a review of dark fermentative hydrogen production from biodegradable municipal waste fractions

$: A review of dark fermentative hydrogen production from biodegradable municipal waste fractions$
Waste Management 33 (2013) 1345–1361

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Waste Management

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Review

A review of dark fermentative hydrogen production from biodegradablemunicipal waste fractions

G. De Gioannis a,c,⇑, A. Muntoni a,c, A. Polettini b, R. Pomi b

a DICAAR – Department of Civil and Environmental Engineering and Architecture, University of Cagliari, Cagliari, Italyb Department of Hydraulics, Transportation and Roads, University of Rome ‘‘La Sapienza’’, Italyc IGAG-CNR, Environmental Geology and Geoengineering Institute of the National Research Council, Italy

a r t i c l e i n f o

Article history:Received 10 July 2012Accepted 19 February 2013Available online 1 April 2013

Keywords:Anaerobic digestionBiological hydrogen productionDark fermentationFood wasteOrganic fractions of municipal solid waste

0956-053X/$ - see front matter � 2013 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.wasman.2013.02.019

Abbreviations: AR, aged refuse; COD, chemical oxuous stirred tank reactor; F/M, food/microorganishydraulic retention time; HST, heat shock treatmentcell; MFC, microbial fuel cell; OLR, organic loading ratemunicipal solid waste; OMW, olive mill wastewater;primary sludge; SBR, sequencing batch reactor; SRsewage sludge (mixture of primary and secondarycarbon; TS, total solids; TKN, total Kjeldahl nitrogen; Ublanket; VFAs, volatile fatty acids; VS, volatile solids;⇑ Corresponding author at: University of Cagliari, D

and Environmental Engineering and Architecture0706755551; fax: +39 0706755523.

E-mail address: [email protected] (G. De Gioannis)

a b s t r a c t

Hydrogen is believed to play a potentially key role in the implementation of sustainable energy produc-tion, particularly when it is produced from renewable sources and low energy-demanding processes. Inthe present paper an attempt was made at critically reviewing more than 80 recent publications, in orderto harmonize and compare the available results from different studies on hydrogen production from FWand OFMSW through dark fermentation, and derive reliable information about process yield and stabilityin view of building related predictive models. The review was focused on the effect of factors, recognizedas potentially affecting process evolution (including type of substrate and co-substrate and relative ratio,type of inoculum, food/microorganisms [F/M] ratio, applied pre-treatment, reactor configuration, tem-perature and pH), on the fermentation yield and kinetics. Statistical analysis of literature data from batchexperiments was also conducted, showing that the variables affecting the H2 production yield wereranked in the order: type of co-substrate, type of pre-treatment, operating pH, control of initial pH andfermentation temperature. However, due to the dispersion of data observed in some instances, the ambi-guity about the presence of additional hidden variables cannot be resolved. The results from the analysisthus suggest that, for reliable predictive models of fermentative hydrogen production to be derived, ahigh level of consistency between data is strictly required, claiming for more systematic and comprehen-sive studies on the subject.

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1. Introduction

In Europe, anaerobic digestion of biodegradable residues has re-ceived renewed attention by the scientific and technical commu-nity over the last decade (Mata-Alvarez et al., 2000, Mata-Alvarez, 2002; De Baere, 2003; Bolzonella et al., 2006; Karagianni-dis and Perkoulidis, 2009), especially for the organic fraction ofmunicipal solid waste. This is due to several reasons that stem

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ygen demand; CSTR, contin-ms; FW, food waste; HRT,; MEC, microbial electrolysis; OFMSW, organic fraction ofPBR, packed bed reactor; PS,

T, solids retention time; SS,sludge); TOC, total organic

ASB, upflow anaerobic sludgeWAS, waste activated sludge.ICAAR – Department of Civil, Cagliari, Italy. Tel.: +39

.

from the EU legislative framework, which has set specific con-straints on landfilling of biodegradable wastes, maximization ofmaterials recycling as well as enhancement of energy productionfrom renewable sources.

Numerous investigators (Han and Shin, 2004a; Liu et al., 2006;Gómez et al., 2006, 2009; Ueno et al., 2007; Chu et al., 2008; Wangand Zhao, 2009; Lee et al., 2010b; Dong et al., 2011) demonstratedthat if fermentation of biodegradable organic substrates is appro-priately operated in a two-staged mode, separation of the acido-genic and methanogenic phases can be accomplished: whileacidogenesis produces hydrogen and carbon dioxide as the gaseousproducts and releases VFAs into the liquid solution, methanogene-sis allows for final conversion of the residual biodegradable organicmatter from the first stage into methane and carbon dioxide. Con-sidering that H2 has the highest calorific value per unit weight ofany known fuel and an improved acidogenic phase has been re-ported to result in enhanced biogas yield in the second stage, sep-arating the phases of the anaerobic digestion process wouldincrease the energy efficiency overall (Liu et al., 2006; Lee andChung, 2010; Dong et al., 2011). Furthermore, proper processingof the digestate to yield a valuable final product for use as a soil

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1346 G. De Gioannis et al. / Waste Management 33 (2013) 1345–1361

amending material would contribute to improved environmentalsustainability of management of biodegradable organic residues.

A number of potentially suitable residual substrates have beenevaluated for biohydrogen generation potential through dark fer-mentation. Among these, fractions of municipal solid waste suchas food waste (FW) and the broader mixture of materials knownas organic fraction of municipal solid waste (OFMSW; basicallyFW combined with non-recoverable paper residues) may representrelatively inexpensive and suitable sources of biodegradable or-ganic matter for H2 production, mainly due to their high carbohy-drate content and wide availability (Okamoto et al., 2000; Layet al., 2003; Kim et al., 2004, 2011a; Liu et al., 2006; Li et al.,2008a,b; Zhu et al., 2008; Wang and Zhao, 2009; Nazlina et al.,2011).

Hydrogen production via fermentation involves either faculta-tive or strict anaerobic bacteria. The various metabolic pathwaysthat may establish can either be promoted or inhibited, dependingon the adopted operating conditions, which govern the productionof specific volatile fatty acids (VFAs) and alcohols including acetate,propionate, butyrate, lactate and ethanol. In carbohydrates fer-mentation, the acetate and butyrate pathways involve the produc-tion of, respectively, 4 and 2 mol of molecular hydrogen per mol ofglucose degraded. However, propionate, ethanol and lactic acid canalso be produced in mixed bacterial cultures, adversely affecting H2

production: propionate is a metabolite of a H2-consuming path-way, while ethanol and lactic acid are associated with zero-H2

pathways (Guo et al., 2010a). The question as to how to achieveoptimal H2 generation while keeping treatment costs low and pro-ducing an effluent suitable for further treatment is probably themain technical issue to be addressed. To this regard, operationalparameters including temperature, pH, reactor configuration, sub-strate concentration and organic loading rate should be the subjectfor optimization of process efficiency. Recent literature studies onH2 production from FW and OFMSW through dark fermentationhave focused on a broad range of operating conditions, implicitlydenoting that for full-scale application of the process a betterunderstanding of the influence of the relevant process parametersis still required.

The aim of this manuscript is to present an updated overview ofH2 production from FW or OFMSW through dark fermentation,based on more than 80 recent related publications. Although anumber of review papers has been published on fermentative H2

production from various biodegradable wastes, to the authors’knowledge a critical overview of literature studies with a specificfocus on FW/OFMSW is still missing. The analysis conducted inthe present study was focused on the following issues: (a) typeof inoculum and applied pre-treatment, (b) type of fermentationreactor, (c) organic loading rate (OLR), (d) solids retention time(SRT), (e) temperature and pH. Since the numerous literature stud-ies on this subject have adopted different approaches focusing onseveral specific aspects of the fermentation process, the reportedresults are diverse and sometimes even conflicting. On account ofthis, an effort was made in the present manuscript to statisticallyanalyzing literature data to derive information on the relativeimportance of the main parameters of concern, as well as on theirpotential mutual relationships.

2. Process yield and conversion efficiency

An important issue related to fermentative hydrogen produc-tion from biodegradable wastes involves how to appropriatelyevaluate and express process efficiency. To this regard, the ex-pected hydrogen production yield may be conveniently convertedinto a parameter representing the conversion efficiency attainedupon fermentation, which may in turn be expressed either in terms

of mass or energy units. The concept of conversion efficiency de-rives from the existence of a fermentation barrier to hydrogen pro-duction from organic substrates, which may be elucidatedconsidering the conversion of a simple carbohydrate such as glu-cose. If the complete conversion reaction to hydrogen is taken intoaccount (Eq. (1)), it turns out that theoretically 12 mol H2 can beextracted from 1 mol of glucose:

C6H12O6 þ 6H2O! 12H2 þ 6CO2 ð1Þ

However, this reaction is energetically unfavorable with respectto biomass growth and would also only occur at extremely low H2

concentrations, so that the real conversion potential is in fact lowerthan this theoretical value. At the best, the optimized conversion ofglucose into hydrogen is limited to acetate production and is there-fore practically limited by the existence of an upper threshold – theso-called Thauer limit (Thauer et al., 1977) – of 4 mol H2/mol glu-cose (Eq. (2)). As a result, only one third of the theoretical hydrogenproduction can be achieved in practice, since part of the reducingequivalents in the original substrate remains as acetate.

C6H12O6 þ 2H2O! 4H2 þ 2CO2 þ 2CH3COOH ð2Þ

In practice, however, organic intermediates also act as electronscavengers, which gives rise to the production of more reduced fer-mentation products compared to acetate, including propionate,butyrate and longer aliphatic acids, lactate, formate, alcohols andketones, with an associated decrease in the H2 generation yield.In case the butyrate fermentation pathway is established, the con-version efficiency is reduced to 2 mol H2/mol glucose:

C6H12O6 ! 2H2 þ 2CO2 þ CH3CH2CH2COOH ð3Þ

It has also been shown (Nath and Das, 2004; Davila-Vazquezet al., 2008; Hallenbeck and Ghosh, 2009) that, since in nature fer-mentation processes have been optimized not to produce hydro-gen but to sustain microbial growth, hydrogen represents awaste of energy during metabolism and is therefore preferentiallyrecycled within the metabolic pathways. As a result, a number ofreduced products are formed to sustain microbial cell synthesis,including ethanol, butanol, butyrate and lactate, which allow forNADH re-oxidation. This explains how in real practice, even underoptimal process conditions, conversion efficiencies to H2 of higherthan 15% of the original electrons in the substrate are hardly at-tained (Angenent et al., 2004).

On account of the considerations above, the conversion effi-ciency may be calculated on a mass basis as follows:

Em ¼mol H2 produced=mass of substrate

Theoretical mol H2produced=mass of substrate� 100 ð4Þ

Table 1 reports conversion efficiency data according to the def-inition provided above, as derived from different literature sources.

