Application of molecular techniques on heterotrophic hydrogen production research
R.Y.Li b ,T.Zhang a ,?,H.H.P.Fang a
a Department of Civil Engineering,The University of Hong Kong,Hong Kong,China b
School of Environmental Science and Engineering,Tianjin University,Tianjin,China
a r t i c l e i n f o Article history:
Received 29November 2010
Received in revised form 16February 2011Accepted 17February 2011
Available online 21February 2011Keywords:
Hydrogen production Molecular techniques 16S rDNA
Fe-hydrogenase gene PCR
a b s t r a c t
This paper reviews the application of molecular techniques in heterotrophic hydrogen production https://www.doczj.com/doc/e610023587.html,monly used molecular techniques are introduced brie?y ?rst,including cloning-sequencing after poly-merase chain reaction (PCR),denaturing gradient gel electrophoresis (DGGE),terminal-restriction frag-ment length polymorphism (T-RFLP),?uorescence in situ hybridization (FISH)and quantitative real-time PCR.Application of the molecular techniques in heterotrophic hydrogen production studies are discussed in details,focusing on identi?cation of new isolates for hydrogen production,characterization of microbial compositions in bioreactors,monitoring microbial diversity variation,visualization of microbial distribu-tion in hydrogen-producing granular sludge,and quanti?cation of various microbial populations.Some sig-ni?cant ?ndings in recent hydrogen production studies with the application of molecular techniques are discussed,followed by a research outlook of the heterotrophic biohydrogen ?eld.
ó2011Elsevier Ltd.All rights reserved.
1.Introduction
Energy and environment are essential for sustainable develop-ment of the global prosperity.Currently,over 80%of energy supply is dependent on fossil fuels,which cause the deterioration of envi-ronment and rapid exhaustion of natural energy sources (Guo et al.,2010).This has led to the search for alternative energy sources,among which hydrogen has attracted much attention re-cently.As a clean energy,producing only water after combustion,hydrogen may become an alternative to fossil fuels in the future.It also has a high energy yield of 122kJ/g,which is about 2.75times that of fossil fuels (Kim et al.,2006a ).
At present,hydrogen is commercially produced by either thermocatalytic reformation of hydrocarbons or electrolysis of water,both of which are highly energy consuming and unsustain-able processes (Das and Veziroglu,2008).Heterotrophic biological production of hydrogen has,however,attracted research interests due to its potential ability of degrading organic pollutants which serve as carbon and energy sources for the microbes during har-vesting hydrogen (Li and Fang,2009).Heterotrophic hydrogen pro-duction is often classi?ed into two categories depending on whether light is required,i.e.dark fermentation and photo fermen-tation (Levin et al.,2004).Dark fermentation converts organic pol-lutants into hydrogen by dark fermentative bacteria in the absence of light,producing organic acids,mainly acetate and butyrate,and alcohols as by-products.Photo fermentation is potentially able to convert acids and alcohols,which are the by-products of dark fer-mentation,into hydrogen by photosynthetic bacteria using light as energy source.Dark fermentation has a high production rate of hydrogen,but with low hydrogen yield,converting no more than 40%of the chemical energy in the organic pollutants into hydrogen (Li and Fang,2007).In comparison,photo fermentation produces little organic residues,resulting in higher hydrogen yield,but has much lower hydrogen production rate than dark fermentation (Lee et al.,2010).
Various factors have been studied for heterotrophic hydrogen production including source of inoculation,feeding substrates,reactor design and operating conditions such as pH,temperature and hydraulic retention time (HRT)etc.With the development of molecular techniques,identi?cation and quanti?cation of microor-ganism communities involved in hydrogen production become more convenient,effective and accurate.The nucleic acid based techniques have been widely used in heterotrophic hydrogen pro-duction studies in the past decade,which contributed much to identi?cation of the new isolated hydrogen-producing bacteria,exploration of the metabolic functions and interactions of different species in hydrogen production system,and investigation on the effects of operational factors on microbial communities.The appli-cation of molecular techniques will thus help to optimize the oper-ational conditions of the bioreactors,improve the reactor stability,and increase the hydrogen production rate and yield.
This article aims to review the application of molecular tech-niques in heterotrophic hydrogen production https://www.doczj.com/doc/e610023587.html,monly
0960-8524/$-see front matter ó2011Elsevier Ltd.All rights reserved.doi:10.1016/j.biortech.2011.02.072
Corresponding author.Tel.:+852********;fax:+852********.
