Progress 09/01/04 to 08/31/08
Outputs
OUTPUTS: The overall goal of this work was to develop and apply new methods for monitoring and analyzing microbial diversity in anaerobic digesters. Fundamental understanding of digester microbial diversity and interactions is needed to develop predictive models to manage and engineer digesters. Objective 1: Construct DNA microarrays that represent total microbial diversity in the thermophilic digester. The strength of DNA microarrays is that they allow the simultaneous detection and quantification of thousands of genes. We could not anticipate at that time how rapidly DNA sequencing technologies would advance in terms of both high-throughput capacity and economy. Because new pyrosequencing technology became available, we tested it as a competitive alternative method to microarrays. Success of this experiment prompted us to adopt the newer technology rather than continue toward developing DNA microarrays. In fact, we have demonstrated that 454 sequencing is clearly a superior technology for these research goals. In contrast to DNA microarrays, high-throughput pyrosequencing can be used to detect new organisms and for very deep sampling of genetic diversity. Another limitation of microarrays is the hybridization and quantification of the fluorescent signal on the chips. This problem is eliminated by pyrosequencing where quantification can be done by counting sequences generated either by PCR or as cDNA. Equally important is economics of analyses. The relatively low cost of pyrosequencing makes it more economical per sample than using DNA microarray chips. Results of pyrosequencing analysis were outstanding. More than 28 Mb of DNA were sequenced using this method, providing an unprecedented view of anaerobic digester microbial community structure. More than 9500 pyrosequences were obtained for a pilot plant digester, and more than 40,000 pyrosequences were generated for a biofilm digester. Representation of total microbial diversity from the pyrosequencing was compared to that derived from traditional PCR-based random cloning approach. Whereas a rarefaction analysis of the pilot plant 16S rDNA clone library yielded about 100 OTUs (99% level), pyrosequencing yielded more than 2500 OTUs. Furthermore, pyrosequence data allows a comparison of DNA sequence variation which permits the detection of populations with microdiverse variation. Such variation could be essential to the performance of any environmental biotechnology system. Objective 2: was to test whether the biofilm microbial populations in a digester differentiate from the planktonic populations in the same reactor. Both 16S rRNA gene clone libraries and pyrosequencing were used on the planktonic and biofilm populations over time. The biofilm digester contained predominantly novel phylotypes including probable new species, genera, families and possibly higher taxonomic groups. This data was analyzed with a computational evolutionary method that we applied for the first time to 16S rDNA community data. We found that the biofilm community differentiated over time from the planktonic community and that distinct populations showed a habitat preference for either the biofilm or planktonic environments. PARTICIPANTS: The following research scientists and organizations participated in this project: Dr. Hilary Lappin-Scott, Department of Biosciences, University of Exeter (UK); Dr. David Stafford, Enviro Control Ltd (Cardiff, UK). This grant funded the research of a PhD student, several undergraduate students, and provided partial funding for a MS student at WVSU. The WVSU Gus R. Douglass Institute (Land Grant programs) provided support through the operation of the pilot plant anaerobic digester. TARGET AUDIENCES: The target audiences for this project are as follows: 1. environmental biotechnologists, environmental engineers, 2. microbiologists, 3. poultry industry (poultry farms) and other animal farms, 4. bioenergy industry, 6. waste management industry. PROJECT MODIFICATIONS: The primary goal of this grant was to apply new technology to the problem of detecting, quantifying, and economically monitoring microbial population dynamics in anaerobic digesters. The original technology chosen for the project was DNA microarrays. The goal was to develop DNA microarrays that represent the microbial diversity of a model thermophilic anaerobic digester. However, during this grant award period, the unexpected and rapid development of high-throughput, low-cost DNA sequencing technology occurred. Pyrosequencing was tested as an alternative technology for reaching the primary goal. Experiments with pyrosequencing demonstrated that it is superior to DNA microarrays in terms of the objectives of this research. The power of pyrosequencing exceeded the potential of DNA microarrays. Therefore, we prudently adopted the new technology to reach the grant goals of developing an economical, comprehensive, quantifiable method for simultaneously monitoring the dynamics of hundreds of microbial populations in a bioreactor.
