大麻沤麻液细菌种群多样性

从分子水平分析大麻沤麻液中细菌种类及其优势菌群,为筛选出大麻微生物脱胶的生产菌种奠定基础。采用PCR-DGGE技术研究大麻沤麻液中细菌种群的多样性及动态变化,使用Gel-Pro Analyzer 4.5软件分析DGGE指纹图谱,并对优势条带进行分析。结果表明:从发酵初期过渡到主发酵期细菌种群多样性上升,群落演替迅速,从主发酵期进入到发酵末期,细菌种群多样性下降,群落演替缓慢;在整个发酵过程中,细菌种群多样性指数在发酵中期(72h)达到高峰,说明发酵中期细菌种群数量、优势菌群种类达到了最高值,发酵初期和末期以不可培养细菌为主,主发酵期以成团泛菌属为主。     

传统分离培养结合DGGE法检测榨菜腌制过程的细菌多样性

采用传统分离培养和基于16S rRNA 作为分子标记的变性梯度凝胶电泳(Denaturing gradient gel electrophoresis, DGGE)的方法, 分析榨菜腌制过程中不同时期的可培养细菌数量、多样性及其群落结构。结果表明, 用传统分离与分子鉴定方法获得7个属的细菌类群, 其中乳杆菌属(Acidobacterium)是优势菌群, 明串珠菌属(Leuconostoc)是次优势菌群。对通过DGGE方法得到的11条16S rRNA优势条带序列进行了比对, 结果表明明串珠菌属(Leucon     

The influence of urea feeding on the bacterial and archaeal community in the forestomach of collared peccary (Artiodactyla, Tayassuidae)

Aims:  This study was carried out to test whether bacterial and archaeal populations, and products of fermentation in each compartment of collared peccary stomach, vary significantly with urea feeding. Bacteria and archaeal population variation among the four stomach compartments were also compared.
Methods and Results:  Archaeal and bacterial communities in the forestomach of four individuals per treatment – peccaries fed diets with and without urea – were analysed at molecular level using PCR followed by denaturing gradient gel electrophoresis. Volatile fatty acids profiles in the three different compartments of the forestomach were also compared. The bacterial community composition varied considerably among each compartment and with urea provision, but no variation was observed between archaeal populations. Differences in bacterial communities between treatments – with and without urea – were greater than amongst stomach compartments. The acetate: propionate proportion decreased with urea provision in diet. Some differences in bacterial but not archaeal community composition were observed in each compartment of the collared peccary forestomach.
Conclusions:  There are some differences in bacterial but not archaeal populations in each compartment of collared peccary stomach. Use of urea in the diet of peccary can substantially modify the profile of volatile fatty acids released in its forestomach, but does not influence the archaeal community composition. Urea has an important effect on bacterial population DGGE profile present in the peccary's forestomach.
Significance and Impact of the Study:  These results demonstrate the ability of the collared peccary to use urea as source of nonprotein nitrogen, and confirm a hypothesis that the collared peccary has a digestive physiology more similar to ruminant than nonruminant animals.     

Community analysis of a full-scale anaerobic bioreactor treating paper mill wastewater

To get insight into the microbial community of an Upflow Anaerobic Sludge Blanket reactor treating paper mill wastewater, conventional microbiological methods were combined with 16S rRNA gene analyses. Particular attention was paid to microorganisms able to degrade propionate or butyrate in the presence or absence of sulphate. Serial enrichment dilutions allowed estimating the number of microorganisms per ml sludge that could use butyrate with or without sulphate (10(5)), propionate without sulphate (10(6)), or propionate and sulphate (10(8)). Quantitative RNA dot-blot hybridisation indicated that Archaea were two-times more abundant in the microbial community of anaerobic sludge than Bacteria. The microbial community composition was further characterised by 16S rRNA-gene-targeted Denaturing Gradient Gel Electrophoresis (DGGE) fingerprinting, and via cloning and sequencing of dominant amplicons from the bacterial and archaeal patterns. Most of the nearly full length (approximately 1.45 kb) bacterial 16S rRNA gene sequences showed less than 97% similarity to sequences present in public databases, in contrast to the archaeal clones (approximately. 1.3 kb) that were highly similar to known sequences. While Methanosaeta was found as the most abundant genus, also Crenarchaeote-relatives were identified. The microbial community was relatively stable over a period of 3 years (samples taken in July 1999, May 2001, March 2002 and June 2002) as indicated by the high similarity index calculated from DGGE profiles (81.9+/-2.7% for Bacteria and 75.1+/-3.1% for Archaea). 16S rRNA gene sequence analysis indicated the presence of unknown and yet uncultured microorganisms, but also showed that known sulphate-reducing bacteria and syntrophic fatty acid-oxidising microorganisms dominated the enrichments.     

