Research Article Biogenic Hydrogen Conversion of De-Oiled ...downloads.hindawi.com › journals ›...

Research ArticleBiogenic Hydrogen Conversion of De-OiledJatropha Waste via Anaerobic Sequencing Batch ReactorOperation Process Performance Microbial Insightsand CO2 Reduction Efficiency

Gopalakrishnan Kumar12 and Chiu-Yue Lin134

1 Department of Environmental Engineering and Science Feng Chia University Taichung 40724 Taiwan2 Laboratory for Research on Advanced Processes for Water Treatment Instituto de IngenierıaUnidad Academica Juriquilla Universidad Nacional Autonoma de Mexico Boulevard Juriquilla 3001 QRO 76230 Mexico

3 Green Energy Development Center Feng Chia University Taichung 40724 Taiwan4Masterrsquos Program of Green Energy Science and Technology Feng Chia University Taichung 40724 Taiwan

Correspondence should be addressed to Chiu-Yue Lin cylinfcuedutw

Received 20 August 2013 Accepted 24 December 2013 Published 5 February 2014

Academic Editors M Ameri and M Q Fan

Copyright copy 2014 G Kumar and C-Y Lin This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

We report the semicontinuous direct (anaerobic sequencing batch reactor operation) hydrogen fermentation of de-oiled jatrophawaste (DJW) The effect of hydraulic retention time (HRT) was studied and results show that the stable and peak hydrogenproduction rate of 148 LLlowastd and hydrogen yield of 87mLH

2g volatile solid added were attained when the reactor was operated

at HRT 2 days (d) with a DJW concentration of 200 gL temperature 55∘C and pH 65 Reduced HRT enhanced the productionperformance until 175 d Further reduction has lowered the process efficiency in terms of biogas production and hydrogen gascontent The effluent from hydrogen fermentor was utilized for methane fermentation in batch reactors using pig slurry andcow dung as seed sources The results revealed that pig slurry was a feasible seed source for methane generation Peak methaneproduction rate of 043 L CH

4Llowastd and methane yield of 205mL CH

4g COD were observed at substrate concentration of

10 g CODL temperature 30∘C and pH 70 PCR-DGGE analysis revealed that combination of celluloytic and fermentative bacteriawere present in the hydrogen producing ASBR

1 Introduction

Two crucial factors that play important role towards sus-tainable development for the global prosperity are continu-ous energy supply and environmental-related issues Energyderived from fossil fuels is dominating the energy sector inrecent decades however depletion of these reservoirs hasmade an urge to find alternative fuel sources to fulfill theworldrsquos energy demand which would become a big issue inthe near future Among the proposed alternative fuels such ashydrogen ethanol butanol andmethane hydrogen stands asan extraordinary and promising fuel mainly due to its uniquecharacters like high energy yield (122 kJg) and water vapor

release upon combustion which are representing the carbonneutral property In addition hydrogen use in fuel cells forthe production of electricity has been demonstrated widelyaround the globe [1 2]

Lignocellulose or solid wastes are proven to be a promis-ing feedstock for biological hydrogen production by vari-ous research groups because of their vast availability easycollection process and high content of cellulose (a feasiblesubstrate for hydrogen producing microorganisms) [3ndash6]The biodiesel energy sector generates an enormous amountof solid waste especially when jatropha biomass is used asfeedstock The recalcitrant nature and poisonous substancespresent in this solid waste make the treatment process not

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 946503 9 pageshttpdxdoiorg1011552014946503

2 The Scientific World Journal

economically feasible [7 8] Thus the utilization of thiswaste for hydrogen fermentation has dual benefits wastemanagement and energy generation (mainly hydrogen gasadditionally ethanol and volatile fatty acids would be alsogenerated as fermentation coproducts)

Development of hydrogen fermentation process for lig-nocellulose waste needs proper operational conditions andstrategies for example with such solid nature performingcontinuous operation (CSTR) would be of great difficultydue to the possibilities of improper flow in the pumpandor pipelines in other terms the accumulation of solidsin a period of time Thus ASBR (anaerobic sequencingbatch reactor) operation has been suggested and shownas a promising way to treat such solid biomass feedstockand pure substrate such as starch [9ndash11] The generationof DJW is abundant due to the expansion of biodieselindustries Besides a solid portion of 3 ton remained in everyton of biodiesel extraction So development of a hydrogenfermentation form DJW could attain more interest thanother lignocellulose feedstock The main advantages of thisfeedstock are the easy collection and desizing processeswhereas other feed stocks like rice straw wheat strawcorn and so on require energy during the size reductionMoreover development of hydrogen fermentation for DJWincreases the commercial benefits of the biodiesel industrywhereas both biodiesel and biohydrogen could be obtainedfrom a low-cost waste that is jatropha at one time inputThus here we demonstrated a process to treat DJW usingASBR operation In addition the feasibility of the effluentfrom H

2fermentor to generate methane to enhance the total

energy production is also evaluated

2 Materials and Methods

21 DJW Substrate and Anaerobic Mixed Microflora DJWused in this study was collected from a biodiesel industryusing Jatropha biomass and located in central Taiwan Thecellulosic content was analyzed as 423 of fermentablesugars (141 cellulose and 282 of hemicellulose) usingFIBERTEC 1020 (M6) analyzer as mentioned elsewhere [12]Anaerobic mixed microflora was obtained from a municipalwastewater treatment plant Substrate and seed sludge werestored in a refrigerator at 4∘C before being used in the exper-iments To inactivate the hydrogen consuming methanogensthe sludge was heat treated for 30minutes at 95∘C in a boilingwater bath Characteristics of the DJW and seed sludge weredescribed in our previous study [12]

22 Reactor (ASBR) Startup and Operation A schematicdiagram of the ASBR is shown in Figure 1 The reactor wasstarted up by feeding glucose (10 gL) initially to enrichthe hydrogen producers (Run 1) There forward DJW ata concentration of 100 gL was fed for the adaptation ofhydrogen producers at an agitation speed of 150 rpm andoperational temperature of 55∘CMeanwhile the effluent wascollected and fed into the reactor along with fresh substrateto avoid the loss of biomass in the form of effluent (Run 2ndash4)After Run 4 it was stopped due to the accumulation of more

amounts of solid particles inside the reactor Thereafter onlyfresh substrate was fed into the reactor The nutrient solutionadded was prepared by following the Endo formulation [13]with slight modifications as mentioned [11] ORP and pHwere monitored using an automated pH ORP panel pH wascontrolled using 1N NaOH buffer