Impacts
The research funded by this grant has made significant contributions to understanding the microbial ecology of anaerobic digestion. First, this study has demonstrated the power of pyrosequencing for revealing previously unknown microbial diversity and for quantitatively analyzing the dynamics of microbial community structure. This technology was applied to anaerobic digesters for the first time and enabled new insights into the relationship of microbial community structure to digester performance. In addition, this grant has provided a database consisting of 16S rRNA genes, comprising 28 Mb of DNA that represents a thermophilic anaerobic digester. This data will be available to other researchers interested in anaerobic digestion. Third, this study has refuted conventional wisdom concerning biofilm digesters which states that the primary benefit of biofilm growth is the retention of microbial cells when effluent is released. Fourth, the methods developed with this grant can be utilized to include microbial population diversity as another performance parameter for studying, monitoring, and engineering waste treatment systems. In summary, this research has demonstrated that pyrosequencing is superior to DNA microarray analysis of microbial communities in terms of the detection of novel microbial diversity, analysis of fine-scale population genetic variation, quantification of populations, and cost. Pyrosequencing will allow the analysis of the microbial ecology of digesters and other waste treatments systems in an unprecedented way, thereby advancing a predictive science of anaerobic biodegradation and bioenergy production.
Publications
- No publications reported this period
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Research Results: The overall objective of this research is to develop DNA microarrays for the functional analysis of microbial populations in thermophilic anaerobic digesters. The specific goals are to create a 16S rDNA diversity collection that represents a thermophilic anaerobic digester and to construct DNA microarrays that represent the phylogenetic diversity of the digester community. The third year of the grant continued to develop a comprehensive collection of bacterial and archaeal 16S rDNA clones from a pilot plant thermophilic digester and a derived biofilm digester. The pilot plant digester is a 10,000 gallon continuous stir tank reactor located on the campus of West Virginia State University. The digester is fed poultry litter and operated at 55C. In addition, the operation and sampling of a 15 liter thermophilic biofilm digester was continued. The biofilm digester was previously started with inoculum from the pilot plant. The analysis of 16S rDNA libraries from
the pilot plant and biofilm digesters progressed, including resequencing of clones to improve quality. The diversity of the methanogenesis operon gene mcrA was sampled in the biofilm digester via cloning. The analysis to date has shown that the biofilm and pilot plant digesters contain similar, but distinct, microbial communities. The biofilm community contains significantly more biodiversity than the suspended community. The Chao 1 diversity estimator projects that the biofilm contains a minimum of 320 operational taxonomic units (OTUs) at the 97% similarity level, while the pilot plant contains a minimum of 180 OTUs. The structure of the pilot plant community in terms of taxon rank-abundance shows few OTUs of high abundance and most OTUs of low abundance. Approximately 85% of the clones in the pilot plant fall into the Firmicutes with 35% of these being Clostridia and 40% being unclassified. The majority of the Clostridia cannot be classified more specifically than family with the
RDP. This demonstrates a large percentage of novel bacterial diversity in the WVSU thermophilic digesters. Dissemination, Events, Services: The Principal Investigator included new teaching material on digesters in his class Environmental Microbiology, and also in the team-taught graduate class Current Concepts in Biotechnology, which were taught spring, 2007. In addition, the PI provided consultation to engineers working on two different projects that were involved in designing thermophilic digesters. One project is based in Pennsylvania, the other in West Virginia. One undergraduate student researcher made a presentation at the West Virginia State University Spring Research Symposium, April 2007, concerning the cloning of mcrA genes from the WVSU digester.
PARTICIPANTS: The principal investigator of the grant directed the research activities and was involved in all aspects of the research. The primary research scientist working to accomplish the experimental goals was a PhD student who worked in the PI's laboratory at WVSU. This grant also supported the research of two undergraduate students during the past year. One student was the recipient of a NASA scholarship that enabled him to work on this project during spring semester 2007. The other undergraduate student was employed as a technician who worked on the research funded by this grant as well as other related digester microbiology research. A full-time technician was assigned to the PI's laboratory through the WVSU Land Grant programs during the past year. The technician assisted in a variety of support roles for this project, including gene library construction, DNA sequencing, etc. The PI maintained non-formal collaborations with Dr. Teodoro Espinosa-Solares (University of
Chapingo, Mexico) and Dr. David Stafford (Enviro Control Ltd, Monmouth, England) concerning anaerobic digester research.