E-mail address:zhangt@hkucc.hku.hk (T.Zhang).
used molecular techniques are introduced brie?y?rst,including cloning-sequencing after polymerase chain reaction(PCR),dena-turing gradient gel electrophoresis(DGGE)(Muyzer et al., 1993),terminal-restriction fragment length polymorphism (T-RFLP)(Liu et al.,1997),?uorescence in situ hybridization (FISH)(Wagner et al.,2003)and quantitative real-time PCR (qRT-PCR)(Zhang and Fang,2006).Applications of the above molecular techniques in analysis of microbial communities in hydrogen production bioreactors are discussed in details,focus-ing on identi?cation of new isolates for hydrogen production, characterization of microbial compositions in bioreactors,moni-toring microbial diversity variation,visualization of microbial distribution in hydrogen-producing granular sludge,and quanti?-cation of various microbial populations.Four tables were compiled for heterotrophic hydrogen production data scattered in literature in terms of aforementioned respective applications except visualization of microbial distribution in granular sludge, which had very limited reports.Data summarized in tables in-clude fermentation type(dark or photo-fermentation),seed sludge(or isolation source for Table1),reactor design(batch or type of continuous reactor),culture volume(l),substrate,maxi-mum volumetric hydrogen production rates(l-H2/l/h),hydrogen yield(mol H2/mol carbohydrates consumed for dark fermentation and compared to stoichiometry(%)),cell density(g-VSS/l),molec-ular techniques used and microbial analysis results.
2.Molecular techniques
The starting point for the molecular methods and related proce-dures is the extraction of nucleic acids.The reliability of the molec-ular techniques depends on quality and representativeness of RNA/ DNA extracted from sludge samples in the reactors.The DNA extraction process is composed of cell lysis,contamination re-moval,solvent extraction,precipitation and puri?cation(Miller et al.,1999).The amount of nucleic acid(as expressed by A260) and purity(as expressed by the ratios of A260/A280for protein con-tamination and A260/A230for salt contamination)are measured spectrophotometrically using the conventional UV–visible spec-trometer or the more advanced spectrometers,such as Nanodrop. The integrity of rRNA(containing5S,16S and23S)and genomic DNA could be visually checked by conducting electrophoresis using agarose gel.The most commonly used phylogenetic biomarker is 16S rRNA gene(16S rDNA)which has a huge database available (over338,193of16S rDNA sequences in GenBank as of Nov.27, 2010).
The extracted DNA is subjected to PCR ampli?cation using‘‘uni-versal’’primers or primers designed to amplify rRNA genes from particular group of organisms.The PCR cycle takes place in three steps:denaturing,annealing and extension.The crucial part of a PCR is the selected primer set targeting genes of interest which cover special taxonomic or functional groups.These primers may be individually designed based on the alignments of relevant DNA sequences,or simply adapted from the literature.The bacte-ria-speci?c primers,such as the sets of EUB8F and UNIV1492R (Zhu et al.,2008),EUB968F and UNIV1392R(Hung et al.,2008), EUB341F and UNIV518R(Akutsu et al.,2008),and EUB357F and UNIV518R(Shin et al.,2004),were commonly used in the micro-bial analysis in hydrogen production studies.
The selected primer sets may target the sequences at various taxonomic levels,from domain to division,subdivision,class,fam-ily,genus,species,and even strain.The PCR products contain a mixture of multiple copies of the same segment ampli?ed at the selected taxonomic level.The PCR products can be cloned and then sequenced to identify species.They can also be analyzed by various techniques,such as DGGE or T-RFLP,which may separate PCR products originating from different DNA sequences of various spe-cies in the sample.
2.1.Cloning and sequencing
Cloning of PCR products is to separate the PCR fragments of the same length but of different sequences.Cloning is composed of three steps:ligation,transformation and host cell reproduction. Several commercial kits are available for DNA cloning,such as the pGEM-T cloning kit and the TA cloning kit.
Taking the TA cloning kit as an example,PCR products are?rstly inserted into the plasmids under the action of the ligase.After liga-tion,the plasmids with PCR product inserts are transformed into Escherichia coli competent cells.Each cell carrying the plasmid with the PCR product insert forms a single white colony(called the clone)on the solid discriminative medium.Whereas,each cell car-rying the plasmid without the PCR product insert forms a blue one, and each cell carrying no plasmid does not form any colony.A DNA library consists of the selected white colonies.The insert in plas-mids of the white colonies may be recovered using PCR with a pri-mer set targeting the sequence on the plasmids which locate at the two sides of the insert or the original primer set which is used to generate PCR products initially.The whole plasmid could be ex-tracted after further cultivation of the colonies in liquid medium. The PCR product obtained using the?rst method,or the plasmids obtained using the second methods,will be sequenced for further identi?cation of the sequences.
Sequencing of the full16S rDNA(about1540bp)is preferred for microbial identi?cation,especially for oligonucleotide probe de-sign and classi?cation of the pure culture.Once a sequence data-base of a clone library is established,the microbial diversity can be determined with reference to the published sequences of the pure cultures and environmental samples.The similarity analysis of DNA sequences is greatly facilitated by a number of rDNA se-quence databases,such as GenBank(http://www.ncbi.nlm.nih.-gov/blast/)and the ribosomal database project(RDP,http:// https://www.doczj.com/doc/e610023587.html,/),and powerful software packages,including MEGA 2.1(Kumar et al.,1993)and ARB(Strunk and Ludwig, 1997).By using the Blast program in GenBank,species closely re-lated to the obtained sequences are listed in the similarity order (Altschul et al.,1990).A primary taxonomy position of the species represented by obtained sequences may also be given.More accu-rate taxonomy analysis of DNA sequences could be conducted by construction of phylogenetic trees.
