Genetic Analysis of Type E Botulinum Toxin-Producing Clostridium butyricum Strains

Type E botulinum toxin (BoNT/E)-producing Clostridium butyricum strains isolated from botulism cases or soil specimens in Italy and China were analyzed by using nucleotide sequencing of the bont/E gene, random amplified polymorphic DNA (RAPD) assay, pulsed-field gel electrophoresis (PFGE), and Southern blot hybridization for the bont/E gene. Nucleotide sequences of the bont/E genes of 11 Chinese isolates and of the Italian strain BL 6340 were determined. The nucleotide sequences of the bont/E genes of 11 C. butyricum isolates from China were identical. The deduced amino acid sequence of BoNT/E from the Chinese isolates showed 95.0 and 96.9% identity with those of BoNT/E from C. butyricum BL 6340 and Clostridium botulinum type E, respectively. The BoNT/E-producing C. butyricum strains were divided into the following three clusters based on the results of RAPD assay, PFGE profiles of genomic DNA digested with SmaI or XhoI, and Southern blot hybridization: strains associated with infant botulism in Italy, strains associated with food-borne botulism in China, and isolates from soil specimens of the Weishan lake area in China. A DNA probe for the bont/E gene hybridized with the nondigested chromosomal DNA of all toxigenic strains tested, indicating chromosomal localization of the bont/E gene in C. butyricum. The present results suggest that BoNT/E-producing C. butyricum is clonally distributed over a vast area.     

Identification of Novel Linear Megaplasmids Carrying a ?-Lactamase Gene in Neurotoxigenic Clostridium butyricum Type E Strains

Since the first isolation of type E botulinum toxin-producing Clostridium butyricum from two infant botulism cases in Italy in 1984, this peculiar microorganism has been implicated in different forms of botulism worldwide. By applying particular pulsed-field gel electrophoresis run conditions, we were able to show for the first time that ten neurotoxigenic C. butyricum type E strains originated from Italy and China have linear megaplasmids in their genomes. At least four different megaplasmid sizes were identified among the ten neurotoxigenic C. butyricum type E strains. Each isolate displayed a single sized megaplasmid that was shown to possess a linear structure by ATP-dependent exonuclease digestion. Some of the neurotoxigenic C. butyricum type E strains possessed additional smaller circular plasmids. In order to investigate the genetic content of the newly identified megaplasmids, selected gene probes were designed and used in Southern hybridization experiments. Our results revealed that the type E botulinum neurotoxin gene was chromosome-located in all neurotoxigenic C. butyricum type E strains. Similar results were obtained with the 16S rRNA, the tetracycline tet(P) and the lincomycin resistance protein lmrB gene probes. A specific mobA gene probe only hybridized to the smaller plasmids of the Italian C. butyricum type E strains. Of note, a ß-lactamase gene probe hybridized to the megaplasmids of eight neurotoxigenic C. butyricum type E strains, of which seven from clinical sources and the remaining one from a food implicated in foodborne botulism, whereas this ß-lactam antibiotic resistance gene was absent form the megaplasmids of the two soil strains examined. The widespread occurrence among C. butyricum type E strains associated to human disease of linear megaplasmids harboring an antibiotic resistance gene strongly suggests that the megaplasmids could have played an important role in the emergence of C. butyricum type E as a human pathogen.     

Genetic Diversity of the Flagellin Genes of Clostridium botulinum Groups I and II

Botulinum neurotoxins (BoNTs) are produced by phenotypically and genetically different Clostridium species, including Clostridium botulinum and some strains of Clostridium baratii (serotype F) and Clostridium butyricum (serotype E). BoNT-producing clostridia responsible for human botulism encompass strains of group I (secreting proteases, producing toxin serotype A, B, or F, and growing optimally at 37°C) and group II (nonproteolytic, producing toxin serotype E, B, or F, and growing optimally at 30°C). Here we report the development of real-time PCR assays for genotyping C. botulinum strains of groups I and II based on flaVR (variable region sequence of flaA) sequences and the flaB gene. Real-time PCR typing of regions flaVR1 to flaVR10 and flaB was optimized and validated with 62 historical and Canadian C. botulinum strains that had been previously typed. Analysis of 210 isolates of European origin allowed the identification of four new C. botulinum flaVR types (flaVR11 to flaVR14) and one new flaVR type specific to C. butyricum type E (flaVR15). The genetic diversity of the flaVR among C. botulinum strains investigated in the present study reveals the clustering of flaVR types into 5 major subgroups. Subgroups 1, 3, and 4 contain proteolytic Clostridium botulinum, subgroup 2 is made up of nonproteolytic C. botulinum only, and subgroup 5 is specific to C. butyricum type E. The genetic variability of the flagellin genes carried by C. botulinum and the possible association of flaVR types with certain geographical areas make gene profiling of flaVR and flaB promising in molecular surveillance and epidemiology of C. botulinum.     