23 Batch Methane Fermentation Methane production wasstudied in batch vials (capacity-125mL) with a workingvolume of 60mL by adding 40mL of hydrogen fermentationeffluent (substrate concentration 10 g CODL) 12mL of seedsludge (either cow dung or pig slurry) 1ndash3mL for pHadjustment (either 1 NNaOHorHCl) 5mL nutrient solution(to enhance the growth of anaerobic bacteria) and the restwas tap water The nutrient solution added was prepared byfollowing the Endo formulation as mentioned [13] The finalpH was adjusted to neutral (70) Then the batch vials werekept in a reciprocal air-bath shaker at 150 rpm at 30∘C Thevolume and composition of gas were monitored daily Fer-mentation was carried out until the gas production becomeszero All experiments were carried out at least triplicates andthe values represented are the mean of triplicate values

24 Analytical Procedures The analytical procedures usedto determine pH total solid (TS) COD and VSS werefollowed by APHA 1995 [14] The volatile fatty acids (VFAs)and ethanol concentrations were analyzed by HPLC Thegas composition was analyzed with a gas chromatographhaving a thermal conductivity detector (China Chromato-graph 8700T)Other experimental procedureswere indicatedin our previous studies [15 16] For total carbohydrateconcentration anthrone-sulphuric acid method was adapted[17] Biogas production was measured in a periodic intervalusing various volumes of glass syringes depending on theexpected biogas production fitted with hypodermic needlesas described by Owen et al [18]

25 Calculations ThemodifiedGompertz equationwas usedto estimate methane production potential and methaneproduction rate (Sigma plot software 100 Systat SoftwareInc USA)

119867(119905) = 119875 sdot exp minus exp [119877119898sdot 119890

119875

(120582 minus 119905) + 1] (1)

where119867(119905) is the cumulative methane production (mL) 119875 isthe methane production potential (mL) 119877

119898is the maximum

methane production rate (mLh) 119890 is 271828 120582 is the lagphase time (h) and 119905 is the cultivation time (h) Methaneproduction rate (MPR L H

2L-d) was defined as the value

of 119877119898divided by the reactor volume (006 L) and multiplied

by one day (24 h) methane yield (MY mL CH4g COD)

was defined as methane produced per gram chemical oxygendemand (COD)

26 PCR-DGGE DNA Sequencing and Phylogenetic Anal-ysis Total genomic DNA was isolated using the Blood ampTissue Genomic DNA Extraction Miniprep System (Vio-gene Taiwan) following themanufacturerrsquos instructionsThe

The Scientific World Journal 3

Effluent and sampling

Influent

Gas collector

3N NaOHsolution

pH panel

Figure 1 ASBR schematic diagram

isolated DNA was confirmed by agarose gel electrophoresis(075) and the samples were stored at minus20∘C for furtherPCR reactions The PCR mixtures (50 120583L) contained eachdeoxynucleoside triphosphate at a concentration of 200mM15mM MgCl

2 each primer at a concentration of 02mM

125U of Taq DNA polymerase (Promega Madison WIUSA) and the PCR buffer provided with the enzyme Theamplification consisted of a DNA denaturing step at 94∘Cfor 5min followed by 30 cycles of denaturation at 94∘C for1min 1min annealing at 55∘C for EUB968gc-UNIV1392rand extension at 72∘C for 1min The cycling included a finalextension step at 72∘C for 10min to ensure full extension ofthe product All PCR operations were performed with anautomatic thermal cycler iCyclerTM (Bio-Rad CA USA)PCR products were analyzed by electrophoresis at 100V for30min through 15 (wtvol) agarose gel The amplified PCRproductswere used for denatured gradient gel electrophoresis(DGGE) analysis The DGGE profile of the PCR-amplifiedDNA was obtained following the method mentioned [19]using a DCode Universal Mutation Detection System (Bio-Rad USA) The 6 (wv) of acrylamide solution was usedto cast a gel with denaturant gradients ranging from 40 to60 Electrophoresis was conducted in a 1X TAE (TrisaceticacidEDTA) buffer solution at 80V and 60∘C for 12 h Thegels were stained for 10min with ethidium bromide andvisualized under UV radiation The number of operationaltaxonomic units (OTU) for each sample was defined asthe number of DGGE bands The selected DGGE bandson the gel was excised with a sterile razor blade placedin 15mL centrifuge tube and add 50 120583L of sterile 1X TAEbuffer and then incubated overnight at 4∘C to reclaim theDNA Additional PCR-DGGE analyses were performed toensure the purity of reclaimed DNA Analysis of targetedDNA sequences was performed in a DNA sequencer (TriBiotech Taiwan) The bioinformatic analysis was carriedout by using the tool BLASTN facility available from NCBIwebsite (httpwwwncbinlmnihgovBLAST) to align thepartial 16S rDNA sequences with the reference microorgan-isms available in the GenBank database These sequences

were further aligned with the closest matches found in theGenBank Database with the CLUSTALW function of MEGA4 [20]

27 Accession Numbers 16s rDNA (4) sequences were sub-mitted to GenBank and the accession numbers of thegene sequences submitted to GenBank included KC503758-KC503761

3 Results

31 Hydrogen Production Performance in ASBR Effect ofHRT The production performance of the ASBR is shown inFigure 2The performance has shown fluctuations during thestart-up period However a steady state condition has beenreached after 30 days while the HRT was 2 days Initiallythe reactor was fed with glucose and DJW (100 gL) in abatch mode to stabilize and enrich the microbial populationin order to utilize the complex substrate The adaptation ofmicrobes to the new environment after 15 days attributed tothe gradual increase in hydrogen production The operationstrategies were presented in Table 1 Shortened HRT hasenhanced performance of the hydrogen production rate(HPR) from 056 plusmn 013 LL-d to 148 plusmn 004 LL-d

The biogas production rate (BPR) and hydrogen contentwere shown in Table 2 ASBR strategy has been previouslyreported as it could increase the biomass concentrationUsually biomass loss occurs in the CSTR when operated atlower HRT This is the reason many authors have suggestedASBR operation for better performances especially whilesolid substrate is used in order to avoid the blockings in thepump during the continuous feeding [9ndash11]Themain advan-tage of thermophilic temperature in hydrogen production isthe reduced solubility of hydrogen which would lower thehydrogen inhibition Thus thermophilic temperature optedin this study also contributes towards the better performanceThe feed provided intermittently to the reactor had avoidedthe oxygen inhibition from the influent This proved the