The obtained sequence information in a clone library can be used to evaluate species diversity or richness of microbial commu-nities in bioreactors,and to design speci?c probes/primers for characterization/quanti?cation of a certain microorganism.But the cloning method is time-demanding and less applicable for analysis of a larger set of samples,such as monitoring the changes of a microbial community over time(Sanz and K?chling,2007).
2.2.Denaturing gradient gel electrophoresis(DGGE)
PCR ampli?cation produces DNA segments with the same size but different sequences.These segments may be separated in an acrylamide gel,but not in an agarose gel,having a linear ascending gradient of denaturants,usually urea and formamide(Muyzer et al.,1993).This so-called DGGE method is based on the differ-ences of the electrophoresis mobility of the partially denatured double-stranded DNA fragments in the polyacrylamide gel.
The universal primer for Bacteria domain341F-518R(Fang et al.,2006a)is usually used in hydrogen production studies.After staining,DNA fragments will appear as separated bands on the gel. Each DGGE band is derived from one speci?c species in the original samples.Thus the band number of a DGGE pro?le provides a quick
8446R.Y.Li et al./Bioresource Technology102(2011)8445–8456
estimate of species richness.The intensity of a band may be used as a rough indication of the relative abundance of a species.
The separated DNA fragments may be recovered from the DGGE bands in the gel and sequenced to identify the corresponding microbial https://www.doczj.com/doc/e610023587.html,bined with sequencing and similarity-based phylogenetic analysis,DGGE can give an overview of the composi-tion of a microbial community.However,it is not easy to slice the bands and purify the carried DNA fragments in some cases.For a complex community,to get DNA sequence of a cut band often de-mands another cloning-sequencing step due to co-migration or poor band separation.The separation could be optimized by chang-ing the electrophoresis conditions,such as the denaturing gradient range,running time,voltage,etc.
Comparing patterns across the gel,especially for those includ-ing a large number of bands,is a dif?cult job.Different combina-tions of samples should be carefully designed if numerous samples are being investigated and multiple gels are required. DNA fragments of different sequences may co-migrate,and minor populations in the samples may be overlooked due to the limited detection sensitivity(Vallaeys et al.,1997).Additionally,the short sequences of the excised bands(usually200–400bp)are less infor-mative for sequencing and comparing with database,and probe and primer designs,and make the resulting phylogenetic analysis less reliable than those using the full16S rDNA sequences from cloning,especially for those novel sequences having less than 85%similarity to known sequences(Hugenholtz et al.,1998).Thus, it might need combination of DGGE with another method such as 16S rDNA sequence analysis to identify the types of bacteria.Sin-gle-stranded DNA fragments generated in PCR may appear as bands and result in an overestimation of microbial diversity in a sample(Gilbride et al.,2006).Moreover,DGGE band intensity does not always quantitatively correlate with the abundance of a spe-ci?c species,as DNA copy number in PCR product depends on both the abundance of species and the ease of ampli?cation.It is also dif?cult to analyze the DGGE pro?le with too many bands,and only the predominant species could be https://www.doczj.com/doc/e610023587.html,stly,the repro-ducibility of DGGE is relative low,compared to other?ngerprint methods,such as T-RFLP.
However,DGGE method has been extensively applied to moni-toring microbial diversity,investigate the composition of a micro-bial community and estimate relative abundance of a species due to its following advantages:(1)rapid and simple monitoring of microbial communities variation based on band patterns,(2)quick overview of the predominant populations in a sample,and(3)cost effective for a large number of samples.
2.3.Terminal-restriction fragment length polymorphism(T-RFLP)
Terminal-restriction fragment length polymorphism(T-RFLP) analysis is a?ngerprinting technique based on restriction digestion of PCR products obtained using a?uorescently labeled primer.To apply this method,one primer?uorescently labeled at50end en-ables a single species to generate a speci?c?uorescent terminal-restriction fragments(T-RFs)of a given size after enzymatic restrictive digestion.The segments are then separated by non-denaturing polyacrylamide gel electrophoresis or capillary electrophoresis,and distinguished by laser-induced?uorescence detection.The detailed principles of T-RFLP have been extensively described by Liu et al.(1997).
With high reproducibility,T-RFLP could be applied to conduct both quantitative and qualitative analyses of a gene(such as16S rDNA)in a microbial community.Its advantage is the ability to de-tect even rare population of a sample.In addition,phylogenetic information can be inferred from the T-RF sizes of the sequences of known bacteria in the databases,including TRFMA,T-Align,PAT,and TAP.More important,T-RFLP can be standardized and be used to compare results between different researchers.
However,compared to DGGE,T-RFLP is more expensive and time-consuming,as PCR fragments need to be puri?ed prior to enzymatic restriction digestion.Another disadvantage of T-RFLP is that the experimental fragment size may not be exactly same as the theoretical length,a1–4bps difference is commonly ob-served.As a result,T-RFLP seems to be useful in characterizing microbial communities with low-to-intermediate diversity,but may not for samples with high diversity.Additionally,the T-RFLP reproducibility may be affected by incomplete restriction diges-tion.Formation of pseudo TRFs,was also reported(Egert and Fried-rich,2003),resulting in overestimation of biodiversity.More information about the limitation of T-RFLP could be found in the paper of Nocker et al.(2007).