Bacterial community associated with ensilage process of wilted guinea grass

Aims: To determine the effects of wilting, storage period and bacterial inoculant on the bacterial community and ensiling fermentation of guinea grass silage. Methods and Results: Fermentation products, colony counts and denaturing gradient gel electrophoresis (DGGE) profiles were determined. There was more lactic acid than acetic acid in all silages, but the lactic acid to acetic acid ratio decreased with storage time. This shift from lactic to acetic acid was not prevented even with a combination of wilting and bacterial inoculant. The DGGE analyses suggest that facultatively heterofermentative lactic acid bacteria (Lactobacillus plantarum, Lactobacillus brevis and Lactobacillus pentosus) were involved in the shift to acetic acid fermentation. Conclusions: Lactic acid can dominate the fermentation in tropical grass silage with sufficient wilting prior to ensiling. Prolonged storage may lead to high levels of acetic acid without distinctive changes in the bacterial community. Significance and Impact of the Study: The bacterial community looks stable compared to fermentation products over the course of long storage periods in tropical grass silage. Acetic acid fermentation in tropical grass silage can be a result of the changes in bacterial metabolism rather than community structure.     

Bacterial and archaeal assemblages in sediments of a large shallow freshwater lake, Lake Taihu, as revealed by denaturing gradient gel electrophoresis

Aims:  To explore the association of microbial community structure with the development of eutrophication in a large shallow freshwater lake, Lake Taihu.
Methods and Results:  The bacterial and archaeal assemblages in sediments of different lake areas were analysed using denaturing gradient gel electrophoresis (DGGE) of amplified 16S rDNA fragments. The bacterial DGGE profiles showed that eutrophied sites, grass-bottom areas and relatively clean sites with a eutrophic (albeit dredged) site are three respective clusters. Fifty-one dominant bacterial DGGE bands were detected and 92 corresponding clones were sequenced, most of which were affiliated with bacterial phylotypes commonly found in freshwater ecosystems. Actinobacteria were detected in the centre of the lake and not at eutrophied sites whereas the opposite was found with respect to Verrucomicrobiales . Twenty-five dominant archaeal DGGE bands were detected and 31 corresponding clones were sequenced, most of which were affiliated with freshwater archaeal phylotypes.
Conclusions:  The bacterial community structures in the sediments of different areas with similar water quality and situation tend to be similar in Taihu Lake.
Significance and Impact of the Study:  This study may expand our knowledge on the relationship between the overall microbial assemblages and the development of eutrophication in the shallow freshwater lake.     

Direct comparison of single-strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) to characterize a microbial community on the basis of 16S rRNA gene fragments

Characterization of microbial communities using single-strand conformation polymorphism (SSCP) was compared with that using denaturing gradient gel electrophoresis (DGGE). This comparison was based on the V3-4 region (Escherichia coli positions: 341-806) of 16S rRNA gene of bacterial or archaeal communities obtained from a methanogenic bioreactor. Significant differences in the bacterial banding profiles were observed while attempting to detect the diversity of the community and its succession during the reactor operation. The SSCP produced a number of sharp bands and differentiated the bacterial community structures to which the DGGE gave an identical pattern. On the other hand, the SSCP and DGGE provided similar succession patterns for archaeal community.     