Days0 10 20 30 40

Days0 10 20 30 40

Days0 10 20 30 40

Days0 10 20 30 40

Days0 10 20 30 40

0

1

2

3

4

0

1

2

3

BPRHPR

01020304050

0

20

40

60

02468

101214

pH

0

2

4

6

ORP

(mV

)

0100200300400500

pHORP

Batch mode

ASBR

HRT

(day

s)

000510152025

Prod

uctio

n ra

te (L

L

HPR

(LL

lowastd)

lowastd)

H2

H2

CO2

CO2

HY

(mL

H2g

VS

adde

d)

Figure 2 ASBR reactor performances

potential of this system and turned as the peak HPR of148 plusmn 004 LLlowastd However the yield is still low at steadystate conditions and it is similar to our previous study whichprovided the same results in batch experiments [12] Peakhydrogen yield (HY) of 87 plusmn 03mLH

2g VS added was

observed at steady state conditions

Table 1 Operation strategies of the ASBR

Run Operationtime HRT (d) Substrate loading rate

(SLR)

1 1ndash48 hrs(2 days) Batch (2 days) Glucose (10 gL)

2 2 days Batch (2 days) DJW (100 gL)3 5 days 25 DJW (200 gL)4 5 days 25 DJW (200 gL)5 10 days 2 DJW (200 gL)6 10 days 175 DJW (200 gL)7 10 days 15 DJW (200 gL)

Table 2 Biogas production performance of the ASBR

Run BPR(LLlowastd) H2

HPR(LLlowastd)

HY(mL H2g VS added)

1 432 plusmn 011 5161 plusmn 070 22 plusmn 010 2231 plusmn 89lowast

2 280 plusmn 012 4463 plusmn 070 12 plusmn 010 146 plusmn 03






lowastRepresents mL H2g Glucoseadded

Table 3 Effluent and SMP analysis at steady state

Conditions HRT 2 days SC 200 gL pH 65 T 55∘CEffluent analysis (gL)

TCOD SCOD TC TS144 plusmn 21 112 plusmn 13 93 plusmn 18 23 plusmn 08

SMP analysis (gL)EtOH HAc HBu HPr068 plusmn 04 18 plusmn 02 23 plusmn 04 084 plusmn 06

TCOD total chemical oxygen demand SCOD soluble COD TC totalcarbohydrate TS total solids EtOH ethanol HAc acetate HBu BuytrateHPr Propionate

32 Soluble Metabolites The soluble metabolic products(SMP) analysis at the steady state condition revealed thatacetic and butyric acids were the main intermediates pro-duced during the DJW fermentation besides propionate andethanol were detected at low level Mainly propionic acid andlactic acid are considered as an undesirable side-product ofdark-fermentative biohydrogen technology Butyric acid wasdetected at higher amount (23 plusmn 04 gL) revealing that thefermentation was mediated through butyrate which is favor-able for hydrogen production as reported in other studies(Table 3) Such an acid dominated pathway led to the efficientbioH2production in many other studies as well in [15 16]

The effluent has been collected and it comprised of about93 plusmn 18 gL of total carbohydrate and 144 plusmn 21 gCODL astotal chemical oxygen demand Total solids was also shown(Table 3) Similar kinds of results were reported during ASBRoperation of marine algae Besides CH

4fermentation of the


Table 4 Methane fermentation of H2 fermentor effluent

Seed source Final pH Cumulative biogas Cumulative CH4 MPR (mLLlowastd) MY (mL CH4g COD)Pig slurry 68 plusmn 12 368 plusmn 44 2010 plusmn 26 4253 plusmn 51 205 plusmn 05

Cow dung 70 plusmn 10 259 plusmn 121 1393 plusmn 57 3486 plusmn 42 137 plusmn 08

Fermentation time (hrs)0 200 400 600

Volu

me

(mL)

0

50

100

150

200

250

BiogasCH4

(CD)

(a)

Fermentation time (hrs)

0 200 400 600

Volu

me

(mL)

0

100

200

300

BiogasCH4

(PS)

(b)

Figure 3 Batch CH4fermentation profiles of pig slurry (PS) and cow dung (CD)

H2fermentation effluent is suggested in order to increase the

total energy production of the process [9]

33 CH4Fermentation via Hydrogen Fermentation Effluent

The CH4batch fermentation results showed that the effluent

could be digested and converted intomethane PeakMPRandMYof 4253plusmn51mLCH

4Llowastd and 205plusmn05mLCH

4g COD

were attainedwhile using pig slurry as a seed source (Table 4)The results are presented in Table 4 Cow dung also provideda MPR and MY of 3486 plusmn 42mL CH

4Llowastd and 137 plusmn

08mL CH4g COD respectively The biogas and methane

production profile of both the seed sources (cow dung andpig slurry) were shown in Figure 3 Pig slurry has been shownas a good seed source for methane fermentation than cowdung for the effluent from hydrogen producing ASBR usingDJW These results indicate that the process proposed couldenhance the energy production of the total process

34 Microbial Community Composition In order to detectthe dominant microorganisms present in the reactor duringthe steady state operation samples were taken at 34th dayof operation while the hydrogen production was shown asthe maximal DGGE band pattern has been obtained byusing the primer set EU968gc-UNIV1392r could reveal thestructure composition of the microbial communities in themixed cultures and is based on the V6 region of the 16srRNA gene Based on DGGE profile 4 distinct bands werenoted These bands were excised and purified to determinetheir 16s rRNA sequencing analysis as shown in Table 5and Figure 4 The evolutionary history was inferred using

the Neighbor-Joining method [21] A total of 4 operationaltaxonomic units (OTU) were obtained (Table 5) in which 2of them belonged to the phyla Firmicutes All the bacteriawere distantly related with gt95 to Clostridium sensu strictosuch asClostridium thermopalmariumTheother 2 bands alsobelonged to the same phyla but the species are identified asBacillus ginsengihumi and Bacillus coagulans

4 Discussions

41 Effect of HRT on Process Performance During ASBRoperation the gaseous components were analyzed as H

2

and CO2and methane was not detected until the end of

fermentationThis indicates that the heat treatment of sewagesludge strongly suppressed the methanogenic activity as wementioned in our previous study [22] Reducing hydraulicretention time (HRT) resulted in the enhanced hydrogenproduction performance [11] and is also one of the methodsto develop a particular group of stable hydrogen producersThe production performance shown in this study could besupported by other studies that employed similar ASBRoperation for lignocellulose-basedwaste such asmarine algaeand sweet sorghum syrup [9 23]The yield is relatively low inthe fermentative hydrogen production process as discussedearlier [24] Generally in dark fermentation themaxima yieldthat could be achieved is only 33 even pure sugar such asglucose is used the main reason for this drawback is thedistribution of electrons to other intermediate products suchas acetate where only 10 of the stoichiometry could beachieved while the substrate conversion rate is more than