2.4.Fluorescence in situ hybridization(FISH)
FISH is a visualization technique based on microscopic exami-nation of a given species or groups of bacteria after staining cells using speci?c?uorogenic oligonucleotide probes which bind RNA molecules in the cells.As a method without DNA extraction and PCR,FISH is an excellent means to overcome problems associated with PCR-based molecular methods,such as DGGE,T-RFLP,cloning and sequencing.
FISH probes are short DNA sequences(about20nucleotides)la-beled with one or two?uorescent dyes.The cells are typically examined by epi?uorescence microscope or laser scanning micro-scopes after staining.The speci?city of the probe enables detec-tion/identi?cation on any desired taxonomic level,from domain down to species,depending on the probe applied.
In addition to visualization,FISH can also be used for microbial quanti?cation in the hydrogen production reactor,basing on either the?uorescence-emitting area(Fang et al.,2005)or the number of individual cells(Fang et al.,2006c).However,the cell count meth-od is only applicable to evenly distributed and homogeneous microbial https://www.doczj.com/doc/e610023587.html,ually,the bacterial count in a microscopic view?eld is required to be in the range of30–150.Cell counts have to be performed at more ten regions in order to have statistical signi?cance.
Compared to PCR-based molecular techniques,FISH has the fol-lowing advantages:(1)fast and simple to have results in a couple of hours,(2)direct observation of uncultured microbes,(3)semi-quantitative,(4)preferential/differential examination of popula-tions,(5)structural analysis of aggregates(?ocs,granular sludge, bio?lms)when combined with a confocal laser scanning micros-copy,(6)easy for routine application,requiring only basic knowl-edge of microscope,and(7)possible to measure metabolic activity of cells by analyzing the intensity of?uorescence of posi-tive cells.
However,for the mixed culture in an anaerobic reactor,strong background auto?uorescence often affects the interpretation of the FISH images,subject to individual judgment and experience about the samples;the problem may partially resolved by using a standardized and automatic procedure.Other disadvantages of FISH mainly include:(1)RNA gene sequence must be known for a target microorganism if the special probe has not yet been re-ported yet;(2)not always possible to design an unambiguously restrictive probe for a microbial group,especially for those groups classi?ed based on metabolic criteria;(3)dif?cult to optimize hybridization conditions of a newly-designed probe;and(4)hard to quantify accurately due to subjective nature involved.
Overall,FISH is a useful method in combination with either?n-gerprint methods(DGGE or T-RFLP)or cloning-sequencing meth-ods to quantify and visualize the OTUs of interest.Details of the FISH technique have been reviewed by Wagner et al.(2003).
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2.5.Quantitative real-time polymerase chain reaction(qRT-PCR)
Hybridization-based techniques such as FISH,and PCR-based techniques such as DGGE and cloning-sequencing,have long been used to quantify microorganisms.Hybridization methods,which have detection limits in the order of105DNA/RNA copies or great-er,are in general less sensitive.They can thus only be used for environmental samples of relatively high microbial concentrations. PCR-based methods,on the other hand,are capable of detecting DNA/RNA at low concentrations.However,the precision of PCR-based methods may be compromised due to a number of factors, including reagent depletion,competition of amplicons with prim-ers,and the loss of polymerase activity as the number of ampli?ca-tion cycle increases(Zhang and Fang,2006).
Due to the advanced development of?uorogenic chemistry, qRT-PCR has become an emerging technique for the detection and quanti?cation of microorganisms in the environment. Compared to the conventional hybridization-and PCR-based tech-niques,qRT-PCR not only has better sensitivity and reproducibility, but it is also quicker to perform and has a minimum risk of amplicon carryover contamination.Details of qRT-PCR have been reviewed by Zhang and Fang(2006).
3.Applications of molecular techniques on heterotrophic hydrogen production research
Although heterotrophic hydrogen production has been studied for several decades,most of related studies were conducted using dark fermentation(Li and Fang,2007).Studies of photo fermenta-tion were conducted mostly for suspended pure cultures with a few exceptions for mixed cultures(Li and Fang,2009).The applica-tion of nucleic acid based techniques has thus focused on dark fer-mentation,with limited reports related to photo fermentation,as shown in Tables1–4.Some signi?cant?ndings in recent hydrogen production studies with the application of molecular techniques are discussed as follows.
3.1.Identi?cation of new isolates
Anaerobic bacteria that produce hydrogen during dark fermen-tation are classi?ed into either strict or facultative anaerobes.Clos-tridium,Ethanoligenens and Desulfovibrio are representatives of strict anaerobes,whereas Enterobacter,Citrobacter,Klebsiella, E.coli and Bacillus are facultative anaerobes(Lee et al.,2010). Among the dark fermentative bacteria,Clostridium and Enterobac-ter are most widely studied(Li and Fang,2007).Photo fermenta-tion is generally carried out by purple non-sulfur bacteria, including the fresh water species Rhodobacter sphaeroides,Rhodob-acter capsulatus,Rhodopseudomonas palustris and Rhodospirillum ru-brum,and the marine species Rhodovulum sp.,Rhodovulum sul?dophilum,and Rhodobacter marinus,with the fresh water spe-cies are most widely used(Li and Fang,2009).
