolysate initially subjected

Here, we can expect a maximum of 65 % of the energy as to H2 producers and subsequently by methanogens, BC7

CH4, with respect to CH4 yield observed via direct bio- resulted in 3.53-fold and BC6 led to 1.2-fold enhancement

methanation as 100 %. In VWS (3 % TS) and BC7 com- in CH4 yields. Thus under all conditions, hydrolytic bac-

bination, we observed 63.3 l CH4/kg TS fed via direct terial mixture proved effective in enhancing the processes

biomethanation. Via indirect biomethanation, we can for generating bioenergy. Secondly, the split of H2 stage

expect a CH yield of 41.1 l. Since we could observe VWS and CH stage allowed us to overcome the problem of H -

4 4 2

(3 % TS) and BC7 combination to generate 31.7 l CH4, it energy transfer [8, 23]. These ﬁndings provide an evidence

is equivalent to 77 % of the expected value. On the other that hydrolysate of organic matter can be easily converted

hand, with FWS (3 % TS) and BC6 combination, we could into bioproducts of high economic values. Combining these

generate 61.7 l CH4/kg TS fed via direct biomethanation metabolic pathways may enable complete and efﬁcient

and 28.3 l CH4/kg TS fed via indirect biomethanation. degradation along with sustainability.

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. Conclusion

Hydrolysis of biowastes with deﬁned bacterial cultures

Discussion helps to improve H2 and CH4 production from VW at 3 %

by 1.9- and 3.1-fold, whereas with FW the corresponding

Bioprocesses involving single bacterial cultures are always enhancements were 2.83- and 1.15-fold, respectively. FW

at the risk of getting contaminated [14]. In order to run the is a better feed for H2 (5-fold) compared to VW. 3 % TS is

process continuously there is a need to maintain conditions the best concentration observed for H2 and CH4 generation

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Indian J Microbiol (July–Sept 2014) 54(3):262–267 267

with both VW and FW. The effective H2 yields were 17 10. Molino A, Nanna F, Ding Y, Bikson B, Braccio G (2013) Bi-

and 85 l/kg TS fed, where as effective CH4 yields were omethane production by anaerobic digestion of organic waste.

Fuel 103:1003–1009. doi: 10.1016/j.fuel.2012.07.070

61.7 and 63.3 l/kg TS fed from VWS and FWS, respec- 11. Patterson T, Esteves S, Dinsdale R, Guwy A, Maddy J (2013)

tively. Hydrolysis thus proved beneﬁcial in achieving cost- Life cycle assessment of biohydrogen and biomethane production

effective conversion of waste to energy. and utilisation as a vehicle fuel. Bioresour Technol 131:235–245.

doi:10.1016/j.biortech.2012.12.109

Acknowledgments The authors wish to thank the Director of CSIR- 12. Premier GC, Kim JR, Massanet-Nicolau J, Kyazze G, Esteves

´

Institute of Genomics and Integrative Biology (IGIB), Delhi, CSIR- SRR, Penumathsa BKV, Rodrıguez J, Maddya J, Dinsdale RM,

WUM (ESC0108) and Department of Biotechnology (DBT-BT/PR- Guwya AJ (2013) Integration of biohydrogen, biomethane and

11517/BCE/08/709/2008) Government of India for providing neces- bioelectrochemical systems. Renew Energy 49:188–192. doi: 10.

sary funds and facilities. PK is thankful to CSIR for granting Senior 1016/j.renene.2012.01.035

Research Fellowship. 13. Xiao L, Deng Z, Fung KY, Ng KM (2013) Biohydrogen gener-

ation from anaerobic digestion of food waste. Int J Hydrogen

Energy 38:13907–13913. doi: 10.1016/j.ijhydene.2013.08.072

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9. Chuang YS, Lay CH, Sen B, Chen CC, Gopalakrishnan K, Wu 0960-8524(92)90003-G

JH, Lin CS, Lin CY (2011) Biohydrogen and biomethane from 23. Kalia VC, Joshi AP (1995) Conversion of waste biomass (pea-shells)

water hyacinth (Eichhornia crassipes) fermentation: effects of into hydrogen and methane through anaerobic digestion. Bioresour

substrate concentration and incubation temperature. Int J Technol 53:165–168. doi: 10.1016/0960-8524(95)00077-R

Hydrogen Energy 36:14195–14203. doi: 10.1016/j.ijhydene.2011.

04.188

123

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2014年5月24日星期六

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