TARGET AUDIENCES: The primary audience who has benefited from this research to date is students at WVSU. A PhD student and two undergraduate students worked directly on this research and learned microbiology and molecular biology methods. The knowledge acquired by the PI through the analysis of project data as well as collateral research and literature-based learning has directly enhanced the topics of environmental biotechnology and environmental genomics which he presents in his classes Microbiology, Environmental Microbiology, and Microbial Genetics. The grant helped to support the training of a new research technician in his laboratory during 2007. The primary target audience for this project will be research scientists and environmental engineers who work with anaerobic digestion. This audience will be reached when the grant research reaches publication which will occur next year.
PROJECT MODIFICATIONS: The termination date of the project was extended for one year (8/31/08). The extension is needed to complete the goals of the grant. The bulk of the research funded by this grant has been accomplished by one PhD student. This student has made excellent progress, but an additional year was needed to complete the experimental goals. The DNA microarray applications will occur in the final year of the grant.
Impacts
Anaerobic digesters impact the environment by diminishing and stabilizing high organic content waste, such as animal and other agricultural residuals, and by producing bioenergy (methane). The performance of digesters requires the synergistic metabolism of many species of bacteria, most of which are unknown. The performance of digesters requires managing these microbial populations. The standard operation of digesters involves monitoring biochemical variables such as volatile fatty acids and pH, but has not been based on knowledge of the composition and dynamics of the bacterial populations. In order to develop predictive models of digester metabolism and to assist in digester operation, this grant will utilize DNA microarrays to track and measure the activity of the major bacterial populations. This research will utilize DNA microarrays to monitor the bacterial populations in a thermophilic anaerobic digester. This new tool will be applied to improving the operation
and maintenance of digesters and will permit the comparison of different digesters to ascertain which key populations are needed for optimal and diverse digester applications. The research during the past year continued to build the database that will enable this long-term goal to be reached. Participants in the project have benefited through an increase in knowledge and skills. This grant supported the research of one graduate student and two undergraduate students at WVSU during the past year. The graduate student has continued to learn advanced molecular microbial ecology methods, and the undergraduate students learned basic microbiology and molecular biology methods. A new laboratory technician was also trained during the past year to use molecular microbiology methods.
Publications
- No publications reported this period
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Progress 01/01/06 to 12/31/06
Outputs
The overall objective of this research is to develop DNA microarrays for the functional analysis of microbial populations in thermophilic anaerobic digesters. The specific goals are to 1) expand an existing anaerobic digester 16S rDNA diversity collection, and include new functional gene diversity, 2) construct DNA microarrays that represent the phylogenetic and functional diversity of a methanogenic digester community, and 3) compare the biofilm microbial populations to the planktonic cell populations in a filter (biofilm) reactor. The second year of the grant continued to develop a comprehensive collection of bacterial and archaeal 16S rDNA clones from a pilot plant thermophilic digester and a biofilm digester. The pilot plant digester is a 10,000 gallon continuous stir tank reactor located on the campus of West Virginia State University. During this time period, a 15 liter thermophilic biofilm digester was sampled in accordance with objective 3. This digester was
begun with inoculum from the pilot plant. Both digesters were fed poultry litter substrate. The analysis of 16S rDNA libraries from the pilot plant continued. Following a year of growth, the biofilm and suspended cells in the digester were also sampled and 16S rDNA bacterial libraries were created for both samples. At the time of sampling, the biofilm reactor feed rate was 1 liter/day (20g/liter COD) and COD removal was 60-70%. More than 800 clones have been collected and 400 of these have been analyzed to date. Preliminary analysis has shown that the biofilm and suspended cell environments within the digester harbor similar, but distinct, microbial communities. The biofilm community contains about two times as much biodiversity as the suspended community. The Chao 1 diversity estimator projects that the biofilm contains 204 operational taxonomic units (OTUs) at the 97% similarity level, while the suspended cell environment contains 100 OTUs. The most abundant phylotypes are found
within the Bacillales, Clostridiales, and Bacteroidetes. The single largest group of phylotypes within both digesters can only be identified by the RDP as unclassified Firmicutes, indicating a large percentage of novel bacterial diversity in the CSTR and biofilm digesters.