The hydrogen production efficiency may alternatively be evalu-ated from an energetic perspective, considering the fraction of thetotal energy content of the substrate recovered in the form ofhydrogen, as expressed by Eq. (5):

Ee ¼Energy content of the H2 produced

Energy content of the original substrate� 100 ð5Þ

Assuming 2888 kJ/mol glucose and 242 kJ/mol H2 (Dong et al.,2009b) as the lower heating values of glucose and hydrogen, en-ergy conversion efficiencies of 33.5% and 16.8% are calculated ifthe acetate (Eq. (2)) or butyrate (Eq. (3)) fermentation pathwaysare assumed to occur, respectively.

Alternatively, the amount of energy converted into hydrogenmay also be derived considering the COD equivalent of H2, whichis equal to 16 g COD/mol H2; accordingly, if the specific hydrogenproduction is expressed per unit mass of input COD, it may be easy

Table 1Conversion efficiency data documented in the literature.

Type of substrate Conversion efficiency Specific H2 production Reference

OFMSW 1.98 ± 0.03 mol H2/molglucose 127 ml/g VSremoved Alzate-Gaviria et al. (2007)FW 9.3% of influent COD converted to H2 205 ml/gVSadded Chu et al. (2008)FW 2.1 mol H2/molhexose consumed – Elbeshbishy et al. (2011)FW – 27–28 ml/g VSadded Gómez et al. (2009)

– 19–26 ml/g VSadded

FW 5.1–19.3% of influent COD converted to H2 70–263 ml/g VSaddeda Han and Shin (2004b)

FW 1.12 mol H2/molhexose 80.9 ml/g VSadded Kim et al. (2008b)FW 0.87 mol H2/molhexose added 62.6 ml/g VSadded Kim and Shin (2008)FW 2.05 mol H2/molhexose consumed 153.5 ml/g VSadded Kim et al. (2009)FW 0.35–0.54 mol H2/molhexose – Kim et al. (2010)FW + sewage sludge 9.35% of influent COD converted to H2 165 ml/g VSadded Kim et al. (2011a)

2.11 mol H2/molhexose added

FW 1.79 mol H2/molhexose 137.2 ml/g VSadded Kim et al. (2011b)FW (liquid phase) 1.82 mol H2/molglucose – Lee and Chung (2010)OFMSW 2.5 mol H2/molhexose 114 ml/g VSadded Lee et al. (2010b)FW 1–2 mmol/gCOD – Li et al. (2008b)FW 0.6–0.9 mol H2/molhexose 28.4–46.3 ml/g VSadded Shin et al. (2004)

0.03–0.1 mol H2/molhexose 1.3–5.0 ml/g VSadded

FW 1.8 mol H2/molhexose 91.5 ml/g VSadded Shin et al. (2004)FW + paper 1.48–3.2 mol H2/molhexose 165–360 ml/g VSremoved Valdez-Vazquez et al. (2005)

a Calculated from data provided in the manuscript.

Table 2Type of inoculum used and applied pre-treatment conditions.

Type of inoculum Type of pre-treatment

Reference

Mixture of deep soil, excretesvaccine, pig excretes

HST (100 �C,15 min)

Alzate-Gaviriaet al. (2007)

Activated sludge HST (110 �C,30 min)

Cappai et al.(2009, 2010,2011)

Anaerobic sludge HST (boiling,15 min)

Dong et al.(2009a,b)


Han and Shin(2004a)


Han and Shin(2004b)

Anaerobic sludge HST (90 �C, 10 min) Kim et al. (2004)Anaerobic sludge HST (90 �C, 10 min) Kim et al. (2008b)Anaerobic sludge HST (90 �C, 15 min) Kim et al. (2010)Anaerobic sludge HST (90 �C, 10 min) Kim and Shin

(2008)H2-producing microbial

consortiumCultivation Lay et al. (1999)


Anaerobic sludge HST (100 �C, 2 h) Lay et al. (2003)Anaerobic grass compost HST (60–120 �C, 2–

8 h)Lay et al. (2005)

Anaerobic sludge HST (80 �C, 20 min) Lee and Chung(2010)

Compost Acclimation Lee et al. (2010a)Anaerobic sludge HST (90 �C, 30 min) Lee et al. (2010b)Anaerobic sludge HST (90 �C, 10 min) Shin et al. (2003)H2-producing microbial

consortiumCultivation Shin et al. (2004)

Anaerobic sludge HST (105 �C,3 h) + acclimation

Sreela-or et al.(2011a)

Anaerobic sludge HST (105 �C,3 h) + acclimation

Sreela-or et al.(2011b)

Anaerobic sludge Cultivation Zhu et al. (2008)

G. De Gioannis et al. / Waste Management 33 (2013) 1345–1361 1347

to estimate the fraction of the substrate’s energy content which isactually converted into H2 (Kim et al., 2011a).

It has been widely documented that in practice, even underoptimized process conditions, a considerable portion of the carbon,reducing equivalents and energy content of the original substrateremains in the effluent from the hydrogenogenic phase. In orderto maximize the overall conversion yield and ensure adequate sub-strate degradation, the biohydrogen production process shouldthus be thought as a part of a combined process where additionalenergy production and enhanced substrate conversion are attainedin different processing stages, which is discussed further inSection 6.

3. Observed influence of process conditions

As will be shown later in this review, literature studies on bio-logical hydrogen production from residual substrates including FWand OFMSW report considerably wide ranges of variation in thespecific production yield observed. The large differences docu-mented in the literature reflect the underlying influence of numer-ous process parameters, including substrate composition andpresence of co-substrates, type of inoculum and applied pre-treat-ment, reactor type, mode of reactor operation (batch, semi-contin-uous or continuous), operating variables such as temperature,hydraulic retention time (HRT), OLR and pH. These are recognizedto be the most relevant factors affecting the evolution of the fer-mentative pathways and the associated hydrogen generation yield.

The effects of the above mentioned factors are also recognizedto be strictly interrelated and mutually interactive, so that achange in one parameter may affect hydrogen production not onlydue to its individual effect, but also as a result of combined inter-actions with other process variables. Elucidating and predicting theindividual influence of single process conditions and their mutualinteractions (which may be either synergistic or antagonistic) isprobably one of the main challenges of the research on biologicalhydrogen production, especially in those cases where complexsubstrates (for which the metabolic reactions are not fully knownin advance) are concerned.

In the following sections a discussion of literature findingsabout the influence of the relevant process parameters is provided,also highlighting where the effects of the operating variables havebeen found to be mutually dependent.

3.1. Type and pre-treatment of inoculum

The seed microorganisms to be used in the process and the needfor inoculum pre-treatment are among the most debated issues inhydrogen production from dark fermentation. A variety of data isavailable in the literature as for the type of inocula used in FW/


OFMSW fermentation experiments and the nature of pre-treat-ments applied to improve the process yield (please refer to thesummary data provided in Table 2).

In general, various pure cultures have been explored to producehydrogen from a variety of substrates (mainly mono-substrates). Infermentative H2 production from FW/OFMSW, researchers usemixed microbial cultures for practical reasons, since a mixed cul-ture system would be cheaper to operate, easier to control, andcapable of digesting a variety of feedstock materials (Li and Fang,2007; Valdez-Vazquez et al., 2005). Anaerobic sludge from full-scale anaerobic digesters, either with or without specific pre-treat-ment, has been used in numerous investigations as a supplier of amixed anaerobic consortium. Among the most notable exceptions,Han and Shin (2002), Lee et al. (2008, 2010a) and Cappai et al.(2009) used, respectively, rumen microorganisms from the stom-ach of cows, an enriched culture from FW compost, and waste acti-vated sludge (WAS) with no specific pre-treatment. In such studies,rumen microorganisms were investigated due to their enhancedcellulolytic capability, while facultative anaerobic bacteria inWAS were considered suitable to enhance the fermentative stageof the process due to their high growth rate and capability to rap-idly recover from accidental oxygen intrusion.

Alzate-Gaviria et al. (2007) adopted mixtures of inocula (deepsoil and vaccine and pig excretes), while Zong et al. (2009) testedcattle dung compost; in both cases a thermal pre-treatment(15 min at 100 �C) was applied.

Aged refuse (AR) excavated from a solid waste landfill with over10 years of placement was also used to enhance biohydrogen pro-duction from FW (Li et al., 2008a). The best results (194 N l H2/kg VS, 94.3 N l H2/kg VS h) were attained when FW was mixed withsewage sludge (SS) or AR at mixing ratios of 100:30 (dry weight)and 100:50 (wet weight), respectively; the FW and SS were ther-mally pre-treated (2 h at 160 �C), while AR was used as receivedassuming that exposure to air upon mining reduced strictly anaer-obic hydrogen utilizing methanogens. In general, the addition ofAR was believed to significantly promote the hydrogen generationyield in comparison to SS, due to the enrichment in effective bio-mass population and to the alkaline characteristics which createdfavorable conditions for the metabolism of hydrogen-producingbacteria.

Although the use of mixed microflora for fermentative hydro-gen production is practically more viable, important limitationsarise from the coexistence of H2-producing and consuming bacte-ria in nature. A solution which is often practiced involves pre-treat-ment by various methods to harvest hydrogen producers, onaccount of their larger chances to survive when a mixed cultureis treated by harsh conditions due to the ability of some bacterialspecies, including Bacillus and Clostridium, to sporulate as a reac-tion to adverse environmental conditions. To this regard, the mainoptions available are heat-shock treatment (HST), acid treatment,aeration, freezing and thawing, as well as addition of specificchemical compounds.