Genetic diversity and amplification of different clostridial [FeFe] hydrogenases by group-specific degenerate primers

Aims: The aim of this study was to explore and characterize the genetic diversity of [FeFe] hydrogenases in a representative set of strains from Clostridium sp. and to reveal the existence of neither yet detected nor characterized [FeFe] hydrogenases in hydrogen‐producing strains. Methods and Results: The genomes of 57 Clostridium strains (34 different genotypic species), representing six phylogenetic clusters based on their 16S rRNA sequence analysis (cluster I, III, XIa, XIb, XIV and XVIII), were screened for different [FeFe] hydrogenases. Based on the obtained alignments, ten pairs of [FeFe] hydrogenase cluster‐specific degenerate primers were newly designed. Ten Clostridium strains were screened by PCRs to assess the specificity of the primers designed and to examine the genetic diversity of [FeFe] hydrogenases. Using this approach, a diversity of hydrogenase genes was discovered in several species previously shown to produce hydrogen in bioreactors: Clostridium sartagoforme, Clostridium felsineum, Clostridium roseum and Clostridium pasteurianum. Conclusions: The newly designed [FeFe] hydrogenase cluster‐specific primers, targeting the cluster‐conserved regions, allow for a direct amplification of a specific hydrogenase gene from the species of interest. Significance and Impact of the Study: Using this strategy for a screening of different Clostridium ssp. will provide new insights into the diversity of hydrogenase genes and should be a first step to study a complex hydrogen metabolism of this genus.     

梭菌属分类研究进展:现状和展望

目前构成梭菌属的微生物在系统发育和表型特征上不一致。多相分类数据表明,梭菌属物种之间差异大。大量基于16S rRNA 基因的系统发育研究表明,梭菌属应被限定为梭菌属类群I,作为狭义梭菌属(Clostridium sensu stricto)。尽管有这方面认识,梭菌属新物种仍持续增加,这些新物种并不能与梭菌属类群I 和标准种丁酸梭菌(C. butryicum)形成一致分支,引发梭菌属分类上的混乱。本文明确了梭菌属物种的范围,即只包括模式种和梭菌属类群I。此外,4 个物种念珠状真杆菌(Eubacterium moniliforme)、旋舞真杆菌(Eubacterium tarantellae)、最大八叠球菌(Sarcina maxima)和胃八叠球菌(Sarcina ventriculi)应被调至梭菌属,分别命名为念珠状梭菌(Clostridium moniliforme)、旋舞梭菌(Clostridium tarantellae)、最大梭菌(Clostridium maximum)和胃梭菌(Clostridium ventriculi)。一个新属哈撒韦氏菌属(Hathewaya)被提议成立,3 个梭菌属物种溶组织梭菌(Clostridium histolyticum)、泥渣梭菌(Clostridium limosum)和解朊梭菌(Clostridiumproteolyticum)重新归为溶组织哈撒韦氏菌(Hathewaya histolytica)、泥渣哈撒韦氏菌(Hathewaya limosa)和解朊哈撒韦氏菌(Hathewaya proteolytica),其中Hathewaya histolytica 为模式种。     

Gene arrangement in the upstream region of Clostridium botulinum type E and Clostridium butyricum BL6340 progenitor toxin genes is different from that of other types

The cluster of genes encoding the botulinum progenitor toxin and the upstream region including p21 and p47 were divided into three different gene arrangements (class I–III). To determine the gene similarity of the type E neurotoxin (BoNT/E) complex to other types, the gene organization in the upstream region of the nontoxic-nonhemagglutinin gene (ntnh) was investigated in chromosomal DNA from Clostridium botulinum type E strain Iwanai and C. butyricum strain BL6340. The gene cluster of type E progenitor toxin (Iwanai and BL6340) was similar to those of type F and type A (from infant botulism in Japan), but not to those of types A, B, and C. Though genes for the hemagglutinin component and P21 were not discovered, genes encoding P47, NTNH, and BoNT were found in type E strain Iwanai and C. butyricum strain BL6340. However, the genes of ORF-X₁ (435 bp) and ORF-X₂ (partially sequenced) were present just upstream of that of P47. The orientation of these genes was in inverted direction to that of p47. The gene cluster of type E progenitor toxin (Iwanai and BL6340) is, therefore, a specific arrangement (class IV) among the genes encoding components of the BoNT complex.     