Isolation of new hydrogen-producing bacteria and investigation of their hydrogen production characteristics provided important microbial species with high capacity or unique properties in hydro-gen production.Table1shows that16S rRNA gene sequence anal-ysis,i.e.sequencing of the full16S rDNA followed by phylogenetic analysis,is preferred for identi?cation of the new isolates.Some of the new isolates were?rst reported to have hydrogen production ability by fermentation of organic pollutants,such as Megasphaera elsdenii RG1(Ohnishi et al.,2010)and Rubrivivax gelatinosus L31(Li and Fang,2008).Some isolates demonstrated high hydrogen-pro-ducing capacity.Klebsiella pneumoniae ECU-15,which was isolated from anaerobic sewage sludge,showed highest hydrogen produc-tion rate of482ml/l/h compared with other reported Klebsiella species(Niu et al.,2010).Another isolate Enterobacter https://www.doczj.com/doc/e610023587.html,1 was very ef?cient in using xylose to produce hydrogen with a yield (2.0±0.05mol H2/mol xylose)higher than most of other species (Long et al.,2010).Also,some new isolated species displayed un-ique properties in hydrogen production,such as Pantoea agglomer-ans BH-18,which was a salt-tolerant anaerobe with a wide range of initial pH(pH5–10)in hydrogen production(Zhu et al.,2008),and Enterobacter https://www.doczj.com/doc/e610023587.html,1,by which hydrogen production from xylose was superior to glucose and sucrose(Long et al.,2010).
3.2.Characterization of microbial compositions
With the development of biohydrogen research,mixed cultures have been commonly used in dark fermentation,especially in re-cent studies,although photo fermentation is conducted mostly using pure cultures.Besides the hydrogen-producing bacteria (HPB)in a mixed culture system for dark fermentation,hydrogen consumers and metabolic competitors were also identi?ed(Guo et al.,2010).Hydrogen consumers mainly include sulfate-reducing bacteria,methane-producing bacteria,and homoacetogenic https://www.doczj.com/doc/e610023587.html,ctic acid bacteria were often found in dark fermentation sys-tem as a competitor(Guo et al.,2010).
In order to better understand the process,the microbial com-munity compositions were investigated and linked to the hydrogen production performance.Table2shows that the most commonly used method for characterization of microbial compositions in dark fermentation reactors is16S rRNA based DGGE combined with sequencing and similarity-based phylogenetic analysis.Clon-ing-sequencing and T-RFLP were also used in some reports.FISH was reported to be applied for detecting the presence of speci?c species,such as methanogens using Archaea probe(Castellóet al., 2009).
Table2also shows that most of the predominant HPB in dark fermentation system were Clostridium genus under mesophilic conditions and Thermoanaerobacterium genus under thermophilic conditions.The predominance of Clostridium species is likely due to the heat shock treatment on the inoculum(Guo et al.,2010). In the hydrogen production reactors without heat pre-treatment for seed sludge,quite different microbial compositions were re-ported.In a hydrogen fermentation system treating food waste by micro?ora from leaf-litter cattle-waste compost without heat pre-treatment,Clostridium was absent from this system.Instead, M.elsdenii was the dominant HPB(Ohnishi et al.,2010).Microbial analysis results in another study without pre-treatment for seed sludge,also showed the absence of Clostridium,whereas Anaero-truncus,Megasphaera,and Pectinatus were HPB in the system(Cas-tellóet al.,2009).
The16S rRNA gene has been widely used as a universal molec-ular biomarker to characterize microbial communities.However,it is dif?cult to reveal the practical phylogenetic diversity of the com-munity only based on16S rRNA gene sequences,such as the genus Clostridium,which is a large and phenotypically heterogeneous bacterial group and is yet not phylogenetically well de?ned (Quéméneur et al.,2010).Recently,functional genes,such as Fe-hydrogenase genes have been used as speci?c biomarker for char-acterization of Clostridium HPB in dark fermentation bioreactors using cloning-sequencing method(Fang et al.,2006b;Huang et al.,2010).In addition to Clostridium,other HPB such as Ethano-ligenens,Megasphaera,Syntrophomonas and Syntrophobacter spe-cies were also identi?ed and characterized using Fe-hydrogenase gene based cloning-sequencing method(Xing et al.,2008).
Characterization of microbial community compositions using molecular techniques,helped to explain hydrogen production per-formance.The low hydrogen yields were often related to the pres-ence of non-hydrogen producers that consumed hydrogen or competed for the substrates,such as methanogens(Castelló
R.Y.Li et al./Bioresource Technology102(2011)8445–84568449
et al.,2009),Bacillus racemilacticus (Kim et al.,2006b )and Strepto-coccus bovis (Kim et al.,2008),etc.