Table 5 Affiliation of DGGE fragments determined by their 16S rDNA and isolated microorganisms

Sequence no Family Closest match Homology () Sequence length (bp)

1 FirmicutesBacillus ginsengihumistrain Gsoil 114 99 422(Accession no NR 041378)

2 FirmicutesBacillus coagulansstrain NBRC 12583 98 419(Accession no NR 041523)

3 FirmicutesClostridium thermopalmariumstrain BVP 99 411(Accession no NR 026112)


S4

S3

S2

S1

40

60

Bacillus shackletonii strain LMG 18435

Bacillus ginsengihumi strain Gsoil 114

Bacillus coagulans 2ndash6 strain

Bacillus coagulans strain NBRC 12583

Bacillus methanolicus strain NCIMB 13113

Clostridium thermopalmarium strain BVP

Clostridium thermobutyricum

Clostridium haemolyticum strain ATCC 9650

Clostridium noyi NT strain

Clostridium noyi strain JCM 1406

S2

S1

S3

S4

100

97

83

100

98

51

100

100

100

100

100100

01

EColi

Figure 4 PCR-DGGE profile of the microbial community and Phylogenetic tree of the respective OTUs

98 In our study the substrate conversion rate in termsof total carbohydrate is only about 50 as indicated in ourprevious report [12]

Generally hydrogen fermentation is associated with theproduction of intermediate acid production The productionof VFAs or solvents during the anaerobic fermentationprocess is often a crucial signal inmonitoring the feasibility ofhydrogen producing cultures [25 26] While glucose is usedas a substrate the maximum theoretical yields of 4mol and

2mol hydrogenwould be produced via acetic and butyric acidpathways as shown in (2) and (3) respectively

C6H12O6+ 2H2O 997888rarr 2CH

3COOH + 2CO

2+ 4H2 (2)

C6H12O6997888rarr CH

3CH2CH2COOH + 2CO

2+ 2H2 (3)

In recent years the hydrogen fermentation effluent isutilized for the production of methane or hydrogen bymeans of anaerobic digestion or photofermentation as it


could effectively add more amount of energy to the process[9 27] The effluent (rich in organic acids) was utilized formethane fermentation since many studies reveal that VFAsare a good source formethane fermentation especially in two-stage fermentation [28] Thus the effluent was employed inbatch reactors to generate methane using two types of seedinoculum as cow dung and pig slurry In fact cow dungand pig slurry are good source for methane generation asindicated in other studies [28 29] In our study also we havedemonstrated that hydrogen fermentation effluent has thepotential for themethane generation which in turn increasesthe total energy efficiency of the process

42 Microbial Insights during the ASBR Operation In themicrobial insights responsible for hydrogen fermentationtwo bands were closely related to Clostridium thermopalmar-ium reported as the potential hydrogen producing bacterium[30] which produces hydrogen from cellulose since it con-tains cellulolytic enzymes The strains of genus Clostridiumare able to produce acetate and butyrate as well as hydrogenduring anaerobic fermentation using glucose as substrate [31]Besides composition of DJW is mainly of cellulose (polymerof glucose) and hemicellulose (such as xylose arabinose andcellobiose) [22] The other genus belongs to Bacillus and thesame phyla Firmicutes and the species were identified asBacillus ginsengihumi and Bacillus coagulans Bacillus coagu-lans is reported as the producer of lactic acid from hemicellu-lose extracts [32] at slightly thermophilic temperature due tothese reasons only these twomajor organisms were present inthe reactor during the steady state conditions The inoculumsource (sewage sludge) selected in this present study was arich source of hemicelluloytic and cellulolytic bacteria asreported previously [33] The PCR-DGGE based sequenceanalysis revealed the presence of dominant butyratemediatedhydrogen producing bacteria present in the reactor at steadystate However the PCR-DGGE does not reveal the quantityof the microbial populations used for the qualitative analysisof the organismrsquos identification The heat treatment methodhas been applied mostly for eliminating homoacetogens withconsequent microbial community reduction Though manynonspore hydrogen producers could be destroyed by heat itenhances the growth of Clostridial spp which in turn resultsin higher hydrogen production efficiency Moreover Bacillusis also reported as spore formers while the unfavorable con-ditions occurred Thus the combination of these cellulolyticand fermentative bacteria supported the possible pathway ofhydrogen generation

43 COD Balance and CO2Reduction Efficiency The COD

balance of the system has been shown in the Figure 5 It canbe seen that nearly 85 of the COD has been balanced theremaining percentage would be the trace amount of SMPs(like butanol vareate etc) which were not detected by theGC-FID Peak HPR and HY of 73 LKg-d and 83 LKg DJWwere attained during the ASBR operation which accountsfor the energy production of 94 Gjhay for the biohydrogenproduction (carbon neutral) According to our previousstudy [28] by replacing the hydrogen energy produced fromthis process the amount of CO

2reduction was analyzed as

H2fermentor (ASBR)

H2 1 g COD

Effluent

By products

85 CODbalance

Influent (200 gL)TCOD 286 g COD TCOD 144 g COD

(SMP) (887 gCOD)

Figure 5 COD balance of the ASBR system

025 tons for coal 02 tons for fuel oil and 017 tons for thenatural gas respectivelyThis proves that the ASBR operationof DJW to produce hydrogen is an environmentally friendlyprocess with the possibilities towards the greener and cleanerenvironment

44 Significance of the Results Outlook and Suggestions Theoperation strategy for a lignocellulose based waste is animportant step towards its commercialization of the technol-ogy especially industrial waste like DJW Increasing the totalenergy values (biodiesel biohydrogen and biomethane) froma single input low-cost waste that is jatropha biomass wouldbe a feasible solution for the future energy demands Addi-tionally biodiesel production from jatropha biomass wasdemonstrated previously by various authors as a promisingfuture energy carrier [34ndash36]

Comparison of the various lignocellulose biomass con-versions to hydrogen using ASBR operation was given inTable 6 It could be seen that in ASBR operation HRT ismostly dependent on the substrate nature For example DJWand marine algae are solid in nature have long HRT dueto the long adaptation period of microbes to utilize thesubstrate The production performance varied significantlyamong the substrates and themicrobial source usedTheHPRvalue of the DJW-ASBR operation is comparable with theother cellulosic wastes However the HPR and HY valuesof POME are higher than other values reported This ismainly due to the high amount of nutrients present in itThus hydrogen fermentation is directly proportional to theamount of sugars present in it So recovering the sugars in theform of hydrolysate is suggested to enhance the productionperformance of DJW