The strategy for H2-consumers inhibition should be selected onthe basis of capital and operational costs, feasibility and complex-ity of the process layout, time needed for inoculum stabilization,effectiveness over the entire fermentation process, and secondaryeffects/degree of compatibility with further process steps (e.g.,methanogenesis, aerobic composting). It should also be mentionedthat when non-sterile substrates are used, proliferation of newnon-inhibited methanogens is possible, therefore continuousapplication of the inhibition method is typically required (Val-dez-Vazquez and Poggi-Varaldo, 2009). The main chemical inhibi-tors that have been used for H2 utilizing methanogens are sodium-2-bromoethanesulfonate, 2-bromoethanesulphonic acid, iodopro-paneacetylene, ethylene, ethane, methyl chloride, methyl fluoride,lumazine, nitrate and chloroform. Chemical inhibitors may be

either specific or non-specific towards methanogens, that can in-clude both H2 consumers and other types of methanogens. TheCoenzyme M (CoM) is involved in the terminal stage of methanebiosynthesis, where the methyl group carried by CoM is reducedto methane by the methylCoM reductase. BES (sodium-2-bromoe-thanesulfonate), BESA (2-bromoethanesulphonic acid) and lum-azine (C6H4N4O2) are structural analogues of CoM specificallyfound in methanogens only but not in other bacteria or Archea.They can competitively inhibit the methyl transfer reaction atthe terminal reducing stage of methane formation from H2 andCO2. Ethylene is recommended as a reversible selective inhibitorof methanogenesis; methanogenic activity has been reported tocompletely recover after ethylene removal. Acetylene was alsoused as a non-specific inhibitor of methanogens. It has been as-sumed that acetylene destroys the proton gradient across the cellmembrane and thus leads to a breakdown of energy metabolism.Chloroform (CHCl3) is known to inhibit the function of corrinoidenzymes and the methylCoM reductase. CHCl3 can inhibit bothacetoclastic and hydrogenotrophic methanogens. However, CHCl3

investigation has been found not only to inhibit the activity ofmethanogenic Archaea but also that of homoacetogenic bacteriaand acetate-consuming sulfate-reducing bacteria. Iodopropane isanother corrinoid antagonist and prevents the function of B12 en-zymes as methyl group carriers. The effect of methyl fluoride isquite controversial: it has been reported to inhibit effectively aer-obic CH4 oxidation, while not to affect methanogenesis. Howeverin some experiments, methanogenesis was reduced by about 75%compared to the control without the inhibitor. Methanogenesiscould partly be recovered when CH3F was flushed with N2. Nitro-compounds such as nitrate, nitrite, nitroethane, 2-nitropropanoland phosphate can be used as alternative electron acceptors thatmore effectively consume the reducing equivalents produced dur-ing fermentation, redirecting the electron flow away from thereduction of carbon dioxide to methane (Chidthaisong and Conrad,2000; Liu et al., 2011).

The most common approach to harvest hydrogenogenic micro-organisms reported in the literature is however by far HST, whichis based on the ability of some bacterial species, including Bacillusand Clostridium, to sporulate as a reaction to adverse environmen-tal conditions. Typically, HST requires temperatures around 100 �Cfor durations of 15–120 min in order to suppress non-spore-form-ing bacteria, leaving spores of acidogenic bacteria that will germi-nate back to their active vegetative state when suitable growthconditions get re-established (Lay et al., 2003; Lin and Lay, 2004;Fang et al., 2006; Alzate-Gaviria et al., 2007; Argun et al., 2008;Bhaskar et al., 2005). In recent studies on FW, the pre-treatmentconditions adopted ranged from 20 min at 80 �C (Lee and Chung,2010) to 2 h at 100 �C (Lay et al., 2003).

Concerns about the net energy gain of HST, however, make thisbiomass selection method controversial and claims for furtherinvestigation. Enrichment of hydrogen producers by HST is an en-ergy-intensive practice, and the degree of energy consumption canonly be partially reduced by adopting temperatures as low as 75–85 �C. On account of this, studies have also focused on hydrogenproduction from FW with no inoculum pre-treatment with theaim of reducing costs and simplifying the process (Chu et al.,2008; Hong and Haiyun, 2010; Lee et al., 2008, 2010a; Li et al.,2008b; Shin and Youn, 2005; Pan et al., 2008; Zhu et al., 2008;Kim et al., 2011b).

An approach defined as biokinetic control has been introducedby Valdez-Vazquez et al. (2005). This is based on maintaining suchenvironmental conditions as to hinder methanogens growth,including low pH, appropriate temperatures or short HRTs causingthe wash-out of methanogens (Valdez-Vazquez et al., 2005; Val-dez-Vazquez and Poggi-Varaldo, 2009; Cappai et al., 2010; Kimet al., 2011b).


There are also studies in the literature where no inoculum wasadded to the feed material, the evolution of the fermentation pro-cess relying in such cases on the indigenous biomass present in thewaste only. In a two-stage fermentation process for combinedhydrogen and methane production from an unsterilized mixtureof OFMSW and slaughterhouse waste, Gómez et al. (2006) foundthat the hydrogen-producing stage had a stable performance(52.5–71.3 N l H2/kg VSremoved). Wang and Zhao (2009) also applieda two-stage process, which was performed in a semi-continuousrotating drum in which the indigenous mixed microbial culturescontained in food waste were used for hydrogen production; inthe hydrogen production stage (operated at an OLR of22.65 kg VS/m3 d and a solids retention time [SRT] of 160 h) a max-imum hydrogen yield of 0.065 Nm3 H2/kg VS was attained. In thework by Kim et al. (2011b) temperature control in the range 35–60 �C was adopted as a biokinetic control strategy to optimize H2

production from FW. The optimal condition for both the H2 pro-duction yield and rate was found at an operating temperature of50 �C, with values of, respectively, 1.8 mol H2/mol hexoseadded (or137 N ml H2/g VSadded) and 369 N ml H2/l h (or 18.8 N ml H2/g VS h). When FW was preliminarily heat-shocked (90 �C, 20 min)but the fermentation temperature was maintained at 35 �C (Kimet al., 2011a), the H2 production yield (1.8 mol H2/mol hexoseadded,or 165 N ml H2/g VSadded) was comparatively similar to the previ-ous case, however the process rate (300 N ml H2/l h, or 7.0 N ml H2/g VS h) was appreciably reduced.

3.2. Type of reactor and influence of retention time and OLR

Different reactor configurations have been used to treat FW/OFMSW, mostly consisting of small-scale (100–500 ml) vesselsand stirred fermenters of 2–10 l, operated under batch, semi-con-tinuous or continuous conditions.

In fermentative hydrogen production, the SRT, and in turn theOLR, affect the substrate conversion efficiency, the type of activemicrobial population as well as the metabolic pathways estab-lished in the system. The influence of SRT and OLR on hydrogenyield is controversial in the literature. It is generally acknowledgedthat long SRTs favor the buildup of H2 consumers, such as metha-nogens, and competitors for substrate, such as non-H2-producingacidogens (Wang and Zhao, 2009). On the other hand, a low SRTmay reduce the substrate utilization efficiency, in particular inthe case of complex substrates which need an adequate hydrolysisperiod, and cause the washout of the active biomass, in turnimpairing the conversion yield. It should be noted that, since mostof the reactors used in the reviewed literature were often operatedwith no biomass recycle, HRT and SRT coincide. In view of full-scale implementation of the process, HRT is of particular concernsince it is clearly related to capital costs. The OLR of the systemmay affect a number of operating issues, including VFA accumula-tion and pH changes (which in turn is a function of the system’salkalinity), as well as variations in the composition of the activebiomass, with consequent modifications of the associated meta-bolic pathways. A comparison of the OLR effects on hydrogen pro-duction documented in the literature is complicated by the factthat this parameter is often expressed in inconsistent units in dif-ferent studies (kg VS/m3 d, kg COD/m3 d, kg TOC/m3 d), and har-monization of the units used is not always possible due to lack ofthe required conversion factors.

Most studies that used stirred reactors with continuous orsemi-continuous operation adopted HRT values between 21 h(Lee and Chung, 2010, who worked on the liquid phase extractedfrom FW) and 4 d (Lee et al., 2010a). The reported OLRs values fallwithin the ranges 8–38 kg VS/m3 d (Hong and Haiyun, 2010; Chuet al., 2008) or 20–64 kg COD/m3 d (Li et al., 2008b; Chu et al.,2008).

Shin and Youn (2005) found that prolonging the HRT of a semi-continuous system from 2 to 5 d and reducing the OLR from 10 to8 kg VS/m3 d more than doubled the hydrogen yield (2.2 vs.1 mol H2/mol hexose); the change in the OLR was found to preventVFAs to accumulate in excess of 20,000 mg COD/l. Similar findingswere reported by Wang and Zhao (2009) who operated an inte-grated two-stage fermentation system; a significant reduction inVS removal and H2 yield was observed as OLR progressively in-creased from 15.10 to 37.75 kg VS/m3 d and SRT decreased from10 to 6.6 d. Such negative effects on the fermentation process wereascribed to the associated reduced timespan of substrate hydroly-sis. Furthermore, increased OLRs were found to result in reducedacetate and butyrate production with an associated increase inpropionate and lactate concentrations; at an OLR of 37.75 kg VS/m3 d, the lactate concentration attained a maximum, accountingfor �30% of the total COD of the measured metabolites (ethanol,acetate, propionate, butyrate and lactate). A significant increasein the hydrogen yield was recorded when SRT increased from 5to 6.6 d (corresponding to a decrease in OLR from 30.20 to22.65 kg VS/m3 d), while a further increase in SRT from 6.6(OLR = 22.65 kg VS/m3 d) to 10 d (OLR = 15.10 kg VS/m3 d) onlyslightly increased hydrogen production.

In an optimization study of semi-continuous digestion of FWand dewatered WAS (Hong and Haiyun, 2010), long HRTs (8.9 d)were found to result in improved hydrogen generation yield. Pro-longed solids retention times were also adopted by Valdez-Vaz-quez et al. (2005): an OLR of 11 g VS/kg d corresponded to a SRTof 21 days, and the hydrogen production yield obtained was 165and 360 N ml/g VSrem under mesophilic and thermophilic condi-tions, respectively; the same SRT value is also reported by Val-dez-Vazquez and Poggi-Varaldo (2009), with an associatedmaximum H2 production of 51.2 N ml/g VSrem.

Different conclusions were suggested by the operation of a two-phase hydrogen + methane production plant studied by Lee andChung (2010); in this case an OLR increase from 7.4 to71.3 g COD/l h, with an associated HRT decrease from 66 to 21 h,resulted in a significant increase in the hydrogen production rate,which varied from 0.62 to 3.88 l/m3 d.

In addition to the widely used continuous-flow stirred reactor(CSTR), other types of reactors have also been investigated in orderto improve the efficiency of biohydrogen production. A packed bedreactor (PBR) was used by Alzate-Gaviria et al. (2007) to obtainhigh hydrogen production yields in the short HRT required formethanogenesis inhibition; a yield of 99 N ml H2/g VSremoved wasattained.

An anaerobic sequencing batch reactor (SBR) was used by Kimet al. (2008b) for FW fermentation, working at different SRTs andHRTs; the maximum hydrogen yield (80.9 N ml H2/g VS, or1.12 mol H2/mol hexose) was displayed at an SRT of 126 h and anHRT of 33 h.

A leaching-bed reactor operated in a sequential batch mode atan SRT of 6 d was used by Han and Shin (2004b) for FW fermenta-tion; the outflow from the leaching-bed reactor was then fed to anupflow anaerobic sludge blanket (UASB) reactor for methane pro-duction; with an OLR of 11.9 kg VS/m3 d, a hydrogen yield of0.31 Nm3 H2/kg VS was achieved.