Reports of molecular techniques used for microbial composition analysis in photo fermentation were very limited.Zhang et al.(2002)found that the predominant population was R.capsulatus in the mixed phototrophic sludge using cloning and sequencing methods.
3.3.Monitoring microbial diversity variation
Table 3shows that DGGE and T-RFLP are effective methods and have been extensively applied to monitor microbial diversity in hydrogen production https://www.doczj.com/doc/e610023587.html,rmation on microbial diversity variation gives deep insight into the function of microorganisms in hydrogen fermentation process and is very helpful to optimize operational conditions.
Microbial diversity varied with many operational conditions,such as HRT,temperature and pH,etc.Different results were re-ported for various microbial communities.Zhang et al.(2006)found that shortening of HRT from 50to 6h reduced the microbial diversity associated with an elimination of propionate production without affecting the existence of dominant species,and increase the hydrogen yield of the glucose-feeding bioreactor.In contrast,variation of HRT had little effect on the microbial diversity for the anaerobic sludge producing hydrogen from sucrose in an up-?ow reactor (Li et al.,2006)and an extreme thermophilic micro-?ora producing hydrogen from glucose at 75°C (Yokoyama et al.,2009).Generally,microbial diversity decreased with the increase of both sludge pre-treatment temperature (Baghchehsaraee et al.,2008)and operation temperature (Karadag and Puhakka,2010),but increased with pH (Fang and Liu,2002;Fang et al.,2006a ).Microbial diversity also varied with seed sludge (Akutsu et al.,2008),organic loading rates (Hafez et al.,2010)and sodium con-centration (Kim et al.,2009)etc.During the operation of hydrogen production system,the diversity may also change without any con-dition variation because the operation of reactor itself is a micro-bial cultivation and selection process.Li et al.(2008)reported that Rhodobacter sp.was the predominant species throughout but R.gelatinosus and R.sphaeroides vanished gradually in the mixed phototrophic sludge during the operation of continuous photo fer-mentation reactor.
3.4.Visualization of microbial distribution in hydrogen-producing granular sludge
FISH combined with ?uorescence microscopy has been applied to investigate the spatial distribution of microorganisms in the hydrogen-producing granular sludge.Since most of the studies on heterotrophic hydrogen production were conducted by sus-pended cultures,reports on microbial analysis for granular sludge were still very limited.
In a sucrose-feeding dark fermentation bioreactor,self-forming granular sludge was formed at HRT of 0.5h,and Clostridium sp.and Streptococcus sp.were predominant microorganisms.FISH images showed that Clostridium sp.formed net-like structures where Streptococcus sp.aggregated to form a solid granular core inside the net,making the net structure more rigid (Hung et al.,2011).Similar spatial distribution of microorganisms was also observed in the glucose-feeding bioreactor using the same seed sludge (Hung et al.,2007).
3.5.Quanti?cation of various microbial populations
As summarized in Table 4,cloning-sequencing,qRT-PCR,FISH,and DGGE can be applied for quanti?cation of various microbial
T a b l e 2(c o n t i n u e d )
F e r m e n t a t i o n p r o c e s s
S e e d s l u d g e
R e a c t o r t y p e
C u l t u r e v o l u m e (l )
S u b s t r a t e s M a x .H 2
r a t e (l /l /h )
Y i e l d C e l l d e n s i t y (g /l )
M o l e c u l a r t e c h n i q u e s u s e d M i c r o b i a l c o m p o s i t i o n s
R e f e r e n c e
C a t t l e d u n g c o m p o s t
B a t c h
7.0S u c r o s e
0.583.11m o l H 2/m o l s u c r o s e
–
C l o n i n g s e q u e n c i n g
13F e -h y d r o g e n a s e g e n e t y p e s w e r e i d e n t i ?e d f r o m c l o n e l i b r a r i e s .C .p e r f r i n g e n s w a s t h e m o s t d o m i n a n t H 2
p r o d u c e r H u a n g e t a l .(2010)
A c i d o p h i l i c s l u d g e
B a t c h
0.15
R i c e s l u r r y 0.04–
–
C l o n i n g s e q u e n c i n g O T U s b a s e d o n F e -h y d r o g e n a s e g e n e f r a g m e n t s w e r e m o r e c l o s e l y r e l a t e d t o C .p a s t e u r i a n u m ,C .p a r a p u t r i ?c u m ,C .p e r f r i n g e n s ,a n d C .s a c c h a r o b u t y l i c u m F a n g e t a l .(2006b )
P h o t o f e r m e n t a t i o n M i x e d p h o t o t r o p h i c s l u d g e
C S T R 0.45A c e t a t e +b u t y r a t e +e t h a n o l 0.017412.0%
3.1
C l o n i n g s e q u e n c i n g P r e d o m i n a n t p o p u l a t i o n w a s R h o d o b a c t e r c a p s u l a t u s ,t w o o t h e r s p e c i e s i n t h e B a c i l l u s /C l o s t r i d i u m g r o u p w e r e a l s o f o u n d i n t h e m i x e d s l u d g e
Z h a n g e t a l .(2002)
a
T h e h y d r o g e n y i e l d i n t h i s s t u d y w a s b a s e d o n c a r b o h y d r a t e a d d e d ,w h i l e o t h e r s b a s e d o n c a r b o h y d r a t e c o n s u m e d .b
C S T R –C o n t i n u o u s S t i r r e d T a n k R e a c t o r ;U A S B –U p ?o w A n a e r o b i c S l u d g e B l a n k e t ;S B R –S e q u e n c i n g B a t c h R e a c t o r .
