Optimization of Biogas Production by Use of a Microbially Enhanced Inoculum

A renewable energy source, biogas, comprises of methane (80%) and carbon dioxide (15%), and is a great alternative to the conventional fossil-based fuels, such as coal, gas and oil. Biogas is created during anaerobic biological digestion of waste materials, such as landfill material, animal manure, wastewater, algal biomass, industrial organic waste etc. A biogas potential from organic waste in the United States is estimated at about 9 million tons per year and technology allows capture of greenhouse gases, such as methane and carbon dioxide, into a form of a fuel. In the light of global climate change and efforts to decrease carbon footprint of fuels in daily life, usage of biogas as an alternative fuel to fossil fuels looks especially promising. The goal of this research was to develop and test an approach for optimization of biogas production by engineering microorganisms digesting organic waste. Specifically, bacteria that can digest algal biomass, collected from the wastewater lagoons or open waterbodies. The research also expands on the previous efforts to analyze microbial interactions in wastewater treatment systems. A computational model is developed to aid with prognosis of microbial consortia ability to form complex aggregates in reactors with upflow mode of feeding substrate. Combining modeling predictions and laboratory experiments in organic matter digestion will lead to more stable engineered systems and higher yields of biogas

Augmenting Anaerobic Digestion of Microalgal Biomass

Anaerobic digestion of microalgal biomass cannot be achieved without specialized hydrolytic microorganisms. Potentially algalytic bacteria belonging to Citrobacter and Alcaligenes species were isolated from a wastewater lagoon system. A combination of two potentially algalytic bacteria was successfully incorporated into the granular anaerobic consortia. A series of anaerobic cultures were prepared with different microbial combinations to test the methane production from algal biomass collected from a local wastewater treating trickling filter. The anaerobic microbial community mixed with two algalytic bacteria produced 10% more methane when compared to the methane produced by a native granular consortium. The presence of the algalytic bacteria of interest was confirmed by PCR using bacteria specific primers at the conclusion of the study. A computer-simulated model was designed to prove the possibility of incorporating algalytic bacteria into a mature methane-producing granule. Future research will address the anaerobic degradation potential of the modified granular consortia on other types of the microalgal biomass

Single-cell genomics of a candidate division TM6 - uncultured bacterial phylum in Zodletone Spring, Oklahoma

M.S.--University of Oklahoma,2014Includes bibliographical references (leaves 45-50).Previously, metagenomic studies of Zodletone Spring (south-western Oklahoma) revealed uncultured and poorly characterized bacterial phyla. Among them, a bacterial phylum "Candidate Division TM6" is present. It is widespread in a variety of natural environments and may contribute to geochemical cycling processes. We used a single-cell approach for targeting and separation of cells belonging to candidate division TM6. This approach includes design of specific primers, fluorescence in situ hybridization and fluorescence activated cell sorting, to obtain bacteria within this novel phylum. Extracted DNA from sorted cells was amplified using multiple displacement amplification and phylogenic analysis of sorted cells demonstrated the presence of candidate division TM6 DNA, based on 16SrRNA analysis. Amplified DNA was used for whole-genome sequencing on a MiSeq Illumina platform and generated 250 bp paired end reads. Genome de nova assembly was done using SPAdes 2.5.0 and Ray 2.2.0 algorithms, with subsequent phylogenetic binning of scaffolds into the TM6 assembly of 64,034 bp. Assembled genes were annotated on the RAST server and manually with the NCBI database. There were nine proteins encoded by assembled genes: ATP-dependent DNA helicase PcrA (EC 3.6.4.12), DNA polymerase III subunit gamma/tau (EC 2.7.7.7), transcriptional regulator OmpR, histidine kinase (EC 2.7.13.3), lysophospholipase (EC 3.1.1.5), cyanophycin synthase (EC 6.3.2.29), acetyltransferase 3 (EC 2.3.-.-), allophanate hydrolase (EC 3.5.1.54), RND multidrug efflux transporter and beta-lactamase (EC 3.5.2.6). All the proteins clustered distinctly from those in other different phyla. Given these results, we suggest that CD TM6 is able to use nitrogen, stored in a form of a cyanophycin polymer, by utilizing it in urea cycle