Impacts
Anaerobic digesters contribute to environmental sustainability by stabilizing high organic content waste, such as farm and other agricultural residuals. The output of anaerobic digestion can be used as fertilizer and bioenergy (methane). The performance of digesters requires the metabolism of many species of bacteria that live in cooperative consortia. The majority of these bacteria are new to science and have unknown metabolisms. The maintenance and control of digesters requires managing these microbial populations. The standard operation of digesters involves monitoring biochemical variables such as volatile fatty acids and pH, but knowledge of the composition and dynamics of the majority of bacterial populations has never been accomplished. In order to develop predictive models of digester operation it is necessary to measure the activity of all of the major bacterial populations. This can be accomplished by applying DNA microarray technology which permits the
simultaneous detection of thousands of gene targets. This research will create DNA microarrays that permit comprehensive monitoring of the bacterial populations in a thermophilic anaerobic digester. This new tool will be applied to improving the operation and maintenance of digesters and will permit the comparison of different digesters to ascertain which key populations are needed for optimal and diverse digester applications.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
The overall objective of this research is to develop DNA microarrays for the functional analysis of microbial populations in thermophilic anaerobic digesters. The specific goals are to 1) expand an existing anaerobic digester 16S rDNA diversity collection, and include new functional gene diversity, 2) construct DNA microarrays that represent the phylogenetic and functional diversity of a methanogenic digester community, and 3) compare the biofilm microbial populations to the planktonic cell populations in a metabolically stable filter reactor using DNA microarrays. The digester utilized in this study is a pilot plant thermophilic anaerobic continuous stir tank reactor located on the campus of West Virginia State University. The digester is fed poultry litter. The first year of the grant focused on objective 1. A library of rpoB genes representing the microbial community of the pilot plant digester was created and sequenced. Two additional 16S rDNA libraries
representing the bacteria in the digester were also created and sequenced using universal primers. Rarefaction analysis of the 16S rDNA collection showed the presence of more than 60 operational taxonomic units at a level of 97% similarity. Phylogenetic analysis showed that the libraries were dominated by Firmicutes in the groups Clostridiaceae, Peptococcaceae, and unclassified Clostridiales. Additional families present were Lachnospiraceae, Peptostreptococcaceae, Syntrophomonadaceae, and Eubacteriaceae. A library of the methanogenesis operon gene mcrA was also created from digester DNA. The library contained clones within the methanogen genera Methanobacterium and Methanoculleus. A 15 liter biofilm anaerobic digester was set up and operated in order to study the microbial biofilm.
Impacts
Anaerobic digesters represent a managed ecosystem that is used for the bioconversion of organic waste into useful or benign products. Digester effluent has been demonstrated to be useful as fertilizer, and biogas (methane) produced by digesters can be used as an energy source. The performance of digesters requires the metabolism of many species of bacteria that live in cooperative consortia. The majority of these bacteria are new to science and have unknown metabolisms. The maintenance and control of digesters requires managing these microbial populations. The standard operation of digesters involves monitoring certain biochemical variables such as volatile fatty acids and pH, but knowledge of the composition and dynamics of the majority of bacterial populations has never been available. In order to develop predictive models of digester operation it is necessary to measure the activity of all of the major bacterial populations. This can be accomplished by applying DNA
microarray technology which permits the simultaneous detection of thousands of gene targets. This research will create DNA microarrays that permit comprehensive monitoring of the bacterial populations in a thermophilic anaerobic digester. This new tool will be applied to improving the operation and maintenance of digesters and will permit the comparison of different digesters to ascertain which key functional populations are needed for optimal and diverse digester applications.
Publications
- No publications reported this period
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