R.Y.Li et al./Bioresource Technology 102(2011)8445–84568451
populations in hydrogen-producing sludge,among which cloning-sequencing has been most extensively used so far.
Based on cloning-sequencing method,relative abundance (%)of various microbial population were investigated and the bacteria species responsible for hydrogen production in the bioreactors could be estimated,such as Thermoanaerobacterium thermosacchar-olyticum (over 80%)in a starch-feeding thermophilic bioreactor (Akutsu et al.,2008)and thermophilic reactors of us (Liu et al.,2003;Zhang et al.,2003),Clostridium sp.(85%)in a sucrose-degrad-ing granular sludge (Hung et al.,2010),and R.capsulatus (81%)in a photo fermentation bioreactor fed with acetate,butyrate and eth-anol as mixed substrates (Fang et al.,2004;Zhang et al.,2002).Cloning-sequencing analysis also revealed the relative abundance of various HPB in a mixed culture system.Chu et al.(2010)?rst re-ported that Clostridium sp.strain Z6(72%)was dominant thermo-philic hydrogen-producing bacteria from food waste,whereas T.thermosaccharolyticum only accounted for 12%in the mixed culture.
More recently,qRT-PCR has emerged in hydrogen production studies as an effective method for quanti?cation of microorgan-isms.A new group of dark fermentative bacteria was monitored quantitatively by qRT-PCR using a designed TaqMan gene probe (Li et al.,2007).The abundance of this HPB in the biomass was found to increase from 0.02%to 72%in a batch reactor treating rice slurry waste at pH 4.5over 130h operation.The corresponding abundances were 4.4%at pH 5.0and 0.01–0.02%at pH 5.5–6.5(Li et al.,2007).A set of primers speci?c for Fe-hydrogenase genes of Clostridium species was identi?ed and used to monitor the change of HPB in the batch reactor treating rice slurry using qRT-PCR.The quantitative analysis results showed the HPB had an aver-age generation time of 4.2h (Fang et al.,2006b ).In a continuous photo fermentation bioreactor using acetate and butyrate as sub-strates,three phototrophic HPB were identi?ed in the seed sludge.The relative abundance of these bacteria were examined by clon-ing-sequencing method,with the results that Rhodobacter sp.,R.gelatinosus and R.sphaeroides accounted for 42.3%,38.5%and 7.7%respectively.Based on qRT-PCR analysis,it was found that hydrogen production rate generally increased with the amount of Rhodobacter sp.in the reactor,but had no clear correlations with the other two hydrogen-producing bacteria.This ?nding helped to con?rm that Rhodobacter sp.was the species most likely respon-sible for hydrogen production in the photo bioreactor (Li et al.,2008).
In addition to cloning-sequencing and qRT-PCR,microbial quan-ti?cation in the hydrogen production reactor could also been ana-lyzed by DGGE band intensity (Fang et al.,2005),and FISH basing on either the ?uorescence-emitting area (Fang et al.,2005;Zhang et al.,2002)or the number of individual cells (Chu et al.,2009;Fang et al.,2006c ).
4.Conclusions
Unlike methanogenic fermentation,which has been commer-cialized for wastewater treatment for two decades with thousands of full-scale installation worldwide,fermentation of wastewater for hydrogen production remains at the infantile stage.Molecular techniques especially 16S rDNA-based methods have contributed much for the development of heterotrophic hydrogen production researches.More recently,Fe-hydrogenase genes has also been more and more used as biomarker to characterize and quantify HPB especially Clostridium species.It can be anticipated that more advanced and further developed molecular techniques may be incorporated into the research on hydrogen fermentation,contrib-uting to the improvement of hydrogen production performance of bioreactors.