传统豆酱发酵过程中细菌多样性动态

细菌在豆酱发酵过程中起到非常重要的作用,并与豆酱的风味和质量密切相关,因此研究豆酱中细菌的多样性具有重要意义。以自然发酵的豆酱样品为研究对象,采用细菌16S rDNA的部分可变区的PCR-DGGE技术对自然发酵豆酱样品的细菌群落组成和优势菌群进行研究。结果表明,传统豆酱发酵过程细菌群体中既有原始种群的减少和增长,也有次级种群的增多和演变。在整个发酵过程中,初期和末期以不可培养细菌为主,初期细菌群体快速演替,细菌种群多样性指数在发酵42 d和56 d达到两次高峰。     

Analysis and comparison of the bacterial community in fermented grains during the fermentation for two different styles of Chinese liquor

Bacterial populations in fermented grains during fermentation may play important roles in Chinese liquor flavor. PCR-based denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene library analysis were performed to analyze the bacterial community structure of two styles of liquor. The results of DGGE profiles showed that bacterial diversity decreased with the fermentation process and Lactobacillus acetotolerans became the predominant species at the end of the fermentation. But the obvious differences of bacterial community appeared in the middle stage of two styles of liquor fermentation, in which the different upstream production techniques were used. Moreover, 16S rRNA gene libraries of two styles were constructed. A total of 125 and 107 clones, chosen from two libraries, were grouped into 46 and 49 operational taxonomic units (OTUs) by amplified ribosomal DNA restriction analysis. According to sequencing results of clones, the predominant bacteria in strong aroma style fermented grains were those from the class Bacilli, Bacteroidetes, and Clostridia, whereas the predominant bacteria in fermented grains of roasted sesame aroma style belonged to Bacilli, Flavobacteria, and Gammaproteobacteria. Molecular analysis of the bacterial diversity of the liquor fermentation will benefit the analysis of important microorganisms playing key roles in the formation of liquor flavor components.     

Dynamics of a pig slurry microbial community during anaerobic storage and management

The microbial community of a pig slurry on a farm was monitored for 6 months using both molecular and cultural approaches. Sampling was carried out at all the different stages of effluent handling, from the rearing build-up to slurry spreading. Total DNA of each sample was extracted and analyzed by PCR-single-strand conformation polymorphism (SSCP) analysis using primers targeting the 16S rRNA genes from the archaeal and bacterial domains and also the Eubacterium-Clostridium, Bacillus-Streptococcus-Lactobacillus, and Bacteroides-Prevotella groups. A comparison of the SSCP profiles showed that there were rapid changes in the dominant bacterial community during the first 2 weeks of anaerobic storage and that the community was relatively stable thereafter. Several bacterial populations, identified as populations closely related to uncultured Clostridium and Porphyromonas and to Lactobacillus and Streptococcus cultured species commonly isolated from pig feces, remained present and dominant from the rearing build-up to the time of spreading. Enumeration of fecal indicators (enterococci and Escherichia coli) performed in parallel using cultural methods revealed the same trends. On the other hand, the archaeal community adapted slowly during pig slurry storage, and its diversity increased. A shift between two hydrogenotrophic methanogenic Methanobrevibacter populations from the storage pit to the pond was observed. Microorganisms present in pig slurry at the time of spreading could not be detected in soil after spreading by either molecular or cultural techniques, probably because of the detection limit inherent in the two techniques.     

Bacterial community structure, compartmentalization and activity in a microbial fuel cell

AIMS: To characterize bacterial populations and their activities within a microbial fuel cell (MFC), using cultivation-independent and cultivation approaches. METHODS AND RESULTS: Electron microscopic observations showed that the fuel cell electrode had a microbial biofilm attached to its surface with loosely associated microbial clumps. Bacterial 16S rRNA gene libraries were constructed and analysed from each of four compartments within the fuel cell: the planktonic community; the membrane biofilm; bacterial clumps (BC) and the anode biofilm. Results showed that the bacterial community structure varied significantly between these compartments. It was observed that Gammaproteobacteria phylotypes were present at higher numbers within libraries from the BC and electrode biofilm compared with other parts of the fuel cell. Community structure of the MFC determined by analyses of bacterial 16S rRNA gene libraries and anaerobic cultivation showed excellent agreement with community profiles from denaturing gradient gel electrophoresis (DGGE) analysis. CONCLUSIONS: Members of the family Enterobacteriaceae, such as Klebsiella sp. and Enterobacter sp. and other Gammaproteobacteria with Fe(III)-reducing and electrochemical activity had a significant potential for energy generation in this system. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has shown that electrochemically active bacteria can be enriched using an electrochemical fuel cell.     