Elbeshbishy et al. (2011) evaluated the performance of a soni-cated biological reactor (basically a CSTR equipped with an ultra-sonic probe at its bottom) by comparison with a conventionalCSTR fed with raw FW and a CSTR fed with sonicated FW. Whilethe CSTR treating the sonicated feed exhibited a 23% increase inhydrogen yield compared to the conventional CSTR, for the soni-cated reactor the observed improvement was as high as 62%; sim-ilarly, the hydrogen production rate increased, in comparison tothe conventional system, by 27% and 90% for the CSTR fed withsonicated waste and for the sonicated reactor, respectively.


Although no data on full-scale hydrogen fermentation plants iscurrently available, some experience has recently been gained onpilot-scale reactors. A pilot-scale (150 l working volume) anaerobicSBR treating FW is described in Kim et al. (2010); the reactor wasoperated at 35 �C and an HRT of 36 h, achieving a hydrogen yield of0.5 mol H2/mol hexose. The largest pilot-scale system workingwith FW is described in Lee and Chung (2010). A two-phase hydro-gen + methane fermentation system with a 500 l CSTR as the firststage was operated at 30 �C and an optimal HRT of 21 h for thehydrogen production stage. The hydrogen fermentation tank al-lowed for a production of �1.82 mol H2/mol hexose.

3.3. Process temperature

The majority of recent studies on hydrogen production from FWand OFMSW was conducted under mesophilic conditions, specifi-cally between 30 (Lee and Chung, 2010) and 40 �C (Wang and Zhao,2009), and typically in the range 35–37 �C (Dong et al., 2009a;Hong and Haiyun, 2010; Kim et al., 2010, 2011a; Li et al.,2008a,b; Zong et al., 2009). The effect of temperature in the meso-philic range (30–45 �C) was investigated by Kim et al. (2008a) inFW fermentation by Clostridium beijerinckii KCTC 1785; hydrogenproduction increased with increasing temperature up to 40 �C(with a maximum in acetate and butyrate production), being thenstrongly inhibited at 45 �C. Studies on thermophilic conditions,mostly at temperatures of 55 �C, are also documented in the liter-ature (Chu et al., 2008; Lee et al., 2010a; Shin and Youn, 2005; Val-dez-Vazquez and Poggi-Varaldo, 2009; Nazlina et al., 2011).Thermophilic conditions are assumed to optimize the enzymaticactivity of hydrogenase during fermentation by Clostridia, to inhibitthe activity of H2 consumers and also to suppress the growth oflactate-forming bacteria (Lay et al., 1999; Oh et al., 2004; Valdez-Vazquez et al., 2005). Kim et al. (2011b) investigated the effect oftemperature in both the mesophilic and thermophilic range (35–60 �C) on FW fermentation in the absence of any specific inoculum,and found that the lowest and highest H2 production yields wereassociated to temperatures of 35 and 50 �C, respectively. Althoughin both cases the amount of organic acids was comparable, lactatewas predominant at 35 �C while butyrate was the main VFA com-ponent at 50 �C. Microbial analysis of the fermentation mediumalso indicated that the dominating species were lactic acid bacteriaat 35 �C and H2-producers at 50 �C, thus confirming the role oftemperature in dictating the nature of microbial consortium duringthe process.

On the other hand, however, high temperatures have also beenreported to induce thermal denaturation of proteins and essentialenzymes, in turn negatively affecting microbial activity (Leeet al., 2006).

Comparative studies on hydrogen production from FW undermesophilic and thermophilic conditions were carried out by Shinet al. (2004), Valdez-Vazquez et al. (2005), Kim et al. (2008a) andPan et al. (2008). Shin et al. (2004) found that the biogas producedfrom a thermophilic (55 �C) culture was free of methane, whilstmethane was detected under mesophilic (35 �C) conditions, withhydrogen yields of 1.8 and 0.1 mol H2/mol hexose, respectively;the improved yield at higher temperatures was mirrored, in addi-tion to the absence of methane, by negligible propionate concen-trations. In semi-continuous fermentation, Valdez-Vazquez et al.(2005) observed that both the hydrogen content in the biogasand the production yield were higher under thermophilic (55 �C)than under mesophilic (35 �C) conditions, with values of 58 vs.42% by vol. and 360 vs. 165 N ml H2/g VSremoved, respectively.

Pan et al. (2008) studied the effect of temperature at differentfood/microorganisms (F/M) ratios on batch hydrogen productionfrom mixed FW using anaerobic digestion sludge as the inoculum.F/M ratios of 7–10 g VS/g VSS were found to be adequate for hydro-

gen production via thermophilic (50 �C) fermentation (maximumyield = 57 ml H2/g VS at F/M = 7), while under mesophilic (35 �C)conditions hydrogen generation was decreased (maximumyield = 39 ml H2/g VS at F/M = 6).

3.4. pH

The influence of pH on hydrogen fermentation is also quite con-troversial in the literature. In general, pH is considered the mostpivotal parameter due to its effects on hydrogenase activity, meta-bolic pathways as well as substrate hydrolysis. The H+ ion concen-tration in the system is also critical for maintaining adequate ATPlevels in the system, since in the presence of an H+ excess ATP isused to ensure cell neutrality rather than to produce H2 (Nazlinaet al., 2011). Several studies have been conducted on the optimalpH range for fermentative hydrogen production, however the re-sults are often inconsistent due to differences in substrate, seedsludge and operating conditions adopted (Luo et al., 2010; Wuet al., 2010). In this regard it should be mentioned that numerousliterature studies report the results of fermentation runs whereonly the initial pH was adjusted, without any further control alongthe process. However, it is noted that the importance of the initialpH may be overlooked when making direct comparison of resultsobtained at given initial pH values, since several factors – amongthe others, substrate characteristics (composition and buffercapacity) and type of inoculum – dictate the prevailing metabolicpathways and in turn pH evolution during the process, thus deter-mining different hydrogen production yields and rates.

It is acknowledged that low pHs result in inhibition of thehydrogenase activity, which is regarded to as a key factor explain-ing the influence of pH on fermentative hydrogen production (Kha-nal et al., 2004; Nazlina et al., 2011). The metabolic pathwaysinvolving acetate and butyrate production appear to be favoredin the pH range 4.5–6.0, while neutral or higher pHs are believedto promote ethanol and propionate production (Guo et al.,2010a; Rechtenbach et al., 2008; Rechtenbach and Stegmann,2009). It should also be mentioned that hydrogen is mainly pro-duced during the exponential growth phase of Clostridia, while inthe stationary growth phase a shift from acidogenesis (with asso-ciated hydrogen generation) to solvent production is observed. Ithas been suggested (Khanal et al., 2004) that the shift occurs belowpH 4.5, more precisely at pHs as low as 4.1, and the cause seems toinvolve the buildup of VFAs and H2 during the exponential growthphase. Solventogenesis is therefore assumed as a detoxificationmethod of the biomass to avoid inhibitory effects caused by highVFA contents and associated low pHs in the liquid solution (Val-dez-Vazquez and Poggi-Varaldo, 2009). However, other research-ers observed a shift to solventogenesis at pH levels above 5.7,due to the synthesis or activation of the enzymes required for sol-vent production (Khanal et al., 2004). To this regard, Fang et al.(2006) observed a switch to solventogenesis occurring atpHs > 6.5. Other authors (Nazlina et al., 2011) indicated thatdecreasing pH below 6.0 increasingly promoted lactate formation,with an associated negative effect on the hydrogen productionyield. The system pH may also affect the degree of biomass activity,with values <6 capable of significantly inhibiting sulfate-reducingand methane-producing microorganisms. As far as homoacetogen-ic microorganisms are concerned, the effect of pH is not clear. Luoet al. (2010) observed homoacetogenesis inhibition at pHs of 4–5;however, since some homoacetogenic bacteria belong to the genusClostridium, pH values in this range do not necessarily lead toinhibition.

The initial pH is known to affect hydrogen production throughits influence on lag phase duration, spore germination (in thosecases where a shock treatment is preliminary applied to the inoc-ulum) and the synthesis of enzymes (Kim et al., 2011c). Khanal


et al. (2004) found that an initial pH of 4.5 gave the highest specifichydrogen production potential for sucrose and starch, but it wasaccompanied by the lowest production rate and longest lag phase;at higher initial pHs, hydrogen production started earlier and witha higher production rate, but with a shorter overall duration. In thesame study it was also observed that the lower the initial pH, thehigher the maximum acetate/butyrate ratio was. However, Abreuet al. (2009) investigated hydrogen production from arabinoseusing four different anaerobic sludge samples at different initialpHs (4.5–8.0), and in all cases observed higher hydrogen produc-tion potentials as the initial pH increased.

In the investigation of the effect of initial pH on hydrogen pro-duction from FW, batch tests are reported in the literature withvalues typically varying between 5 and 9 (Lay et al., 2003; Kimet al., 2008a, 2011c; Zhu et al., 2008; Dong et al., 2009a; Zonget al., 2009), without any further control during the test. However,different batch and semi-continuous/continuous experiments in-volved pH adjustment during operation, with set-point values be-tween 5 (Liu et al., 2006; Kim et al., 2009; Nazlina et al., 2011) and7 (Hong and Haiyun, 2010), but most commonly in the narrowerrange of 5–5.5 (Alzate-Gaviria et al., 2007; Chu et al., 2008; Kimet al., 2008a,b, 2010; Lee and Chung, 2010; Li et al., 2008b; Leeet al., 2010b; Liu et al., 2006). The pH values on FW were oftenadopted by these authors from other studies on simple substratessuch as glucose. In the case of FW and OFMSW, relatively fewexperiments have been conducted to compare the effect of pH onhydrogen production. Shin et al. (2004) investigated fermentativehydrogen production from FW at pH 4.5, 5.5 and 6.5 under meso-philic and thermophilic conditions; the best results in terms ofcumulative H2 production were attained at pHs of 4.5 and 5.5 un-der thermophilic conditions. Shin and Youn (2005) studied the per-formance of a semi-continuous system under thermophilicconditions at pHs of 5.0, 5.5 and 6.0, with a value of 5.5 yieldingthe best performance. The influence of the initial (5.0–8.0) andoperating (5.0–6.5) pH on hydrogen production from FW by Clos-tridium beijerinckii KCTC 1785 was investigated by Kim et al.(2008a); the optimal values were found to be an initial pH of 7.0and an operating pH of 5.5, with associated values of hydrogenyield and rate of 128 N ml H2/g CODdegraded and 108 N ml H2/l h,respectively. Lee et al. (2008) investigated thermophilic hydrogenproduction from vegetable FW at pHs of 5.5–7.0, obtaining a max-imum production rate of 0.48 mmol H2/g VS h at pH = 6, and amaximum yield of 0.57 mmol H2/g COD at pH = 7; no hydrogenproduction was observed at pH = 5.5. Batch tests on a mixture ofFW, olive mill wastewater (OMW) and WAS (the latter with orwithout HST), were performed at pHs in the range 4.5–7.5 (Cappaiet al., 2010). The best performance with the untreated inoculumwas observed at pH = 6.5, with a cumulative hydrogen productionof 42.9 N l H2/kg VS, which was also accompanied by the highesthydrogen content 51% by vol. in the biogas. Using the HST inocu-lum, the best results were obtained at pHs of 6.5 and 7.0 with pro-duction yields of around 60 N l H2/kg VS (5.6 N l H2/lreactor), whilethe highest hydrogen content in the biogas was measured at apH of 7.5, with values up to 80% by vol. The higher hydrogen con-tents at high pHs were likely due to an indirect effect of enhancedCO2 dissolution in the liquid phase under alkaline conditions. A re-cent study by Nazlina et al. (2011) focused on batch thermophilic(55 �C) digestion of FW at controlled pHs of 5.0, 5.5 and 6.0; thelowest H2 production yield (�18 N ml H2/g substrate-COD) wasobserved at pH = 5.0, while comparable results (�63 and�61 N ml H2/g substrate-COD, respectively) were obtained at pHsof 5.5 and 6.0. Higher pH conditions were also found to result inlower lactate production and higher removal of carbohydratesand volatile solids (VS), however these were also accompanied bya decreased concentration of Clostridia in the digestion medium(65% of total biomass as opposed to 92% at pH = 5.5), indicating