T a b l e 3(c o n t i n u e d )
F e r m e n t a t i o n p r o c e s s
S e e d s l u d g e
R e a c t o r t y p e
C u l t u r e v o l u m e (l )
S u b s t r a t e s M a x .H 2r a t e (l /l /h )
Y i e l d
C e l l d e n s i t y (g /l )
M o l e c u l a r t e c h n i q u e s u s e d
M i c r o b i a l a n a l y s i s r e s u l t s
R e f e r e n c e
S l u d g e f r o m C S T R H 2
f e r m e n t o r
B a t c h
0.2S t a r c h
0.009592m l H 2/g s t a r c h
0.625
D G G
E ,
F I S H
E u b a c t e r i a w a s a b u n d a n t i n b o t h t h e m e s o p h i l i c s e e d s l u d g e a n d t h e t h e r m o p h i l i c s t a r c h -d e g r a d i n g s l u d g e .C .h i s t o l y t i c u m g r o u p w a s a b u n d a n t i n t h e m e s o p h i l i c s l u d g e ,b u t n e a r l y a b s e n t i n t h e r m o p h i l i c s l u d g e Z h a n g e t a l .(2003)
A n a e r o b i c d i g e s t e r s l u d g e
C S T R +s e t t l e r
5.0G l u c o s e 0.10–1.48
1.1–
2.9m o l H 2/m o l g l u c o s e
1.5–17.0
P C R -D G G E
T h e m i c r o b i a l d i v e r s i t y i n c r e a s e d w h e n t h e o r g a n i c l o a d i n g r a t e s (O L R )i n c r e a s e d f r o m 6.5t o 25.7g C O D /L /d .A t e x t r e m e l y h i g h O L R o f 154a n d 206g C O D /L /d ,c l e a r m i c r o b i a l s h i f t s w e r e i d e n t i ?e d H a f e z e t a l .(2010)
S l u d g e f r o m C S T R H 2
f e r m e n t o r C S T R
1.7G l u c o s e
–2.1±0.1m o l H 2/m o l g l u c o s e –
P C R -D G G E
T h e d i v e r s i t y o f m i c r o b i a l c o m m u n i t i e s i n c r e a s e d w i t h p H
F a n g a n d L i u (2002)
P h o t o f e r m e n t a t i o n
M i x e d p h o t o t r o p h i c s l u d g e
C S T R
1.0A c e t a t e +b u t y r a t e 0.008±0.0019–0.7±0.1
P C R -D G G E ;
R h o d o b a c t e r s p .w a s t h e p r e d o m i n a n t s p e c i e s t h r o u g h o u t ,R .g e l a t i n o s u s a n d R h o d o b a c t e r s p h a e r o i d e s v a n i s h e d g r a d u a l l y
L i e t a l .(2008)
a
T h e h y d r o g e n y i e l d i n t h i s s t u d y w a s b a s e d o n c a r b o h y d r a t e a d d e d ,w h i l e o t h e r s b a s e d o n c a r b o h y d r a t e c o n s u m e d .b
C S T R –C o n t i n u o u s S t i r r e d T a n k R e a c t o r ;U A S B –U p ?o w A n a e r o b i c S l u d g e B l a n k e t ;A G S B –A g i t a t e d G r a n u l a r S l u d g e B e d .
R.Y.Li et al./Bioresource Technology 102(2011)8445–84568453
Acknowledgements
The authors wish to thank the Hong Kong General Research Fund (7125/09E)and HKU ICEE funding for the ?nancial support of this study.References
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T a b l e 4(c o n t i n u e d )
F e r m e n t a t i o n p r o c e s s
S e e d s l u d g e
R e a c t o r t y p e
C u l t u r e v o l u m e (l )
S u b s t r a t e s
M a x .H 2r a t e (l /l /h )
Y i e l d
C e l l d e n s i t y (g /l )
M o l e c u l a r t e c h n i q u e s u s e d M i c r o b i a l a n a l y s i s r e s u l t s
R e f e r e n c e
M i x e d p h o t o t r o p h i c s l u d g e
B a t c h
0.1
A c e t a t e ;b u t y r a t e
0.0053–0.0067
37.0–62.5%
0.4
P C R -D G G E ;F I S H
P r e d o m i n a n t s p e c i e s r e s e m b l i n g R .c a p s u l a t u s w i t h o v e r 80%r e l a t i v e a b u n d a n c e
F a n g e t a l .(2005)C o -c u l t u r e o f C .b u t y r i c u m a n d R .s p h a e r o i d e s B a t c h
0.2
G l u c o s e +a c e t a t e –
–
–
F I S H
C e l l n u m b e r s o f R .s p h a e r o i d e s a n d C .b u t y r i c u m i n t h e c o -c u l t u r e r e a c h e d t h e m a x i m u m a t 18a n d 40h r e s p e c t i v e l y ,a n d t h e n d e c r e a s e d F a n g e t a l .(2006c )M i x e d p h o t o t r o p h i c s l u d g e C S T R
1.0A c e t a t e +b u t y r a t e 0.008±0.0019
–0.7±0.1C l o n i n g s e q u e n c i n g R h o d o b a c t e r s p .(42.3%);R .g e l a t i n o s u s (38.5%);R .s p h a e r o i d e s (7.7%);P s e u d o m o n a s s p .(7.7%)L i e t a l .(2008)M i x e d p h o t o t r o p h i c s l u d g e
C S T R 1.0
A c e t a t e +b u t y r a t e
0.008±0.0019–0.7±0.1
q R T -P C R
H y d r o g e n p r o d u c t i o n r a t e g e n e r a l l y i n c r e a s e d w i t h t h e a m o u n t o f R h o d o b a c t e r s p .i n t h e r e a c t o r
L i e t a l .(2008)
a
C S T R –C o n t i n u o u s S t i r r e d T a n k R e a c t o r ;A G S B –A g i t a t e d G r a n u l a r S l u d g e B e d .
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