The structure of the bacterial and archaeal community in a biogas digester as revealed by denaturing gradient gel electrophoresis and 16S rDNA sequencing analysis

Aims:  To identify the bacterial and archaeal composition in a mesophilic biogas digester treating pig manure and to compare the consistency of two 16S rDNA-based methods to investigate the microbial structure.
Methods and results:  Sixty-nine bacterial operational taxonomic units (OTU) and 25 archaeal OTU were identified by sequencing two 16S rDNA clone libraries. Most bacterial OTU were identified as phyla of Firmicutes (47·2% of total clones), Bacteroides (35·4%) and Spirochaetes (13·2%). Methanoculleus bourgensis (29·0%), Methanosarcina barkeri (27·4%) and Methanospirillum hungatei (10·8%) were the dominant methanogens. Only 9% of bacterial and 20% of archaeal OTU matched cultured isolates at a similarity index of ≥97%. About 78% of the dominant bacterial (with abundance >3%) and 83% of archaeal OTU were recovered from the denaturing gradient gel electrophoresis (DGGE) bands of V3 regions in 16S rDNAs.
Conclusions:  In the digester, most bacterial and archaeal species were uncultured; bacteria belonging to Firmicutes , Bacteroides and Spirochaetes seem to take charge of cellulolysis, proteolysis, acidogenesis, sulfur-reducing and homoacetogenesis; the most methanogens were typical hydrogenotrophic or hydrogenotrophic/aceticlastic; DGGE profiles reflected the dominant microbiota.
Significance and Impact of the Study:  This study gave a first insight of the overall microbial structure in a rural biogas digester and also indicated DGGE was useful in displaying its dominant microbiota.     

Denaturing gradient gel electrophoresis analysis of bacterial populations in 5-stage biological nutrient removal process with step feed system for wastewater treatment

Changes in the bacterial populations of a 5-stage biological nutrient removal (BNR) process, with a step feed system for wastewater treatment, were monitored by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA fragments. DGGE analysis indicated seasonal community changes were observed, however, community profiles of the total bacteria of each reactor showed only minor differences in the samples obtained from the same season. The number of major bands was higher in the summer samples, and decreased during the winter period, indicating that the microbial community structure became simpler at low temperatures. Since the nitrogen and phosphate removal efficiencies were highly maintained throughout the winter operation period, the bacteria which still remaining in the winter sample can be considered important, playing a key role in the present 5-stage BNR sludge. The prominent DGGE bands were excised, and sequenced to gain insight into the identities of the predominant bacterial populations present, and most were found to not be closely related to previously characterized bacteria. These data suggest the importance of culture-independent methods for the quality control of wastewater treatment.     

Ability of DNA content and DGGE analysis to reflect the performance condition of an anaerobic biowaste fermenter

Molecular-microbiological techniques have delivered insight into microbial populations present in anaerobic fermenters, although much information still remains to be elucidated. In this study, the ability of denaturing gradient gel electrophoresis (DGGE) to throw light on microbial community composition was investigated and latter data were compared with the gas production of a 750,000l anaerobic biogas fermenter. During 1 year, samples were taken from two different sites of the reactor and additionally from the substrate material. After DNA extraction and PCR with archaeal and bacterial primers, PCR products were run on denaturing gradient gels to compare band patterns. Using gel-imaging software (GelComparII), two major clusters could be identified. Dominant bands were excised from the gels, reamplified and sequenced. Most sequences were closely related to Lactobacilli and yet uncultured microorganisms. DNA content of all samples was significantly correlated with the gas production measured online. We concluded that PCR and subsequent DGGE are useful to monitor community shifts in anaerobic fermenter sludge. However, as these changes are not readily detectable via DGGE-pattern analysis, alternative factors influencing the fermenter functioning should be found and investigated. So far DNA-content measurement seems to be a good parameter to quickly determine anaerobic fermenter condition.     