that other microbial phyla may have contributed to H2 productionat higher pH levels. Although bacteria were enumerated, the iden-tification of the other phyla was not performed.

A significant number of studies involving no external pH controlhave also been conducted (Han and Shin, 2002, 2004a,b; Valdez-Vazquez et al., 2005; Pan et al., 2008; Cappai et al., 2009; Valdez-Vazquez and Poggi-Varaldo, 2009; Wang and Zhao, 2009). The con-cept behind this strategy involves appropriate adjustment of theOLR to maintain suitable pH levels for significant and stable hydro-gen production. In semi-continuous experiments using completelymixed reactors, pH was maintained in the range 5.5–6.4 throughbiokinetic control attained by organic overloading (11 g VS/kg d;Valdez-Vazquez et al., 2005; Valdez-Vazquez and Poggi-Varaldo,2009). Wang and Zhao (2009) report that a semi-continuous rotat-ing drum reactor was capable of spontaneously maintaining pHwithin the range 5.2–5.8 at an OLR of 22.65 kg VS/m3 d. In anothersemi-continuous CSTR treating a mixture of FW and OMW, pH wasmaintained in the range 5.0–6.0 by adopting adequate values of thefeed composition (25% w/w FW, 20% w/w OMW, 55% w/w AS), HRT(2 d) and OLR (32.3 kg VS/m3 d) (Cappai et al., 2009). In a thermo-philic two-stage process (combined H2 + CH4 production; Lee et al.,2010b), pH was successfully maintained in the range 5.4–5.7 byrecycling the effluent from the methanogenic stage into the acido-genic reactor.

3.5. Use of co-substrates

FW and OFMSW have often been considered for co-digestionwith other residues, including mainly primary sludge (PS) andWAS, but also wastes from agro-industrial activities. The use ofco-substrates is motivated by other objectives being pursued con-comitantly, including: (a) combined treatment of different wastestreams, (b) ability to treat residues otherwise difficult to manageindividually, (c) dilution of potentially toxic/inhibitory compounds,(d) resulting synergistic effects on biomass, (e) optimization of theconditions for hydrogen production, (f) internal control of pH, and(g) optimization of the carbohydrate/protein ratio. To this regard,although carbohydrates are the preferred substrate for fermenta-tive hydrogen-producing bacteria such as those belonging to theClostridium sp. while hydrogen is hardly produced from proteinsand lipids, some experiences showed that, under some circum-stances, proteins from waste sludge are necessary as a nitrogensource for hydrogen production in both pure and mixed cultures.In particular, Kim et al. (2004) found out that the addition of SSto FW up to 13–19% by weight enhanced the hydrogen productionpotential; lately the same authors (Kim et al., 2011a) also indicatedan optimal FW/sludge ratio for both the hydrogen production po-tential and the generation rate, which was equal to 10:1 w/w ona COD basis for the experimental conditions tested. Shin et al.(2003) showed that the hydrogen production yield decreased asWAS addition increased, due to the presence of methanogens inthe sludge and the low carbohydrate concentration; however,sludge addition also enhanced hydrogen production due to thecontribution of proteins. The maximum hydrogen yield of59.2 ml/g VS was achieved at a FW/sludge ratio of 80:20 w/w.Zhu et al. (2008) tested different mixtures of FW, PS and WASand found appropriate mixing ratios of the three substrates to pro-mote hydrogen production (up to a maximum of 112 ml/g VSadded)due to an improved balance of carbohydrates, nitrogen, phospho-rus and trace metals; moreover, PS and WAS showed a higher buf-fering capacity at low pHs in comparison to FW. Hong and Haiyun(2010) performed semi-continuous tests on mixtures of FW anddewatered sludge for optimization of the FW/sludge ratio andthe operating parameters (HRT, OLR and pH); the best results wereobtained for a FW content of 88% by wt., an HRT of 8.92 days, anOLR of 8.31 g VS/l d and a pH of 6.99. Semi-continuous digestion


runs of mixtures of FW and OMW (Cappai et al., 2009) resulted insignificant and steady hydrogen production when a proper mixingratio between the two was adopted; the best results in terms ofspecific hydrogen production were obtained using 25% w/w FW,20% w/w OMW and 55% w/w WAS; the latter was used as boththe inoculum and the main liquid phase to adjust the water con-tent to wet conditions (�10% TS); the outflow was successfullysubjected to a second methanogenic stage and the final digestedproduct was finally dewatered and composted after mixing witha bulk material.

4. Hydrogen production kinetics

The evolution of hydrogen production over time in batch condi-tions is often described using the Gompertz equation, which hasbeen modified from its original formulation to include parameterswith a biological rather than a mathematical meaning. The modi-fied Gompertz curve describes the time evolution of hydrogen gen-eration using three parameters, namely the H2 productionpotential (Ps), the maximum H2 production rate (Rm) and the lagtime (k), according to Eq. (6)):

P ¼ Ps exp � expRm � e

Psðk� tÞ þ 1

� �� ð6Þ

In general, to compare experimental results obtained using dif-ferent types/amounts of wastes as well as various mixing propor-tions of feed components and F/M ratios, the parameters of themodified Gompertz equation are expressed in specific terms in var-ious ways, typically per unit mass of COD, VS, total solids (TS), hex-ose or carbohydrate-C from the waste in the case of Ps, and per unitmass of volatile suspended solids (VSS) from the inoculum in thecase of Rm. As far as the H2 production potential is concerned, sinceit has been widely demonstrated that hydrogen production mainlyderives from carbohydrates degradation (Lay et al., 2003; Kimet al., 2004; Han and Shin, 2004a,b; Argun et al., 2008; Chu et al.,2008; Dong et al., 2009a; Lee and Chung, 2010; Kim et al.,2011a; Nazlina et al., 2011), meaningful specific units are the massor volume of H2 produced per unit mass of initial or removed car-bohydrates (usually expressed in terms of their hexose equivalent).Other, for instance VS-specific, units may be more useful as designparameters, although their value is commonly substrate-dependent.

Of course, the fact that the hydrogen production yield is ex-pressed with different measures complicates the comparison of re-sults from different studies, since the required conversion factorsare often missing. Typical recently reported values/ranges are:18–205 N l H2/kg VS (Kim et al., 2004, 2011a; Chu et al., 2008);52.5–360 N l H2/kg VSremoved (Gómez et al., 2006; Valdez-Vazquezet al., 2005); 10–133 N ml H2/g COD (Kim et al., 2004, 2011a;Lee et al., 2008; Li et al., 2008b); 0.69–2.10 mol H2/mol hexose(Kim et al., 2010, 2011a; Lee and Chung, 2010); 0.87–1.65 molH2/mol hexoseremoved (Kim et al., 2009); 1.7–5.6 N l H2/lreactor

(Cappai et al., 2010). Further data on process yields derived fromindividual studies are reported in Table 3.

To derive more consistent and comparable values for the kinet-ics of fermentative hydrogen production under varying operatingconditions, in the present review the parameters of the modifiedGompertz equation were derived from several literature studiesavailable; when Gompertz parameters were not directly providedin the papers, they were calculated through least-square fittingof the experimental hydrogen production data with the theoreticalcurve (Eq. (6)). The whole set of kinetic parameters was then con-verted into homogeneous units so as to identify their respectiveranges of variation as a function of the process conditions; thiswas only possible for a reduced number of references, for which

the required information about working volumes, as well as rela-tive amounts and composition of the individual mixture compo-nents, were provided. The results of the analysis of kineticparameters are reported in Table 4. It can be noted that, dependingon the specific type of substrate and inoculum used and the oper-ating conditions adopted (F/M ratio, pH control, temperature, addi-tion of nutrients, etc.), both Ps and Rm differ in the literature by upto three orders of magnitude (with maximum values in the ranges0.2–181 N ml H2/g VS for Ps and 0.1–326 N ml H2/g VSS h for Rm),confirming that for optimization of biohydrogen production therelevant process parameters need to be carefully adjusted.

For an appropriate evaluation of the process kinetics when com-paring data from different experiments, since the values of Ps andRm are interrelated so that the production rate cannot be inter-preted in absolute terms without specifying the value for the asso-ciated production potential, an additional parameter, t95, wasintroduced. This is assumed to be the time required for hydrogenproduction to attain 95% of the maximum yield, and was derivedfrom Ps and Rm rearranging the Gompertz function, as indicatedby Eq. (7). Since t95 provides a measure of how fast the maximumproduction is achieved, it can be usefully adopted to compare, froma kinetic point of view, experimental conditions with differentassociated hydrogen generation yields.

t95 � k ¼ Ps

Rm � eð1� lnð� ln 0:95ÞÞ ð7Þ

As for the lag phase of the hydrogen production process, thedurations reported in recent studies on FW/OFMSW are mostlylower than 20 h (Kim et al., 2004; Shin et al., 2003), with minimumvalues as low as 0.1–1.92 h (Kim et al., 2004; Shin et al., 2004; Panet al., 2008; Cappai et al., 2009). Notably higher values were re-ported by Lay et al. (2003) for a number of individual FW fractions:72 h for rice and potato, 96 h for fat meat. However, shorter dura-tions of the lag phase, namely 2.4 h for lettuce, 4.8 h for potato and14.4 h for rice were reported by Dong et al. (2009a).