In addition in this study the operation strategies usedhave proved that ASBR operation was a good way to treatthe DJW effectively and generate energy meanwhile Theutilization of the H

2fermentor effluent promised that more

amount of bioenergy could be generated in the form ofmethane which is having higher heating value This kind ofapproach to treat the solidwaste ismore suitable for industrialscale applications On the whole bioenergy production fromjatropha biomass and deoiled jatropha waste is the economi-cally feasible and commercially applicable to solve the energy-related issues

From the above results and discussions direct conversionof De-oiled jatropha waste to hydrogen was demonstrated


Table 6 Comparison with other cellulosic materials operated via ASBR operation

Substrate Seed source HRT (h)a Hydrogen production index ReferencePOME Thermoanaerobacterium rich sludge 96 HPR 61 LLlowastd HY 224moL H2moL hexose [37]Sweet sorghum extract Indigenous microflora 12 HPR 35 LLlowastd HY 093moLmoL glucose [38]POME Mixed microflora 72 HPR 67 LLlowastd HY 094 Lg COD [39]Water hyacinth Pig slurry nr HPR 02 LLlowastd HY nr [40]DJW Mixed microflora 36 HPR 148 LLlowastd HY 86mLg VS This studyTequila vinasse Anaerobic granular sludge 12 HPR 212 LLlowastd HY nr [10]Food waste Heat treated sludge 12 HPR 76 LLlowastd HY 112moLmoL hexose [41]Marine algae Mixed microflora 144 HPR nr HY 079moLmoL hexose [9]nr not reported in the source acalculated from the source

via ASBR operation and the effluent from H2fermentor

was efficiently utilized for methane production in batch testsusing pig slurry as seed source and the following conclusionscould be drawn

Stable hydrogen productionsteady state was obser-ved after 30 days of operation The effluent fromthe reactor could be converted into methane gas toincrease the total energy production of the process

Peak HPR and HY were attained as 148 plusmn 004 LLlowastdand 87 plusmn 03mLH

2g volatile solid added when

the reactor was operated at HRT 2 d with DJWconcentration 200 gL temperature 55∘C and pH 65

Peak MPR and MY were achieved as 4253 plusmn51mLLlowastd and a 205 plusmn 05mL CH

4g COD while

Pig slurry was used as seed source with the effluentconcentration of 10 g CODL at 30∘C and pH 70

This system demonstrated that ASBR operation couldbe a feasible method to treat the solid lignocellulosewastes such as DJW PCR-DGGE results revealed thepresence of combination of Clostridium thermopal-marium and Bacillus coagulans which are cellulolyticand fermentative in nature

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors gratefully acknowledge the financial supportby Taiwanrsquos Bureau of Energy (Grant no 101-D0204-3)Taiwanrsquos National Science Council (NSC-99-2221-E-035-024-MY3 NSC-99-2221-E-035-025-MY3 NSC-99-2632-E-035-001-MY3 and NSC-101-2218-E-035-003-MY3) Feng ChiaUniversity (FCU-10G27101) They also thank Hua NengEnvironmental Protection and Energy Technology Ltd Tai-wan for providing them the deoiled Jatropha waste Taiwanfellowship support from MOE of Taiwan to GopalakrishnanKumar is highly acknowledged

References

[1] D Das and T N Veziroglu ldquoHydrogen production by biologicalprocesses a survey of literaturerdquo International Journal of Hydro-gen Energy vol 26 no 1 pp 13ndash28 2001

[2] M Momirlan and T N Veziroglu ldquoCurrent status of hydrogenenergyrdquo Renewable and Sustainable Energy Reviews vol 6 no1-2 pp 141ndash179 2002

[3] O Pakarinen A Lehtomaki and J Rintala ldquoBatch dark fer-mentative hydrogen production from grass silage the effect ofinoculum pH temperature and VS ratiordquo International Journalof Hydrogen Energy vol 33 no 2 pp 594ndash601 2008

[4] M-L Zhang Y-T Fan Y Xing C-M Pan G-S Zhang andJ-J Lay ldquoEnhanced biohydrogen production from cornstalkwastes with acidification pretreatment by mixed anaerobicculturesrdquo Biomass and Bioenergy vol 31 no 4 pp 250ndash2542007

[5] D B Levin C R Carere N Cicek and R Sparling ldquoChallengesfor biohydrogen production via direct lignocellulose fermenta-tionrdquo International Journal of Hydrogen Energy vol 34 no 17pp 7390ndash7403 2009

[6] C-H Lay I-Y Sung G Kumar C-Y Chu C-C Chen and C-Y Lin ldquoOptimizing biohydrogen production from mushroomcultivation waste using anaerobic mixed culturesrdquo InternationalJournal of Hydrogen Energy vol 37 no 21 pp 16473ndash164782012

[7] V Sricharoenchaikul and D Atong ldquoThermal decompositionstudy on Jatropha curcas L waste using TGA and fixed bedreactorrdquo Journal of Analytical and Applied Pyrolysis vol 85 no1-2 pp 155ndash162 2009

[8] K P Srividhya T Tamizharasan and S Jayaraj ldquoCharacteriza-tion and gasification using-Jatropha Curcas Seed Cakerdquo Journalof Biofuels vol 1 no 1 pp 30ndash36 2010

[9] X Shi K-W Jung D-H Kim Y-T Ahn and H-S ShinldquoDirect fermentation of Laminaria japonica for biohydrogenproduction by anaerobic mixed culturesrdquo International Journalof Hydrogen Energy vol 36 no 10 pp 5857ndash5864 2011

[10] G Buitron and C Carvajal ldquoBiohydrogen production fromTequila vinasses in an anaerobic sequencing batch reactor effectof initial substrate concentration temperature and hydraulicretention timerdquo Bioresource Technology vol 101 no 23 pp9071ndash9077 2010

[11] M F Arooj S-K Han S-H Kim D-H Kim and H-SShin ldquoEffect of HRT on ASBR converting starch into biologicalhydrogenrdquo International Journal of Hydrogen Energy vol 33 no22 pp 6509ndash6514 2008