Vertical distribution of bacterial and archaeal communities along discrete layers of a deep-sea cold sediment sample at the East Pacific Rise (∼13°N)

The community structure and vertical distribution of prokaryotes in a deep-sea (ca. 3,191 m) cold sediment sample (ca. 43 cm long) collected at the East Pacific Rise (EPR) approximately 13 degrees N were studied with 16SrDNA-based molecular analyses. Total community DNA was extracted from each of four discrete layers EPRDS-1, -2, -3 and -4 (from top to bottom) and 16S rDNA were amplified by PCR. Cluster analysis of DGGE profiles revealed that the bacterial communities shifted sharply between EPRDS-1 and EPRDS-2 in similarity coefficient at merely 49%. Twenty-three sequences retrieved from DGGE bands fell into 11 groups based on BLAST and bootstrap analysis. The dominant groups in the bacterial communities were Chloroflexi, Gamma proteobacteria, Actinobacterium and unidentified bacteria, with their corresponding percentages varying along discrete layers. Pairwise Fst (F-statistics) values between the archaeal clone libraries indicated that the archaeal communities changed distinctly between EPRDS-2 and EPRDS-3. Sequences from the archaeal libraries were divided to eight groups. Crenarchaea Marine Group I (MGI) was prevalent in EPRDS-1 at 83%, while Uncultured Crenarchaea group II B (UCII B) abounded in EPRDS-4 at 61%. Our results revealed that the vertically stratified distribution of prokaryotic communities might be in response to the geochemical settings and suggested that the sampling area was influenced by hydrothermalism. The copresence of members related to hydrothermalism and cold deep-sea environments in the microbial community indicated that the area might be a transitional region from hydrothermal vents to cold deep-sea sediments.     

Response of microbial community structure to natural and accelerated hydrarch successions in the boreal wetlands in northern Hokkaido, Japan

We estimated the effect of invading Sasa vegetation and accelerated terrestrialization on the microbial community structure in Sarobetsu-genya wetland (SGW) and Nakanominedaira wetland (NW) (original vegetation, Sphagnum). All examined peat-pore water samples were acidic. Electrical conductivity significantly differed between SGW and NW. Nonmetric multidimensional scaling (NMDS) and analysis of similarity based on denaturing gradient gel electrophoresis (DGGE) band patterns revealed differences in the bacterial community structure between the Sasa and Sphagnum vegetations at a depth of 10 cm in NW. In contrast, the bacterial NMDS profiles at all depths differed between the 2 wetlands rather than between the 2 vegetations. The archaeal community structure significantly differed between the wetlands at depths of 30 and 50 cm. The bacterial diversity index derived from the DGGE profiles significantly differed between the wetlands at all depths. The archaeal diversity index significantly differed between the wetlands at a depth of 50 cm. Sasa invasion affected the microbial community structure in the rhizosphere, up to a depth of 10 cm; this effect differed with the terrestrialization speed. These results suggest that in peat bogs subjected to artificially accelerated terrestrialization, the microbial community changes before the occurrence of the natural hydrarch ecological succession involving ground vegetation.     

The impact of grassland management on archaeal community structure in upland pasture rhizosphere soil

The community structure of rhizosphere soil Archaea from three grassland types, associated with different management practices, was examined at a site in the Borders region of Scotland, by analysis of 16S rRNA gene fragments amplified from 16S rDNA and from rRNA. Denaturing gradient gel electrophoresis (DGGE) and sequence analysis of amplified products indicated high relative abundance within the archaeal community of two distinct lineages of non-thermophilic (group 1) Crenarchaeota. Grassland management practices influenced archaeal community structure, as characterized by both 16S rRNA- and 16S rDNA-derived DGGE profiles. One band dominated DGGE profiles in all three grassland types examined, and reproducible differences in the presence and intensity of bands were observed between profiles from managed and natural grassland sites. Analysis of 16S rRNA-derived amplicons from managed and natural grasslands at sites in the north of England and the north of Wales also indicated high relative abundance of non-thermophilic crenarchaeotes within the archaeal community. The band dominating the Scottish grassland site also dominated DGGE profiles from the English and Welsh sites, and similar differences were seen between profiles derived from soils subjected to different management regimes. The study indicates that grassland archaeal communities are dominated by Crenarchaeota, with closely related members of this lineage ubiquitous in distribution in UK upland pasture, and indicate that management practices influence the nature of the crenarchaeotal community.     