The influence of process temperature on the lag phase durationwas discussed by Shin et al. (2004), who observed a shorter (0.1–3.6 h) lag phase under mesophilic compared to thermophilic condi-tions (12–14.4 h). This was ascribed to the fact that the inoculumwas exposed to room temperature before the tests, thus the ther-mophilic biomass activity was possibly affected by this relativelylow temperature. Such findings were confirmed by Pan et al.(2008), who observed lag phase durations of 0.05–4.9 h under mes-ophilic conditions vs. 3.4–5.3 h under thermophilic conditions.

The biomass acclimation period can also be influenced by pH.This issue was discussed by Shin et al. (2004), who found that,while under thermophilic conditions (55 �C) the lag phase durationwas not appreciably affected by pH in the range 4.5–6.5, undermesophilic conditions (35 �C) increasing pHs resulted in shorterlag phase durations (from 0.1 h at pH = 6.5 to 3.6 h at pH = 4.5).These findings were confirmed by Lee et al. (2008), who reportedvalues of 3.8 h at pH = 6.5–7.0 and 7.9 h at pH = 6.0. In mesophilic(39 �C) batch tests on FW and thermally treated WAS (Cappai et al.,2010), relatively slight differences in the lag phase duration wereobserved when pH was varied over the range 6.0–7.0, with valuesranging from 4.6 h at pH 7.0 and 6.3 h at pH 7.5. The same authorshowever found that when digesting a mixture of FW, OMW anduntreated WAS, pH exerted a stronger effect on the lag phase dura-tion, with values decreasing from 15.9 to 6.8 h as pH increasedfrom 4.5 to 6.5.

The influence of different FW pre-treatments on the lag phaseduration was investigated by Kim et al. (2009). Shorter lag phaseswere observed when no pre-treatment was applied (7.3 h) or whenthe waste was maintained at pH = 1 for 1 d before fermentation(8.3 h); longer values (10.0 and 11.9 h, respectively) were found

Table 3Operating parameters and performance data for H2 production from FW and OFMSW fermentation.

Type of substrate Type of inoculum Inoculum pre-treatment

pH Reactor T Specific H2 production Reference

Value/range tested Optimalvalue/range

Type Operationmode

OFMSW Mixture of deep soil, excretesvaccine, pig excretes

HST 5.7 – UASBPBR

Continuous 38 �C 127 ml/g VSremoved

99 ml/g VSremoved

Alzate-Gaviria et al. (2007)

FW Activated sludge HST 6.0–7.5 6.5 Stirred Batch 39 �C 51–81 ml/gVSadded Cappai et al. (2009)FW + OMW Activated sludge – 4.5–6.5 6.5 Stirred Batch 39 �C 3.4–44 ml/gVSadded Cappai et al. (2010)FW Activated sludge HST 6.0–7.0 6.5 Stirred Batch 39 �C 69–114 ml/gVSadded Cappai et al. (2011)FW Activated sludge – 4.5–8.5 6.5 Stirred Batch 39 �C 2.3–58 ml/gVSadded Cappai et al. (2011)FW – – 4.5–8.5 6.5 Stirred Batch 39 �C 5.3–40 ml/gVSadded Cappai et al. (2011)FW Anaerobic sludge – 5.5 (initial) – Stirred Batch 36 �C 37–101 ml/g COD Chen et al. (2006)FW Anaerobic sludge – 5.5 – Stirred Semi-

continuous55 �C 205 ml/gVSadded Chu et al. (2008)

Rice Anaerobic sludge HST 5.5 (initial) – Stirred Batch 37 �C 134 ml/gVSadded Dong et al. (2009a,b)Potatoes 106 ml/gVSadded

Lettuce 50 ml/gVSadded

Lean meat 0Oil 6.25 ml/gVSadded

Fat 0Banyan leaves 1.75 ml/gVSadded

FW Anaerobic sludge Sonication 5.0–6.0 – Stirred Semi-continuous

37 �C 332 ml/g VSadded Elbeshbishy et al. (2011)

OFMSW + slaughterhousewaste

Mesophilic anaerobic sludge – 5.0–6.0 – Stirred Semi-continuous

34 �C 52.5–71.3 ml/g VSremoved Gómez et al. (2006)

FW Anaerobic sludge – 5.0–6.0 – Stirred Semi-continuous

34 �C 27–28 ml/g VSadded Gómez et al. (2009)

Anaerobic sludge 5.0–6.0 – Stirred Semi-continuous

34 �C 19–26 ml/g VSadded

FW Rumen microorganism Acclimation – Leaching-bed

Continuous 37 �C – Han and Shin (2002)

FW Anaerobic sludge HST – Leaching-bed

Continuous 37 �C 310 ml/g VSadded Han and Shin (2004a)

FW Anaerobic sludge HST – Leaching-bed

Continuous 35 �C 34.7-155 ml/g VSaddedb Han and Shin (2004b)

FW (artificial) + dewateredexcess sludge

Anaerobic sludge – Stirred Semi-continuous

35 �C – Hong and Haiyun (2010)

FW + sewage sludge Anaerobic sludge HST 5.0–6.0 – Stirred Batch 35 �C 60.1 ml/g VSadded Kim et al. (2004)FW Selected pure culture – 5.0–8.0 (initial) 7.0 (initial) Stirred Batch 30–

45 �C128 ml/g CODdegraded Kim et al. (2008a)

FW Anaerobic sludge HST 5.3 – Stirred SBR 35 �C 80.9 ml/g VSadded Kim et al. (2008b)FW Anaerobic sludge HST 5.3 – Stirred SBR 35 �C 62.6 ml/g VSadded Kim and Shin (2008)FW – HST 5 – Stirred Batch 35 �C 153.5 ml/g VSadded Kim et al. (2009)FW – HST 5.3 – Stirred SBR 35 �C 0.35–0.54 mol/molhexose Kim et al. (2010)FW + sewage sludge – HST 8.0 (initial), 6.0

(operating value)– Stirred Batch 35 �C 162 ml/g VSadded Kim et al. (2011a)

FW – – 8.0 (initial), 6.0(operating value)

– Stirred Batch 35–60 �C

16.5–137.2 ml/g VSadded Kim et al. (2011b)

OFMSW Hydrogen-producing sludgeanaerobic sludge

HST – – Stirred Batch 37 �C 132 ml/g TVS Lay et al. (1999)

FW Anaerobic compost HST 7 (initial) – Stirred Batch 37 �C 53.4–78.7 N ml/gVS Lay et al. (2005)FW (liquid phase) Anaerobic sludge HST 5.5 – Stirred Continuous 30 �C 1.82 mol/molglucose Lee and Chung (2010)Vegetable FW Compost – 5.5–7.0 6.0–7.0 Stirred Batch 55 �C 0.48 mmol/g CODadded Lee et al. (2008)Vegetable FW Compost Acclimation 6 (initial) – Stirred Semi-

continuous55 �C 1.7 mmol/g CODadded

(72 ml/g VSaddedb)

Lee et al. (2010a)

OFMSW Anaerobic sludge HST 5–5.7 – Stirred Semi- 55 �C 114 ml/g VSadded Lee et al. (2010b)

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Table 3 (continued)

Type of substrate Type of inoculum Inoculum pre-treatment

pH Reactor T Specific H2 production Reference

Value/range tested Optimalvalue/range

Type Operationmode

continuousFW + aged refuse Sewage sludge – 5 – Stirred Batch 36 �C 193.85 ml/g VSadded Li et al. (2008a)FW Acidogenic sludge – 5.3–5.6 – Stirred Semi-

continuous35 �C 0.04–60 mmol/L d (1–

2 mmol/g COD)Li et al. (2008b)

FW Anaerobic sludge HST 3.5–8.5 5–5.5 Stirred Batch 37 �C 0.72–7.4 mmol (pH > 4.5)0 (pH 6 4.5)

Liu et al. (2006)

– – – Stirred Semi-continuous

37 �C 1000–3000 ml/l db

(27–80 ml/g VSaddedb)

FW Anaerobic sludge HST 5.0–6.0 5.5 Stirred Batch 55 �C 18–63 ml/g CODaddedb Nazlina et al. (2011)

FW Thermoph. Anaerobic sludgemesoph. Anaerobic sludge

––

––

6.3–6.6(initial)5.7 (initial)

Stirred Batch 50 �C35 �C

57 ml/g VSadded

39 ml/g VSadded

Pan et al. (2008)

FW Anaerobic sludge Acclimation 5.0–6.0 5.5 Stirred Semi-continuous

55 �C 0.4–1.0 l/l d1–2.4 mol/mol hexosecons

Shin and Youn (2005)

FW + sewage sludge Anaerobic sludge HST 5.0–6.0 – Stirred Batch 35 �C 34.0–59.2 ml/g VS Shin et al. (2003)FW Mesoph. Anaerobic sludge

Thermoph. Anaerobic sludge– 4.5–5.5–6.5 4.5

4.5Stirred Batch 35 �C

55 �C1.3–5.0 ml/g VSadded

28.4–46.3 ml/g VSadded

Shin et al. (2004)

FW Anaerobic sludge HST 5.5 Stirred Batch 30 �C 14.6–104.8 ml/g VSadded Sreela-or et al. (2011a)FW + sludge Anaerobic sludge HST – – Stirred Batch 30 �C 11.57–102.63 ml/g

VSadded

Sreela-or et al. (2011b)

OFMSW + paper waste Compost Acclimation 5.8–6.0 Stirred Continuous 60 �C 2.4–5.4 m3/m3 d Ueno et al. (2007)FW + paper Anaerobic sludge – 5.56–5.95 Unspecified Semi-

continuous55 �C 54.8 N ml/g VSremoved Valdez-Vazquez and Poggi-

Varaldo (2009)FW + paper Thermoph. anaerobic sludge

mesoph. anaerobic sludge– 6.4

5.5––

Unspecified Semi-continuous

55 �C37 �C

360 ml/g VSremoved

165 ml/g VSremoved

Valdez-Vazquez et al.(2005)

FW – – 5.2–5.8 – Rotatingdrum

Semi-continuous

40 �C 49–65 ml/g VSadded Wang and Zhao (2009)

FW + primarysludge + sewage sludge

Anaerobic sludge Acclimation 7.0 (initial) 5.5–6.0 Stirred Batch 35 �C 112 ml/g VSadded Zhu et al. (2008)

a FW collected from a dining hall.b Calculated from data provided by the authors.

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Table 4Observed ranges for the parameters of the modified Gompertz equation as derived from different literature sources.