Diversity of spore‐forming bacteria in cattle manure,slaughterhouse waste and samples from biogas plants

Aims: As biowaste intended for biogas production can contain pathogenic micro‐organisms, the recommended treatment is pasteurization at 70°C for 60 min. This reduces pathogens such as Salmonella spp., whereas spore‐forming bacteria (Bacillus spp. and Clostridium spp.) survive. Most spore‐forming bacteria are harmless, but some can cause diseases such as blackleg, botulism and anthrax. In this study, the effect of the biogas process on Bacillus spp. and Clostridium spp. was investigated. Methods and Results: We analysed 97 faecal samples, 20 slaughterhouse waste samples and 60 samples collected at different stages in the biogas process. Bacillus spp. and Clostridium spp. were quantified and subcultured. The isolates were identified by biochemical methods and by 16S rRNA gene sequencing. Phylogenetic trees were constructed from the sequences obtained from isolates from the samples. Clostridium botulinum/Clostridium spp. and Clostridium sordellii were found both before and after pasteurization, but not after digestion (AD). Some of the isolated strains probably represented new members of the genera Clostridium and Bacillus. Conclusion: After digestion, the numbers of clostridia decreased, but none of the pathogenic bacteria did, whereas Bacillus spp. remained constant during the process. Significance and Impact of the Study: Biogas is gaining in importance as an energy source and because the residues are used as fertilizers, we needed to study the prevalence of pathogenic bacteria in such material.     

Development of a real time PCR Taqman assay based on the TPI gene for simultaneous identification of Clostridium chauvoei and Clostridium septicum

In the present study, a Taqman allelic discrimination assay based on three SNPs of the TPI gene is described. It was used as a differential diagnostic tool to detect blackleg and malignant edema. Sudden deaths of grazing ruminants, such as cattle, sheep and goats, which show clinical signs related to hyperacute infective processes, encouraged the development of a rapid and precise diagnostic molecular method. Specific primers and probes for Clostridium septicum and Clostridium chauvoei were designed on the basis of the TPI gene sequence. The multiplex PCR was tested on the DNA of a total of 57 strains, including 24 Clostridium chauvoei, 20 Clostridium septicum, 1 Bacillus anthracis and 12 other Clostridium spp. The DNA samples from Clostridium chauvoei and Clostridium septicum strains were amplified. Amplification of other DNA samples was not observed, with the exception of Clostridium tertium, which showed a weak positive signal. To avoid misdiagnosis, a confirmatory assay based on a Sybr green real time PCR was proposed. The authors confirmed the efficacy and the specificity of the test used in this study, which proved to be a useful tool for the diagnosis of clostridiosis that are often diagnosed using only traditional tools.     

泸型酒窖泥中梭菌的分离及代谢产物分析

[目的] 分离窖泥中的梭菌微生物并对其代谢产物进行评估。[方法] 对窖泥中梭菌群落的16S rRNA基因进行高通量测序;利用高丰度的梭菌OTU序列在KOMODO数据库进行培养基的预测,定向分离窖泥中梭菌菌株;采用顶空固相微萃取结合气相色谱质谱联用仪对窖泥和代表性梭菌菌株的挥发性代谢产物进行检测。[结果] 利用KOMODO数据库预测的梭菌培养基共计筛选到31株梭菌微生物,分属于梭菌属的14个种;根据风味代谢特性,这些菌株主要分为两大类,一是C.carboxidivorans、C.sporogenes和C.tyrobutyricum等产酸为主的梭菌,二是C.beijerinckii、C.butyricum和C.sphenoides等产醇为主的梭菌。[结论] 利用测序序列预测培养基有助于从窖泥中分离获得丰富的梭菌菌株,其物种和代谢能力的多样性对解析白酒复杂风味形成机理奠定了一定的基础。     