Examination of two years of community dynamics in an anaerobic bioreactor using fluorescence polymerase chain reaction (PCR) single-strand conformation polymorphism analysis

The structures of the bacterial and archaeal communities in an anaerobic digester were monitored over a 2 year period. The study was performed on a fluidized bed reactor fed with vinasse. The objective was to characterize the population dynamics over a long time period under constant environmental parameters. Total bacterial and archaeal populations were measured independently by fluorescence-based polymerase chain reaction (PCR) single-strand conformation polymorphism (SSCP) analysis using an automated DNA sequencer. With the current level of accuracy, the technique was able to monitor 45 bacterial and seven archaeal 16S rDNA molecules. The community dynamics were compared with molecular inventories of the microbial community based on 16S rDNA sequences done at the beginning of the study. The six archaeal and the 22 most frequent bacterial operational taxonomic units (OTUs) identified were associated with their SSCP peak counterparts. Overall, the data indicated that, throughout the period of the study, rapid significant shifts in the species composition of the bacterial community occurred, whereas the archaeal community remained relatively stable.     

Spatial Analysis of Archaeal Community Structure in Grassland Soil

The complex structure of soil and the heterogeneity of resources available to microorganisms have implications for sampling regimens when the structure and diversity of microbial communities are analyzed. To assess the heterogeneity in community structure, archaeal communities, which typically contain sequences belonging to the nonthermophilic Crenarchaeota, were examined at two contrasting spatial scales by using PCR-denaturing gradient gel electrophoresis (DGGE) analysis followed by unweighted pair group method with arithmetic mean analysis of 16S rRNA- and ribosomal DNA-derived profiles. A macroscale analysis was carried out with soil cores taken at 2-m intervals along triplicate 8-m transects from both managed (improved) and natural (unimproved) grassland rhizosphere soils. A microscale analysis was carried out with a single soil core by assessing the effects of both sample size (10, 1, and 0.1 g) and distance between samples. The much reduced complexity of archaeal profiles compared to the complexity typical of the bacterial community facilitated visual comparison of profiles based on band presence and revealed different levels of heterogeneity between sets of samples. At the macroscale level, heterogeneity over the transect could not be related to grassland type. Substantial heterogeneity was observed across both improved and unimproved transects, except for one improved transect that exhibited substantial homogeneity, so that profiles for a single core were largely representative of the entire transect. At the smaller scale, the heterogeneity of the archaeal community structure varied with sample size within a single 8- by 8-cm core. The archaeal DGGE profiles for replicate 10-g soil samples were similar, while those for 1-g samples and 0.1-g samples showed greater heterogeneity. In addition, there was no relationship between the archaeal profiles and the distance between 1- or 0.1-g samples, although relationships between community structure and distance of separation may occur at a smaller scale. Our findings demonstrate the care required when workers attempt to obtain a representative picture of microbial community structure in the soil environment.     

Changes in product formation and bacterial community by dilution rate on carbohydrate fermentation by methanogenic microflora in continuous flow stirred tank reactor

Changes in product formation during carbohydrate fermentation by anaerobic microflora in a continuous flow stirred tank reactor were investigated with respect to the dilution rate in the reactor. In the fermentation by methanogenic microflora, stable methane fermentation, producing methane and carbon dioxide, was observed at relatively low dilution rates (less than 0.33 d(-1) on glucose and 0.20 d(-1) on cellulose). Decomposition of cellulose in the medium was a rate-limiting step in the reaction, because glucose was easily consumed at all applied dilution rates (0.07-4.81 d(-1)). Intermediate metabolites of methane fermentation, such as lactate, ethanol, acetate, butyrate, formate, hydrogen, and carbon dioxide, were accumulated as dilution rate increased. Maximum yield of hydrogen was obtained at 4.81 d(-1) of dilution rate (0.1 mol/mol glucose on glucose or 0.7 mol/mol hexose on cellulose). Lactate was the major product on glucose (1.2 mol/mol glucose), whereas ethanol was predominant on cellulose (0.7 mol/mol hexose). An analysis by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified bacterial 16S rDNA of the microflora indicated that changes in the microbial community took place at various dilution rates, and these changes appeared to correspond to the changes in product distributions. Sequence analyses of the DGGE fragments revealed the probable major population of the microflora. A band closely related to the microorganisms of thermophilic anaerobic bacteria was detected with strong intensity on both glucose and cellulose. Differences in the production yield of hydrogen could have been caused by different populations of microorganisms in each microflora. In the case of cellulose, increasing the dilution rate brought about an accumulation of microorganisms related to Clostridia species that have cellulolytic activity, this being in accordance with the notion of cellulose decomposition being the rate-limiting reaction.