[12] G Kumar and C Y Lin ldquoBio conversion of De-oiled Jatrophawaste to hydrogen and methane gas by anaerobic fermentationinfluence of substrate concentration temperature and pHrdquoInternational Journal of Hydrogen Energy vol 38 no 1 pp 63ndash72 2013

[13] G Endo T Noike and T Matsumoto ldquoCharacteristics ofcellulose and glucose decomposition in acidogenic phase ofanaerobic digestionrdquo Proceedings of the Society For Civil Engi-neers vol 325 no 1 pp 61ndash68 1982 (Japanese)

[14] APHA Standard Methods for the Examination of Water andWastewater American Public Health Association New YorkNY USA 19th edition 1995

[15] C-C Chen C-Y Lin and M-C Lin ldquoAcid-base enrichmentenhances anaerobic hydrogen production processrdquo AppliedMicrobiology and Biotechnology vol 58 no 2 pp 224ndash2282002

[16] C Y Lin and R C Chang ldquoHydrogen production duringthe anaerobic acidogenic conversion of glucoserdquo Journal ofChemical Technology and Biotechnology vol 74 no 1 pp 498ndash500 1999

[17] L H Koehler ldquoDifferentiation of carbohydrates by anthronereaction rate and color intensityrdquo Analytical Chemistry vol 24no 10 pp 1576ndash1579 1952

[18] W F Owen D C Stuckey and J B Healy Jr ldquoBioassayfor monitoring biochemical methane potential and anaerobictoxicityrdquoWater Research vol 13 no 6 pp 485ndash492 1979

[19] G Muyzer E C De Waal and A G Uitterlinden ldquoProfilingof complex microbial populations by denaturing gradient gelelectrophoresis analysis of polymerase chain reaction-amplifiedgenes coding for 16S rRNArdquo Applied and Environmental Micro-biology vol 59 no 3 pp 695ndash700 1993

[20] K Tamura J Dudley M Nei and S Kumar ldquoMEGA4 Molec-ular Evolutionary Genetics Analysis (MEGA) software version40rdquo Molecular Biology and Evolution vol 24 no 8 pp 1596ndash1599 2007

[21] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular biol-ogy and evolution vol 4 no 4 pp 406ndash425 1987

[22] G Kumar C-H Lay C-Y Chu J-H Wu S-C Lee and C-YLin ldquoSeed inocula for biohydrogen production from biodieselsolid residuesrdquo International Journal of Hydrogen Energy vol 37no 20 pp 15489ndash15495 2012

[23] P Saraphirom and A Reungsang ldquoBiological hydrogen pro-duction from sweet sorghum syrup by mixed cultures usingan anaerobic sequencing batch reactor (ASBR)rdquo InternationalJournal of Hydrogen Energy vol 36 no 14 pp 8765ndash8773 2011

[24] R K Thauer K Jungermann and K Decker ldquoEnergy con-servation in chemotrophic anaerobic bacteriardquo BacteriologicalReviews vol 41 no 1 pp 100ndash180 1977

[25] B Dabrock H Bahl and G Gottschalk ldquoParameters affectingsolvent production by Clostridium pasteurianumrdquo Applied andEnvironmental Microbiology vol 58 no 4 pp 1233ndash1239 1992

[26] S K Khanal W-H Chen L Li and S Sung ldquoBiologicalhydrogen production effects of pH and intermediate productsrdquoInternational Journal of Hydrogen Energy vol 29 no 11 pp1123ndash1131 2004

[27] C-Y Chen M-H Yang K-L Yeh C-H Liu and J-S ChangldquoBiohydrogen production using sequential two-stage dark andphoto fermentation processesrdquo International Journal of Hydro-gen Energy vol 33 no 18 pp 4755ndash4762 2008

[28] Y-S Chuang C-H Lay B Sen et al ldquoBiohydrogen andbiomethane from water hyacinth (Eichhornia crassipes) fer-mentation effects of substrate concentration and incubationtemperaturerdquo International Journal of Hydrogen Energy vol 36no 21 pp 14195ndash14203 2011

[29] H Raheman and S Mondal ldquoBiogas production potential ofjatropha seed cakerdquo Biomass and Bioenergy vol 37 pp 25ndash302012

[30] A Geng Y He C Qian X Yan and Z Zhou ldquoEffect of keyfactors on hydrogen production fromcellulose in a co-culture ofClostridium thermocellum and Clostridium thermopalmariumrdquoBioresource Technology vol 101 no 11 pp 4029ndash4033 2010

[31] D B Levin L Pitt and M Love ldquoBiohydrogen productionprospects and limitations to practical applicationrdquo InternationalJournal of Hydrogen Energy vol 29 no 2 pp 173ndash185 2004

[32] S L Walton K M Bischoff A R P Van Heiningen and GP Van Walsum ldquoProduction of lactic acid from hemicelluloseextracts by Bacillus coagulans MXL-9rdquo Journal of IndustrialMicrobiology and Biotechnology vol 37 no 8 pp 823ndash830 2010

[33] R Sleat R A Mah and R Robinson ldquoIsolation and charac-terization of an anaerobic cellulolytic bacterium Clostridiumcellulovorans sp novrdquo Applied and Environmental Microbiologyvol 48 no 1 pp 88ndash93 1984

[34] A Kumar Tiwari A Kumar and H Raheman ldquoBiodieselproduction from jatropha oil (Jatropha curcas) with high freefatty acids an optimized processrdquo Biomass and Bioenergy vol31 no 8 pp 569ndash575 2007

[35] H J Berchmans and S Hirata ldquoBiodiesel production fromcrude Jatropha curcas L seed oil with a high content of free fattyacidsrdquoBioresource Technology vol 99 no 6 pp 1716ndash1721 2008

[36] P K Sahoo and L M Das ldquoProcess optimization for biodieselproduction from Jatropha Karanja and Polanga oilsrdquo Fuel vol88 no 9 pp 1588ndash1594 2009

[37] S O-Thong P Prasertsan N Intrasungkha S Dhamwi-chukorn and N-K Birkeland ldquoImprovement of biohydrogenproduction and treatment efficiency on palm oil mill effluentwith nutrient supplementation at thermophilic condition usingan anaerobic sequencing batch reactorrdquo Enzyme and MicrobialTechnology vol 41 no 5 pp 583ndash590 2007

[38] G Antonopoulou H N Gavala I V Skiadas and G LyberatosldquoInfluence of pH on fermentative hydrogen production fromsweet sorghum extractrdquo International Journal of HydrogenEnergy vol 35 no 5 pp 1921ndash1928 2010