Exploiting Self-Organization in Bioengineered Systems: A Computational Approach

The productivity of bioengineered cell factories is limited by inefficiencies in nutrient delivery and waste and product removal. Current solution approaches explore changes in the physical configurations of the bioreactors. This work investigates the possibilities of exploiting self-organizing vascular networks to support producer cells within the factory. A computational model simulates de novo vascular development of endothelial-like cells and the resultant network functioning to deliver nutrients and extract product and waste from the cell culture. Microbial factories with vascular networks are evaluated for their scalability, robustness, and productivity compared to the cell factories without a vascular network. Initial studies demonstrate that at least an order of magnitude increase in production is possible, the system can be scaled up, and the self-organization of an efficient vascular network is robust. The work suggests that bioengineered multicellularity may offer efficiency improvements difficult to achieve with physical engineering approaches

Qualitative Analysis of Microbial Dynamics during Anaerobic Digestion of Microalgal Biomass in a UASB Reactor

Anaerobic digestion (AD) is a microbiologically coordinated process with dynamic relationships between bacterial players. Current understanding of dynamic changes in the bacterial composition during the AD process is incomplete. The objective of this research was to assess changes in bacterial community composition that coordinates with anaerobic codigestion of microalgal biomass cultivated on municipal wastewater. An upflow anaerobic sludge blanket reactor was used to achieve high rates of microalgae decomposition and biogas production. Samples of the sludge were collected throughout AD and extracted DNA was subjected to next-generation sequencing using methanogen mcrA gene specific and universal bacterial primers. Analysis of the data revealed that samples taken at different stages of AD had varying bacterial composition. A group consisting of Bacteroidales, Pseudomonadales, and Enterobacteriales was identified to be putatively responsible for the hydrolysis of microalgal biomass. The methanogenesis phase was dominated by Methanosarcina mazei. Results of observed changes in the composition of microbial communities during AD can be used as a road map to stimulate key bacterial species identified at each phase of AD to increase yield of biogas and rate of substrate decomposition. This research demonstrates a successful exploitation of methane production from microalgae without any biomass pretreatment

Microbial community dynamics in upflow anaerobic sludge blanket (UASB) reactor

Anaerobic sediment from Logan Lagoons has unique ability to degrade algal biomass. Sediment is comprised of hydrolytic and methanogenic bacterial communities with distinct metabolic preferences. These metabolic preferences result in specific microbial dynamics during the process of anaerobic digestion and biogas production in a laboratory scale upflow anaerobic sludge blanket (UASB) reactor, where each group of anaerobic microorganisms is responsible for a particular stage of digestion of algal biomass. Project is aimed at identification and characterization of microbial consortia in anaerobic sediments and monitoring of the microbial dynamics associated with the anaerobic digestion. Specific methods that are utilized include sequencing of a highly conserved 16S rRNA gene region in microorganisms and molecular characterization via fluorescent in situ hybridization. As a result, we should be able to observe a dynamic pattern of microbial growth regarding the stage of the anaerobic digestion - hydrolysis, acidogenesis, acetogenesis and methanogenesis. Obtained results will provide a roadmap for subsequent growing and isolation of microorganisms of interest to enhance anaerobic digestion and production of biogas from not only algal biomass, but also from various organic wastes, such as pig manure and brewery wastewater

An Experimentally Evaluated Thermodynamic Approach to Estimate Growth of Photoheterotrophic Purple Non-Sulfur Bacteria