Arrangement of the Clostridium baratii F7 Toxin Gene Cluster with Identification of a σ Factor That Recognizes the Botulinum Toxin Gene Cluster Promoters

Botulinum neurotoxin (BoNT) is the most poisonous substances known and its eight toxin types (A to H) are distinguished by the inability of polyclonal antibodies that neutralize one toxin type to neutralize any of the other seven toxin types. Infant botulism, an intestinal toxemia orphan disease, is the most common form of human botulism in the United States. It results from swallowed spores of Clostridium botulinum (or rarely, neurotoxigenic Clostridium butyricum or Clostridium baratii) that germinate and temporarily colonize the lumen of the large intestine, where, as vegetative cells, they produce botulinum toxin. Botulinum neurotoxin is encoded by the bont gene that is part of a toxin gene cluster that includes several accessory genes. We sequenced for the first time the complete botulinum neurotoxin gene cluster of nonproteolytic C. baratii type F7. Like the type E and the nonproteolytic type F6 botulinum toxin gene clusters, the C. baratii type F7 had an orfX toxin gene cluster that lacked the regulatory botR gene which is found in proteolytic C. botulinum strains and codes for an alternative σ factor. In the absence of botR, we identified a putative alternative regulatory gene located upstream of the C. baratii type F7 toxin gene cluster. This putative regulatory gene codes for a predicted σ factor that contains DNA-binding-domain homologues to the DNA-binding domains both of BotR and of other members of the TcdR-related group 5 of the σ⁷⁰ family that are involved in the regulation of toxin gene expression in clostridia. We showed that this TcdR-related protein in association with RNA polymerase core enzyme specifically binds to the C. baratii type F7 botulinum toxin gene cluster promoters. This TcdR-related protein may therefore be involved in regulating the expression of the genes of the botulinum toxin gene cluster in neurotoxigenic C. baratii.     

Clostridiolysin S,a Post-translationally Modified Biotoxin from Clostridium botulinum

Through elaboration of its botulinum toxins, Clostridium botulinum produces clinical syndromes of infant botulism, wound botulism, and other invasive infections. Using comparative genomic analysis, an orphan nine-gene cluster was identified in C. botulinum and the related foodborne pathogen Clostridium sporogenes that resembled the biosynthetic machinery for streptolysin S, a key virulence factor from group A Streptococcus responsible for its hallmark β-hemolytic phenotype. Genetic complementation, in vitro reconstitution, mass spectral analysis, and plasmid intergrational mutagenesis demonstrate that the streptolysin S-like gene cluster from Clostridium sp. is responsible for the biogenesis of a novel post-translationally modified hemolytic toxin, clostridiolysin S.     

24株鸡源丁酸梭菌的分离鉴定及耐药基因与毒力基因携带情况

[目的]旨在对鸡源丁酸梭菌进行分离鉴定与安全性评估.[方法]利用厌氧培养方法对源自汶上芦花鸡与SPF鸡粪便样品进行丁酸梭菌的分离与纯化,挑选可疑菌落进行微生物质谱鉴定,进一步通过16S rRNA基因测序进行鉴定,16S rRNA测序结果与NCBI核苷酸数据库中丁酸梭菌的16S rRNA序列进行同源性分析;同时,进行所有...     

Characterization of Neurotoxigenic Clostridium butyricum Strain by DNA Hybridization Test and by in vivo and in vitro Germination Tests of Spores

The germination of spores of a neurotoxigenic Clostridium butyricum strain (BL 6340), which was isolated from infant botulism in Italy, and that of a non-toxigenic C. butyricum type strain (NCIB 7423) were studied. The spores of BL 6340 strain were killed at 80 C for 10 min, and required the mixture of L-alanine, L-lactate, glucose and bicarbonate for their optimal germination. These characteristics are the same as those of Clostridium botulinum type E strain, but different from those of NCIB 7423 strain. In a hybridization test, however, the labeled DNAs extracted from NCIB 7423 strain highly (98%) hybridized to the DNAs of the BL 6340 strain, but little (45%) to the DNAs of C. botulinum type E strain. The biochemical properties of the BL 6340 and NCIB 7423 strains were identical, but different from those of C. botulinum type E. These data confirmed that the BL 6340 strain belongs to C. butyricum species, but that only its characteristics of toxin production, its minimum requirements for germination, and the behavior of its spores to heat treatment are the same as those of C. botulinum type E. When conventionally raised suckling mice were injected with 5 × 10⁷ spores of BL 6340 strain intra- or orogastrically, botulism was not observed. However, 8- to 13-day-old mice had type E botulinum toxin in the large intestine 3 days after introduction of its spores.     