[39] M Badiei J M Jahim N Anuar and S R Sheikh AbdullahldquoEffect of hydraulic retention time on biohydrogen productionfrom palm oil mill effluent in anaerobic sequencing batchreactorrdquo International Journal of Hydrogen Energy vol 36 no10 pp 5912ndash5919 2011

[40] C H Lay Bioenergy production potential of water hyacinth[PhD dissertation] Feng Chia University Taichung Taiwan2012

[41] S-H Kim S-K Han and H-S Shin ldquoEffect of substrateconcentration on hydrogen production and 16S rDNA-basedanalysis of themicrobial community in a continuous fermenterrdquoProcess Biochemistry vol 41 no 1 pp 199ndash207 2006

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economically feasible [7 8] Thus the utilization of thiswaste for hydrogen fermentation has dual benefits wastemanagement and energy generation (mainly hydrogen gasadditionally ethanol and volatile fatty acids would be alsogenerated as fermentation coproducts)

Development of hydrogen fermentation process for lig-nocellulose waste needs proper operational conditions andstrategies for example with such solid nature performingcontinuous operation (CSTR) would be of great difficultydue to the possibilities of improper flow in the pumpandor pipelines in other terms the accumulation of solidsin a period of time Thus ASBR (anaerobic sequencingbatch reactor) operation has been suggested and shownas a promising way to treat such solid biomass feedstockand pure substrate such as starch [9ndash11] The generationof DJW is abundant due to the expansion of biodieselindustries Besides a solid portion of 3 ton remained in everyton of biodiesel extraction So development of a hydrogenfermentation form DJW could attain more interest thanother lignocellulose feedstock The main advantages of thisfeedstock are the easy collection and desizing processeswhereas other feed stocks like rice straw wheat strawcorn and so on require energy during the size reductionMoreover development of hydrogen fermentation for DJWincreases the commercial benefits of the biodiesel industrywhereas both biodiesel and biohydrogen could be obtainedfrom a low-cost waste that is jatropha at one time inputThus here we demonstrated a process to treat DJW usingASBR operation In addition the feasibility of the effluentfrom H

2fermentor to generate methane to enhance the total

energy production is also evaluated

2 Materials and Methods

21 DJW Substrate and Anaerobic Mixed Microflora DJWused in this study was collected from a biodiesel industryusing Jatropha biomass and located in central Taiwan Thecellulosic content was analyzed as 423 of fermentablesugars (141 cellulose and 282 of hemicellulose) usingFIBERTEC 1020 (M6) analyzer as mentioned elsewhere [12]Anaerobic mixed microflora was obtained from a municipalwastewater treatment plant Substrate and seed sludge werestored in a refrigerator at 4∘C before being used in the exper-iments To inactivate the hydrogen consuming methanogensthe sludge was heat treated for 30minutes at 95∘C in a boilingwater bath Characteristics of the DJW and seed sludge weredescribed in our previous study [12]

22 Reactor (ASBR) Startup and Operation A schematicdiagram of the ASBR is shown in Figure 1 The reactor wasstarted up by feeding glucose (10 gL) initially to enrichthe hydrogen producers (Run 1) There forward DJW ata concentration of 100 gL was fed for the adaptation ofhydrogen producers at an agitation speed of 150 rpm andoperational temperature of 55∘CMeanwhile the effluent wascollected and fed into the reactor along with fresh substrateto avoid the loss of biomass in the form of effluent (Run 2ndash4)After Run 4 it was stopped due to the accumulation of more

amounts of solid particles inside the reactor Thereafter onlyfresh substrate was fed into the reactor The nutrient solutionadded was prepared by following the Endo formulation [13]with slight modifications as mentioned [11] ORP and pHwere monitored using an automated pH ORP panel pH wascontrolled using 1N NaOH buffer

23 Batch Methane Fermentation Methane production wasstudied in batch vials (capacity-125mL) with a workingvolume of 60mL by adding 40mL of hydrogen fermentationeffluent (substrate concentration 10 g CODL) 12mL of seedsludge (either cow dung or pig slurry) 1ndash3mL for pHadjustment (either 1 NNaOHorHCl) 5mL nutrient solution(to enhance the growth of anaerobic bacteria) and the restwas tap water The nutrient solution added was prepared byfollowing the Endo formulation as mentioned [13] The finalpH was adjusted to neutral (70) Then the batch vials werekept in a reciprocal air-bath shaker at 150 rpm at 30∘C Thevolume and composition of gas were monitored daily Fer-mentation was carried out until the gas production becomeszero All experiments were carried out at least triplicates andthe values represented are the mean of triplicate values

24 Analytical Procedures The analytical procedures usedto determine pH total solid (TS) COD and VSS werefollowed by APHA 1995 [14] The volatile fatty acids (VFAs)and ethanol concentrations were analyzed by HPLC Thegas composition was analyzed with a gas chromatographhaving a thermal conductivity detector (China Chromato-graph 8700T)Other experimental procedureswere indicatedin our previous studies [15 16] For total carbohydrateconcentration anthrone-sulphuric acid method was adapted[17] Biogas production was measured in a periodic intervalusing various volumes of glass syringes depending on theexpected biogas production fitted with hypodermic needlesas described by Owen et al [18]

25 Calculations ThemodifiedGompertz equationwas usedto estimate methane production potential and methaneproduction rate (Sigma plot software 100 Systat SoftwareInc USA)

119867(119905) = 119875 sdot exp minus exp [119877119898sdot 119890

119875

(120582 minus 119905) + 1] (1)

where119867(119905) is the cumulative methane production (mL) 119875 isthe methane production potential (mL) 119877

119898is the maximum

methane production rate (mLh) 119890 is 271828 120582 is the lagphase time (h) and 119905 is the cultivation time (h) Methaneproduction rate (MPR L H

2L-d) was defined as the value

of 119877119898divided by the reactor volume (006 L) and multiplied

by one day (24 h) methane yield (MY mL CH4g COD)

was defined as methane produced per gram chemical oxygendemand (COD)

26 PCR-DGGE DNA Sequencing and Phylogenetic Anal-ysis Total genomic DNA was isolated using the Blood ampTissue Genomic DNA Extraction Miniprep System (Vio-gene Taiwan) following themanufacturerrsquos instructionsThe



Influent

Gas collector

3N NaOHsolution

pH panel







3 Results




Days0 10 20 30 40

Days0 10 20 30 40

Days0 10 20 30 40

Days0 10 20 30 40

Days0 10 20 30 40

0

1

2

3

4

0

1

2

3

BPRHPR

01020304050

0

20

40

60

02468

101214

pH

0

2

4

6

ORP

(mV

)