Type of substrate Type of inoculum Ps Rm Rm t95�k Reference

(N ml H2/g VS) (N ml H2/l h) (N ml H2/g VSS h) (h)

OFMSW Anaerobic sludge + acclimated H2-producing culture Min 0.2 – Min 0.1 – Lay et al. (1999)Max 117 Max 16.8

FW Acclimated mesophilic culture Min 1.3 Min 0.19 Min 0.3 Min 12.3 Shin et al. (2004)Max 5.0 Max 0.44 Max 0.7 Max 43.1

FW Acclimated thermophilic culture Min 28.4 Min 1.57 Min 2.5 Min 23.4 Shin et al. (2004)Max 91.5 Max 11.93 Max 19.0 Max 115.1

FW Anaerobic grass compost Min 53.4 – Min 4.4 – Lay et al. (2005)Max 78.7 Max 21.6

FW Anaerobic digester sludge Min 66.1 Min 17.6 Min 39 Min 3.9 Chen et al. (2006)Max 180.6 Max 135.2 Max 286 Max 10.4

FW Compost Min 0 – Min 0 Lee et al. (2008)Max 24.1 Max 12.8

FW Thermophilic anaerobic sludge Min 3.2 – Min 0.3 Min 12.4 Pan et al. (2008)Max 54.2 Max 30.3 Max 36.3

FW Mesophilic anaerobic sludge Min 0.03 – Min 0 Min 17.7 Pan et al. (2008)Max 39.2 Max 12.7 Max 30.1

Rice Acclimated anaerobic sludge 132 37.3 4.7 41.3 Dong et al. (2009a,b)Potato Acclimated anaerobic sludge 102 31 3.9 38.4 Dong et al. (2009a,b)Lettuce Acclimated anaerobic sludge 48 16.3 2.0 34.3 Dong et al. (2009a,b)FW None Min 0 – Min 0a – Kim et al. (2009)

Max 148.7 Max 9.5a

FW Aerobic sludge Min 62.9 Min 503 Min 103 Min 9.8 Cappai et al. (2011)Max 131.9 Max 1320 Max 326 Max 13.5

FW Untreated sewage sludge Min 147.0 Min 300 Min 34.8 Min 15.1 Kim et al. (2011a)Max 175.2 Max 710 Max 118.1 Max 30.8

FW None Min 16.5 Min 27 – Min 11.4 Kim et al. (2011b)Max 137.2 Max 369.1 Max 22.8

FW Anaerobic sludge Min 14.6 Min 3.2 Min 2.2 Min 5.3 Sreela-or et al. (2011a)Max 104.8 Max 38.9 Max 16.9 Max 27.5

FW Anaerobic sludge Min 11.57 Min 5.0 Min 6.8 – Sreela-or et al. (2011b)Max 102.63 Max 163.7 Max 61.6

a Expressed per unit of substrate-VS.


for thermal (90 �C, 20 min) and alkaline (pH = 13 for 1 d) pre-treatment.

The overall duration of the hydrogen production phase was typ-ically observed to range between 1 (Lee et al., 2008; Cappai et al.,2009, 2010; Kim et al., 2009) and 4 d (Han and Shin, 2004b; Kimet al., 2004, 2009; Shin et al. 2004; Zong et al., 2009). Values asshort as �9.5 h were reported by Lee et al. (2008) (vegetable FW,pH controlled at 6.5; thermophilic conditions) and Shin et al.(2004) (FW, pH controlled at 5.5 and 6.5, mesophilic conditions).In the same work, when pH was controlled at 4.5 and thermophilicconditions were adopted, a significantly longer duration (5.8 d) ofhydrogen production was observed; prolonged production periodswere also reported by Lay et al. (2003) for individual FW fractions,with values of 8–9 d for rice and potato and 6.6 d for fat meat.

4.1. Statistical analysis of hydrogen production data

The experimental data of hydrogen production potential (Ps)from the reviewed literature studies were processed to deriveinformation on the relative importance accommodating for theexisting relationships among the main variables of relevance. Tothis aim, literature data were filtered and, when feasible, repro-cessed and converted into homogeneous units as described in Sec-tion 4, resulting in 198 individual data points from 15 differentpublications (Lay et al., 1999; Shin et al., 2003, 2004; Chen et al.,2006; Lee et al., 2008; Pan et al., 2008; Zhu et al., 2008; Cappaiet al., 2009, 2010, 2011; Kim et al., 2009, 2011a,b; Sreela-oret al., 2011a,b) being used for the statistical regression analysis.The input variables used in the analysis and the associated levelsare reported in Table 5, while the response variable was the hydro-gen production potential.

The statistical technique known as recursive partitioning wasapplied for the analysis of data. This was used as a means to builda flexible, parsimonious regression model that can be representedby a binary regression tree; this can also be seen as a way to auto-matically identify the most important variables affecting the re-sponse while accounting for associations among the explanatoryvariables. A regression tree is constructed by recursively partition-ing the data set into two homogeneous groups (son nodes) accord-ing to some criterion, and then splitting the nodes up further oneach of the branches. On each node the response is fitted by thenode average, which implies defining a stepwise constant fitted re-sponse surface. Recursive partitioning (Hothorn et al., 2006) thusinvolves separating statistical groups progressively decreasing insize and increasing in internal homogeneity in terms of the statis-tical distribution of the response variable. In the present case, split-ting was implemented using a conditional inference criterion,implying testing the global null hypothesis of independence be-tween any of the input variables and the response. If the nullhypothesis cannot be rejected, the splitting procedure at that nodeis stopped, and this becomes a terminal node of the tree; other-wise, the input variable exhibiting the strongest association withthe response (as measured by the corresponding p-value) is se-lected and a binary split in it is implemented. The splitting proce-dure continues until each node becomes a terminal node accordingto the above mentioned condition.

The graphical output of the recursive partitioning procedure,which was implemented using the party package in the statisticalsoftware R (Strobl et al., 2009), is shown in Fig. 1. At each terminalnode the number of data points (n) and their statistical distribution(indicated by the associated box plots) are provided. The inputvariables identified as the most important in affecting maximum

Table 5Input variables used for the regression analysis of hydrogen production data.

Variable Symbol No. levelsa Valuesa

Type of substrate Substr_type 2 1 = Food waste2 = Vegetable kitchen waste

Type of co-substrate Co-substr_type 5 1 = No co-substrate added2 = Primary sludge3 = Activated sludge4 = OMW5 = Nightsoil + sewage sludge6 = Primary + activated sludge

Co-substrate/substrate mass ratio Co-substr/substr – –Type of inoculum Inoc_type 6 1 = No inoculum added

2 = Anaerobic sludge3 = Compost4 = Thermophilic anaerobic sludge5 = Pre-selected H2-producing inoculum6 = Activated sludge

F/M mass ratio F/M – –Type of pre-treatment Pretr_type 2 1 = No pre-treatment applied

2 = Thermal pre-treatmentPre-treatment temperature Pretr_T – –Pre-treatment duration Pretr_duration – –Control of initial pH pHin_contr 2 1 = No control

2 = ControlledInitial pH value pHin – –Control of operating pH pH_contr 3 1 = No control

2 = Continuously controlled3 = Controlled with buffer addition

Operating pH value pH – –Buffer type Buffer_type 3 1 = No buffer added

2 = Citrate3 = Disodium hydrogen phosphate

Buffer dosage Buffer_dos – –Fermentation temperature Ferm_T – –Nutrient addition Nut_add 2 1 = No nutrient added

2 = Nutrient added

a Only reported for qualitative (discrete) variables.

Fig. 1. Regression tree obtained from recursive partitioning. The number of data points (n) and the statistical distribution of Ps (indicated by the associated box plots) areshown for each terminal node, while the average value of Ps is provided at each splitting point. All values in N l H2/kg VS.


hydrogen production are: type of co-substrate, type of pre-treat-ment, operating pH, control of initial pH and fermentation temper-ature. Six terminal nodes were identified, which differed both for

the average value and the distribution of the response variable.The type of co-substrate was found at the highest hierarchical levelof the tree, splitting the dataset into two groups, with average Ps


values of �56 N l H2/kg VS on the left branch and �25 N l H2/kg VSon the right branch. When inspecting the tree towards the left, thetype of pre-treatment results in different maximum productionyields, with average values �43 and �66 N l H2/kg VS on the rightand left branches of node 2, respectively.

In synthesis, the highest hydrogen production potential (averagevalue ffi 87 N l H2/kg VS) was found to be attained for the followingcombination of input variables: Co-substr_type = {none, primary +activated sludge, nightsoil + sewage sludge}, pretr_type = {thermal}and pH > 5.0. On the other hand, the combination of input variablesyielding the worst mean response (�14 N l H2/kg VS) was: Co-sub-str_type = {none, primary + activated sludge, nightsoil + sewagesludge}, pretr_type = {none}, pHin_control = {none} andFerm_T 6 39 �C (i.e. mesophilic conditions).

As for the general validity of the results of the statistical analy-sis performed, it should nevertheless be stressed that in some cases(see nodes 4, 5 and 7 in Fig. 1) the dispersion of data around thecorresponding value (the node mean) was appreciable. As theobjective of the analysis was to assess the relative relevance ofthe identified input variables on the yield of the fermentation pro-cess, this feature is not deemed to affect the findings of the analy-sis. Nonetheless, this feature may suggest the presence of hiddenvariables which may explain the residual heterogeneity withinthe individual terminal nodes. Unfortunately, the information thatcan be retrieved from literature data is not sufficient to resolvesuch an ambiguity. However, the obtained results appear to sug-gest that, in order to allow for comparison of data from differentliterature sources and build reliable predictive models for the fer-mentative hydrogen production process, a high level of consistencybetween data is strictly required. This requires both a harmoniza-tion of the way in which the parameters of interest for the process

Fig. 2. Schematization o

are expressed and a more accurate description of the experimentalconditions adopted in the fermentative H2 literature.

5. Hybrid processes

Although according to some authors (Lee and Chung, 2010) fer-mentative hydrogen production from FW may be economicallyviable, it is also generally acknowledged that, since the majorityof the organic content of the original substrate remains unde-graded, the process should be combined with a second treatmentstage to achieve substrate stabilization and increase energy con-version. The second stage may thus be oriented to producing eitheradditional hydrogen or methane, with a variety of potential alter-natives differing in the type of process applied and/or the charac-teristics of the resulting product (Fig. 2).

On one instance the residual organic content of the waste feed,which is mainly in the form of the soluble products from thehydrolytic stage, may be converted into methane in a second-stagereactor where suitable environmental conditions for methanogensare maintained. Alternatively, the hydrogen content still stored inthe effluent from the first stage may be further exploited throughother biological processes including photo-fermentation or micro-bial electro-hydrogenogenesis.