Comparison of polysaccharide glycoconjugates as candidate vaccines to combat Clostridiodes (Clostridium) difficile

Two known Clostridiodes (Clostridium) difficile surface antigens, a lipoteichoic acid (LTA) and a polysaccharide (PS-II) were isolated and purified in order to prepare glycoconjugate vaccines to the carrier protein human serum albumin utilising a reductive amination strategy. Mice and rabbits were immunized with a prime and two boost strategy and the resulting sera were examined for their ability to recognise the purified homologous antigens and subsequently killed whole cells of C. difficile strains and other Clostridia species. Immunisation derived antisera from rabbits and mice, recognised all strains of C. difficile vegetative cells examined, with generally similar titers from animals that received the LTA or the PS-II conjugates. Sera raised to the LTA conjugates were able to recognise other Clostridia species C. butyricum, C. bifermentans and C. subterminale whereas sera raised to the PS-II conjugates were not. These LTA and PS-II sera recognised live cells in an immunofluorescence assay and were also able to recognise the spore form of the bacterium. This study has confirmed that the LTA and PS-II polysaccharides are both highly conserved surface polymers of C. difficile that are easily accessible to the immune system and as such may have potential as vaccine antigens or as targets for therapeutics to combat C. difficile infection.

     

Simple Method for Detection of Clostridium botulinum Type A to F Neurotoxin Genes by Ploymerase Chain Reaction

A polymerase chain reaction (PCR)-based method was established to detect each type of neurotoxin genes of Clostridium botulinum types A to F by employing the oligonucleotide primer sets corresponding to special regions of the light chains of the neurotoxins. In this procedure, the PCR products were easily confirmed by restriction enzyme digestion profiles, and as little as 2.5 pg of template DNAs from toxigenic strains could be detected. The specific PCR products were obtained from toxigenic C. botulinum types A to F, a type E toxin-producing C. butyricum strain, and a type F toxin-producing C. baratii strain, but no PCR product was detected in nontoxigenic strains of C. botulinum and other clostridial species. The neurotoxin genes were also detected in food products of a seasoned dry salmon and a fermented fish (Izushi) which had caused type E outbreaks of botulism. Therefore, it is concluded that this PCR-based detection method can be used for the rapid diagnosis of botulism.     

Utilization of (E)-2-butenoate (Crotonate) by Clostridium kluyveri and some other Clostridium species

Clostridium La 1 obtained from a Clostridium kluyveri culture was compared with a typical C. kluyvery strain (DSM 555). The former grows on crotonate and is unable to use ethanol-acetate as carbon sources. The latter grows on crotonate only after long adaptation periods. Resting cells of both strains show also pronounced differences in the fermentation of crotonate. This holds even for C. kluyveri grown on crotonate. Besides several other differences the most striking is that there is no hybridization between the DNA of both strains.Crotonate seems not to be a very special carbon source since C. butyricum and C. pasteurianum grow on crotonate medium supplemented by peptone and yeast extract.Non Standard Abbreviations EA-medium ethanol and acetate as carbon source - C-medium crotonate as carbon source - DSM Deutsche Sammlung von Mikroorganismen     

New solvent-producing Clostridium sp. strains, hydrolyzing a wide range of polysaccharides, are closely related to Clostridium butyricum

Thirteen new Clostridium strains, previously isolated from soil and found to produce high amounts of solvents from glucose, hydrolyzed a great variety of α- and β-glycans, including raw starch, xylan, pectin, inulin and cellulose. The sequences of the PCR-amplified DNA fragments containing the variable 3′ part of one of the 16S rRNA genes were 99.5% identical. The macrorestriction pattern of two endonucleolytic digests of chromosomal DNA in the pulsed-field gel electrophoresis (PFGE) confirmed their high homogeneity on the DNA level. The complete 16S rRNA gene sequence of three selected strains was 99.8% identical to the 16S rRNA gene sequence from Clostridium butyricum and separates them from C. acetobutylicum. To the closely related four species of solventogenic clostridia a new group of strains has to be added, which has a great potential for the direct fermentation of biomass. Journal of Industrial Microbiology & Biotechnology (2001) 27, 329–335. Received 12 September 2000/ Accepted in revised form 25 July 2001     