0100200300400500

pHORP

Batch mode

ASBR

HRT

(day

s)

000510152025

Prod

uctio

n ra

te (L

L

HPR

(LL

lowastd)

lowastd)

H2

H2

CO2

CO2

HY

(mL

H2g

VS

adde

d)



2g VS added was




(SLR)





HPR(LLlowastd)

HY(mL H2g VS added)






















Volu

me

(mL)

0

50

100

150

200

250

BiogasCH4

(CD)

(a)


0 200 400 600

Volu

me

(mL)

0

100

200

300

BiogasCH4

(PS)

(b)








4g COD







4 Discussions


2










S4

S3

S2

S1

40

60











S2

S1

S3

S4

100

97

83

100

98

51

100

100

100

100

100100

01

EColi





C6H12O6+ 2H2O 997888rarr 2CH

3COOH + 2CO

2+ 4H2 (2)


3CH2CH2COOH + 2CO

2+ 2H2 (3)








H2fermentor (ASBR)

H2 1 g COD

Effluent

By products

85 CODbalance


(SMP) (887 gCOD)



















4g COD while





Acknowledgments


References















































FuelsJournal of







Advances in



Journal of


Renewable Energy





RotatingMachinery


EnergyJournal of



















Influent

Gas collector

3N NaOHsolution

pH panel







3 Results




Days0 10 20 30 40

Days0 10 20 30 40

Days0 10 20 30 40

Days0 10 20 30 40

Days0 10 20 30 40

0

1

2

3

4

0

1

2

3

BPRHPR

01020304050

0

20

40

60

02468

101214

pH

0

2

4

6

ORP

(mV

)

0100200300400500

pHORP

Batch mode

ASBR

HRT

(day

s)

000510152025

Prod

uctio

n ra

te (L

L

HPR

(LL

lowastd)

lowastd)

H2

H2

CO2

CO2

HY

(mL

H2g

VS

adde

d)



2g VS added was




(SLR)





HPR(LLlowastd)

HY(mL H2g VS added)






















Volu

me

(mL)

0

50

100

150

200

250

BiogasCH4

(CD)

(a)


0 200 400 600

Volu

me

(mL)

0

100

200

300

BiogasCH4

(PS)

(b)








4g COD







4 Discussions


2










S4

S3

S2

S1

40

60











S2

S1

S3

S4

100

97

83

100

98

51

100

100

100

100

100100

01

EColi





C6H12O6+ 2H2O 997888rarr 2CH

3COOH + 2CO

2+ 4H2 (2)


3CH2CH2COOH + 2CO

2+ 2H2 (3)








H2fermentor (ASBR)

H2 1 g COD

Effluent

By products

85 CODbalance


(SMP) (887 gCOD)



















4g COD while





Acknowledgments


References















































FuelsJournal of







Advances in



Journal of


Renewable Energy





RotatingMachinery


EnergyJournal of


















Days0 10 20 30 40

Days0 10 20 30 40

Days0 10 20 30 40

Days0 10 20 30 40

Days0 10 20 30 40

0

1

2

3

4

0

1

2

3

BPRHPR

01020304050

0

20

40

60

02468

101214

pH

0

2

4

6

ORP

(mV

)

0100200300400500

pHORP

Batch mode

ASBR

HRT

(day

s)

000510152025

Prod

uctio

n ra

te (L

L

HPR

(LL

lowastd)

lowastd)

H2

H2

CO2

CO2

HY

(mL

H2g

VS

adde

d)



2g VS added was




(SLR)





HPR(LLlowastd)

HY(mL H2g VS added)






















Volu

me

(mL)

0

50

100

150

200

250

BiogasCH4

(CD)

(a)


0 200 400 600

Volu

me

(mL)

0

100

200

300

BiogasCH4

(PS)

(b)








4g COD







4 Discussions


2










S4

S3

S2

S1

40

60











S2

S1

S3

S4

100

97

83

100

98

51

100

100

100

100

100100

01

EColi





C6H12O6+ 2H2O 997888rarr 2CH

3COOH + 2CO

2+ 4H2 (2)


3CH2CH2COOH + 2CO

2+ 2H2 (3)








H2fermentor (ASBR)

H2 1 g COD

Effluent

By products

85 CODbalance


(SMP) (887 gCOD)



















4g COD while





Acknowledgments


References















































FuelsJournal of







Advances in



Journal of


Renewable Energy





RotatingMachinery


EnergyJournal of






















Volu

me

(mL)

0

50

100

150

200

250

BiogasCH4

(CD)

(a)


0 200 400 600

Volu

me

(mL)

0

100

200

300

BiogasCH4

(PS)

(b)








4g COD







4 Discussions


2










S4

S3

S2

S1

40

60











S2

S1

S3

S4

100

97

83

100

98

51

100

100

100

100

100100

01

EColi





C6H12O6+ 2H2O 997888rarr 2CH

3COOH + 2CO

2+ 4H2 (2)


3CH2CH2COOH + 2CO

2+ 2H2 (3)








H2fermentor (ASBR)

H2 1 g COD

Effluent

By products

85 CODbalance


(SMP) (887 gCOD)



















4g COD while





Acknowledgments


References















































FuelsJournal of







Advances in



Journal of


Renewable Energy





RotatingMachinery


EnergyJournal of
























S4

S3

S2

S1

40

60











S2

S1

S3

S4

100

97

83

100

98

51

100

100

100

100

100100

01

EColi





C6H12O6+ 2H2O 997888rarr 2CH

3COOH + 2CO

2+ 4H2 (2)


3CH2CH2COOH + 2CO

2+ 2H2 (3)








H2fermentor (ASBR)

H2 1 g COD

Effluent

By products

85 CODbalance


(SMP) (887 gCOD)



















4g COD while





Acknowledgments


References















































FuelsJournal of







Advances in



Journal of


Renewable Energy





RotatingMachinery


EnergyJournal of























H2fermentor (ASBR)

H2 1 g COD

Effluent

By products

85 CODbalance


(SMP) (887 gCOD)



















4g COD while





Acknowledgments


References















































FuelsJournal of







Advances in



Journal of


Renewable Energy





RotatingMachinery


EnergyJournal of



























4g COD while





Acknowledgments


References















































FuelsJournal of







Advances in



Journal of


Renewable Energy





RotatingMachinery


EnergyJournal of




















































FuelsJournal of







Advances in



Journal of


Renewable Energy





RotatingMachinery


EnergyJournal of

















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