Distribution of energy during the growth and formation of useful chemicals by microorganisms can define the overall performance of a biotechnological system. However, to date, this distribution has not been used to reliably predict growth characteristics of phototrophic microorganisms. The presented research addresses this application by estimating the photon-associated Gibbs energy delivered for the photoheterotrophic growth of purple non-sulfur bacteria and production of dihydrogen. The approach is successfully evaluated with the data from a fed-batch growth of Rhodopseudomonas palustris nifA∗ fixing N2 gas in phototrophic conditions and a reliable prediction of growth characteristics is demonstrated. Additionally, literature-available experimental data is collected and used for evaluation of the presented thermodynamic approach to predict photoheterotrophic growth yields. A proposed thermodynamic framework with modification to account for the phototrophic growth can be used to predict growth rates in broader environmental niches and to assess the possibility for the development of novel biotechnological applications in light-induced biological systems

Development of Archaeal and Algalytic Bacteria Detection Systems

Natural gas (methane) is emerging as a viable power source for many industrial, commercial, and domestic applications. Bio-methane provides a promising replacement for mined natural gas. Methanogenic bacteria produce this bio-methane. These anaerobic bacteria pertain to the Domain Archaea, and are found in extreme environments where few other bacteria survive. They are employed by Up-Flow Anaerobic Sludge Blanket (UASB) reactors in the digestion of wastes to a marketable product (methane). The genome of methanogenic bacteria can be amplified using polymerase chain reaction (PCR), a synthetic DNA replication system. This system employs specific sequences of DNA called primers. The primers employed in this study focused on 16S rRNA amplification providing a fingerprint of the organism’s identity. Previous design of these primers was unsuccessful and resulted in non-specific binding

Qualitative Analysis of Microbial Dynamics during Anaerobic Digestion of Microalgal Biomass in a UASB Reactor

Anaerobic digestion (AD) is a microbiologically coordinated process with dynamic relationships between bacterial players. Current understanding of dynamic changes in the bacterial composition during the AD process is incomplete. The objective of this research was to assess changes in bacterial community composition that coordinates with anaerobic codigestion of microalgal biomass cultivated on municipal wastewater. An upflow anaerobic sludge blanket reactor was used to achieve high rates of microalgae decomposition and biogas production. Samples of the sludge were collected throughout AD and extracted DNA was subjected to next-generation sequencing using methanogen mcrA gene specific and universal bacterial primers. Analysis of the data revealed that samples taken at different stages of AD had varying bacterial composition. A group consisting of Bacteroidales, Pseudomonadales, and Enterobacteriales was identified to be putatively responsible for the hydrolysis of microalgal biomass. The methanogenesis phase was dominated by Methanosarcina mazei. Results of observed changes in the composition of microbial communities during AD can be used as a road map to stimulate key bacterial species identified at each phase of AD to increase yield of biogas and rate of substrate decomposition. This research demonstrates a successful exploitation of methane production from microalgae without any biomass pretreatment

Modeling De Novo Granulation of Anaerobic Sludge

Background: A unique combination of mechanical, physiochemical and biological forces influences granulation during processes of anaerobic digestion. Understanding this process requires a systems biology approach due to the need to consider not just single-cell metabolic processes, but also the multicellular organization and development of the granule. Results: In this computational experiment, we address the role that physiochemical and biological processes play in granulation and provide a literature-validated working model of anaerobic granule de novo formation. The agent-based model developed in a cDynoMiCs simulation environment successfully demonstrated a de novo granulation in a glucose fed system, with the average specific methanogenic activity of 1.11 ml CH4/g biomass and formation of a 0.5 mm mature granule in 33 days. The simulated granules exhibit experimental observations of radial stratification: a central dead core surrounded by methanogens then encased in acidogens. Practical application of the granulation model was assessed on the anaerobic digestion of low-strength wastewater by measuring the changes in methane yield as experimental configuration parameters were systematically searched. Conclusions: In the model, the emergence of multicellular organization of anaerobic granules from randomly mixed population of methanogens and acidogens was observed and validated. The model of anaerobic de novo granulation can be used to predict the morphology of the anaerobic granules in an alternative substrates of interest and to estimate methane potential of the resulting microbial consortia. The study demonstrates a successful integration of a systems biology approach to model multicellular systems with the engineering of an efficient anaerobic digestion system