As for combined two-stage biological H2 + CH4 production,which is one of the most common strategies proposed, the overallanaerobic digestion process can be described by the Eq. (8):

C6H12O6 þ 2H2O! 2CH4 þ 4CO2 þ 4H2 ð8Þ

If the energy conversion efficiency of the process is calculatedthrough Eq. (4), assuming a lower heating value of 801 kJ/molfor methane, a value of 89.0% is obtained. Considering the conven-tional single-stage methane production process (C6H12O6 ?

f hybrid processes.


3CH4 + 3CO2), a conversion yield of 83.2% would be obtained. Thissupports the assumption that combined H2 + CH4 production is,from a theoretical point of view, energetically more favorable thanconventional anaerobic digestion (Dong et al., 2011). CombinedH2 + CH4 production from FW has been studied through semi-con-tinuous or continuous tests (Cappai et al., 2009; Chu et al., 2008;Han and Shin, 2004a,b; Lee and Chung, 2010; Lee et al., 2010b;Liu et al., 2006; Wang and Zhao, 2009). As mentioned above, HRTand OLR values for the methanogenic stage vary in the ranges 4–27 d and 3–8 kg VS/m3 d (Wang and Zhao, 2009) or 4–16 kg COD/m3 d (Lee et al., 2010a,b; Chu et al., 2008). While in most studiessingle-stage methane production was conducted under mesophilicconditions (typically 37 �C) (Han and Shin, 2004a,b; Liu et al., 2006;Zong et al., 2009), combined H2 + CH4 generation from FW was alsoinvestigated under thermophilic (55 �C) conditions (Lee et al.,2010b). Irrespective of whether inoculum was pre-treated (Chuet al., 2008; Liu et al., 2006; Wang and Zhao, 2009) or used as re-ceived (Han and Shin, 2004a,b; Lee and Chung, 2010; Lee et al.,2010b), the two-stage process was found to be capable of signifi-cant H2 + CH4 production. To this regard, Liu et al. (2006) observedthat 100 �C HST of the inoculum did not adversely affect the meth-anogenic stage. CH4 yields were reported to fall within the range460–550 N l/kg VS (Chu et al., 2008; Liu et al., 2006; Wang andZhao, 2009). Although few studies compared conventional single-stage methane production and two-stage H2 + CH4 production,Liu et al. (2006) found that under semi-continuous conditions thelatter process generated 21% more methane, and, as expected, amuch lower amount of VFAs in the final effluent.

Interesting economic considerations were derived by Lee andChung (2010), who managed a two-stage pilot-scale H2 + CH4 fer-mentation process treating FW, connected to a fuel cell. ComparingH2-only fermentation, CH4-only fermentation, and combinedH2 + CH4 fermentation, while negligible differences in productioncosts among the three systems were estimated, an increase in en-ergy production by 12–25% was observed for the combined system.

Further hydrogen production may also be derived by combiningdark fermentation with photo-fermentation. Purple non-sulfur pho-tosynthetic bacteria are capable of using short-chain organic acids aselectron donors to generate H2 through a light-driven metabolism;thus, the metabolic products of dark fermentation represent a poten-tial substrate for photo-fermentative bacteria. Zong et al. (2009)studied a two-stage batch process including dark- and photo-fer-mentation in order to produce hydrogen from FW. Cattle dung com-post mixed with water (1:10 w/v) and heat-treated for 15 min wasused as the inoculum for dark fermentation, whilst R. sphaeroidesZX-5 was used as the inoculum for photo-fermentation. Acetate,butyrate and butanol were found to be consumed during 168-hphoto-fermentation. The hydrogen yield achieved in the individualstages of dark and photo-fermentation was 1.77 and 3.63 mol/molhexose, respectively, with an overall yield of 5.4 mol H2/mol hexose,equivalent to a conversion efficiency of 45%.

Bio-electrochemical systems have also been proposed as meth-ods to couple with fermentative hydrogen production. Amongthese, microbial electrolysis cells (MECs, initially developed byLiu et al., 2005) are based on microbially-mediated oxidation of or-ganic substances in an anodic compartment, with the aid of anexternal circuit where an external power supply is provided. Theelectrons generated by the degradation process are transferredthrough the external circuit to a cathodic compartment, while pro-tons are transferred through the ion exchange membrane that sep-arates the two compartments. In the cathodic compartmentelectrons reduce the protons generated by the biological processor from dissociated water, producing H2 (Liu et al., 2005; Loganet al., 2008; Jeremiasse et al., 2010). For electrochemically-drivenhydrogen production to occur in such systems, it has been demon-strated that a voltage needs to be applied, which theoretically

amounts to 0.14 V if acetate is assumed as the reference organicsubstrate (Logan et al., 2008). Although to the authors’ knowledgethere are no specific examples in the literature of combination ofdark fermentation and MECs for hydrogen production from FW,some studies exist of application to different types of either pureor residual substrates, including pure VFAs (Guo et al., 2010a,b;Kyazze et al., 2010; Manuel et al., 2010; Cheng and Logan, 2011),wastewaters (Lu et al., 2009), lignocellulosic materials (corn stover[Lalaurette et al., 2009], cellulose [Wang et al., 2011]), wheat pow-der (Tuna et al., 2009). However, since the nature of the metabolicproducts of acidogenesis is similar for different types of sourcesubstrates and MECs have been found capable of using differentsubstrates including fermentable and non-fermentable organics,the results of the mentioned studies suggest the feasibility of atwo-stage dark fermentation + MEC process for combined hydro-gen production from FW/OFMSW.

Another candidate process for a second-stage treatment afterdark fermentation is the generation of electric current throughmicrobial fuel cells (MFCs), where bacteria catalyze the oxidationof organic acids from the acidogenic phase of dark fermentation.If the electrons generated by the oxidation reactions are trans-ferred from the anode (biological compartment) to a cathodethrough an external circuit, the electron flow produces electricity(Logan et al., 2006). In the literature, studies are documented ofcombination of dark fermentation and MFCs, using either pure sub-strates such as glucose (Sharma and Li, 2010), synthetic dark fer-mentation effluent (Poggi-Varaldo et al., 2009; Vázquez-Larioset al., 2011), cellulose (Wang et al., 2011) and vegetable waste(Mohanakrishna et al., 2010). In their study on vegetable waste,Mohanakrishna et al. (2010) found that the MFC performancewas improved when the system was fed with the pre-fermentedwaste instead of the untreated waste. This can be ascribed to thefact that adequate MFC operation requires hydrolysis of particulateorganic matter to make this available to the biomass.

6. Conclusions and perspectives

The analysis of over 80 literature references on fermentativehydrogen production from FW and OFMSW has shown that numer-ous process parameters have the potential of affecting the evolu-tion of the metabolic pathways involved, in turn affecting theprocess kinetics and the conversion yield. As indicated by the re-view performed, the main parameters of concern include pH, tem-perature, solids retention time, inoculum addition/type/pre-treatment, presence of co-substrates, reactor configuration, reactoroperation mode, combination with additional processes. Thestrong influence exerted by the individual parameters mentionedas well as the existence of mutual interactions can in fact lead tovariations up to three orders of magnitude in process performancedepending on the specific combination of the operating variablesadopted. At present, given the existing uncertainties about theindividual and joint influence of such parameters, prediction offull-scale reactors performance based on the existing data mayturn out to be unreliable, therefore further systematic study on thisissue is strongly recommended.

As to the process indicators, a variety of parameters have beenproposed in the literature which have been used to evaluate theprocess performance from different perspectives, spanning fromthe production potential to the process kinetics, from the biogascomposition to the energy conversion efficiency. Accordingly,parameters including the specific production potential, the produc-tion rate, the time required to attain a given fraction of the maxi-mum production, the H2 content in the biogas, the energyconversion yield are typically adopted as process performanceindicators. In the authors’ opinion, care should be taken when


adopting a given indicator in place of another to monitor the pro-cess efficiency as well as when using specific parameters to com-pare results obtained under different operating conditions. Forexample, some parameters are strictly dependent on the intrinsiccharacteristics of the substrate concerned, and as such shouldnot be used for comparison purposes unless considering the sametype of substrate. Secondly, for a reliable assessment of the overallperformance of the fermentation process to be attained, the use ofmultiple parameters appears to be recommended, so as to accountfor the complexity and the interrelations between the numerousfactors of concern.

With regard to the above mentioned issues, the authors believethat some considerable effort should be made by the scientificcommunity to harmonize the description methods adopted, withthe aim of allowing comparison of results from different sourcesand gaining an improved understanding of the numerous interrela-tions between the underlying factors which govern the fermenta-tion process. It is believed that this may also help explaining thecontroversies currently found in the results obtained using differ-ent approaches and methods, resolving apparently contrastingconclusions derived from such results.

As to the implementation potential of fermentative hydrogenproduction, although the technical feasibility in case of processingof simple substrates has been demonstrated by many papers, thetechnology still appears to be in an early stage (especially for com-plex substrates), and to the authors’ knowledge no single full scaleplant is operative yet.

For such reasons, considerable efforts appear to be required toassess the potential for full-scale application of the process, fromboth a technical and a global economic perspective. Nevertheless,a number of issues may be mentioned in regard to the technicalfeasibility of the process on the basis of the state of the art ofknowledge to date. In general, it is believed that the technologywould have a rather limited scope if complex organic matter couldnot be effectively used as the substrate; furthermore, an expandednumber of substrate types and the use of mixed cultures as theinoculum would drastically improve the chance of successfuldevelopment of the process. To this regard, several experienceshave demonstrated that appreciable hydrogen production fromFW and OFMSW can be attained using the indigenous biomasspresent in the substrate.

An issue which deserves significant attention specifically froman engineering point of view is the possibility of optimizing theprocess performance by appropriately adjusting process configura-tion and operation, with no need for external control of the operat-ing variables or for application of severe conditions.

In regard to the technical sustainability of biohydrogen produc-tion, it is generally acknowledged that since fermentation aloneconverts, already under optimal conditions, no more than 33% ofthe chemical energy contained in substrate (whatever this maybe), the process should be combined with a second treatment stageaimed at improved substrate stabilization and enhanced energyconversion. The residual organic content of the waste feed, whichis mainly in the form of soluble products of the hydrolytic stage(organic acids and alcohols), may beneficially be converted intomethane in a second-stage reactor. Alternatively, the hydrogencontent which is still stored in the effluent from the first stagemay be recovered through other biological processes includingphoto-fermentation or microbial electro-hydrogenogenesis. Boththe mentioned alternatives to methanogenesis are still at a rela-tively early stage and as such deserve further investigation.

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a review of dark fermentative hydrogen production from biodegradable municipal waste fractions

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