Lipid diversity in clostridia

Studies of the lipidomes of twenty-one species of clostridia have revealed considerable diversity. Even among those species now defined as Clostridium sensu stricto, which are related to Clostridium butyricum, the type species, lipid analysis has shown that a number of distinct clades have characteristic polar lipids. All species of Clostridium sensu stricto have phosphatidylethanolamine, phosphatidylglycerol and cardiolipin which are present as all acyl or alk-1′-enyl acyl (plasmalogen) species. In addition, almost every clade has specialized polar lipids. For example, the group closely related to Clostridium beijerinckii and several other solventogenic species has glycerol acetals of plasmenylethanolamine, which protects the membrane bilayer arrangement when the lipids are highly unsaturated or in the presence of solvents. The group related to Clostridium novyi has aminoacyl-phosphatidylglycerol, which protects these pathogens from cationic antimicrobial peptides (CAMPs) of innate immunity. Clostridium botulinum species, which fall into several groups, align with these clades, and have the same specific lipids. This review will present the current state of knowledge on clostridial lipids.     

Fermentation of raw glycerol to 1,3-propanediol by new strains ofClostridium butyricum

Industrial glycerol obtained through the transesterification process using rapeseed oil did not support growth of several strains ofClostridium butyricum obtained from bacterial culture collections. Ten new strains ofC. butyricum were obtained from mud samples from a river, a stagnant pond, and a dry canal. These new isolates fermented the commercial glycerol and produced 1,3-propanediol as a major fermentation product with concomitant production of acetic and butyric acids. Four of the ten isolates were able to grow on industrial glycerol obtained from rapeseed oil. One strain,C. butyricum E5, was very resistant to high levels of glycerol and 1,3-propanediol. Using fed-batch fermentation, 109 g L^–1 of industrial glycerol were converted into 58 g of 1,3-propanediol, 2.2 g of acetate and 6.1 g of butyrate per liter.     

Fermentative hydrogen production from glucose and starch using pure strains and artificial co-cultures of Clostridium spp.

Background

Pure bacterial strains give better yields when producing H₂ than mixed, natural communities. However the main drawback with the pure cultures is the need to perform the fermentations under sterile conditions. Therefore, H₂ production using artificial co-cultures, composed of well characterized strains, is one of the directions currently undertaken in the field of biohydrogen research.

Results

Four pure Clostridium cultures, including C. butyricum CWBI1009, C. pasteurianum DSM525, C. beijerinckii DSM1820 and C. felsineum DSM749, and three different co-cultures composed of (1) C. pasteurianum and C. felsineum, (2) C. butyricum and C. felsineum, (3) C. butyricum and C. pasteurianum, were grown in 20?L batch bioreactors. In the first part of the study a strategy composed of three-culture sequences was developed to determine the optimal pH for H₂ production (sequence 1); and the H₂-producing potential of each pure strain and co-culture, during glucose (sequence 2) and starch (sequence 3) fermentations at the optimal pH. The best H₂ yields were obtained for starch fermentations, and the highest yield of 2.91?mol?H₂/ mol hexose was reported for C. butyricum. By contrast, the biogas production rates were higher for glucose fermentations and the highest value of 1.5?L biogas/ h was observed for the co-culture (1). In general co-cultures produced H₂ at higher rates than the pure Clostridium cultures, without negatively affecting the H₂ yields. Interestingly, all the Clostridium strains and co-cultures were shown to utilize lactate (present in a starch-containing medium), and C. beijerinckii was able to re-consume formate producing additional H₂. In the second part of the study the co-culture (3) was used to produce H₂ during 13?days of glucose fermentation in a sequencing batch reactor (SBR). In addition, the species dynamics, as monitored by qPCR (quantitative real-time PCR), showed a stable coexistence of C. pasteurianum and C. butyricum during this fermentation.

Conclusions

The four pure Clostridium strains and the artificial co-cultures tested in this study were shown to efficiently produce H₂ using glucose and starch as carbon sources. The artificial co-cultures produced H₂ at higher rates than the pure strains, while the H₂ yields were only slightly affected.