Sage R. Hiibel

Detection and Quantification of Functional Genes of Cellulose- Degrading, Fermentative, and Sulfate-Reducing Bacteria and Methanogenic Archaea

ABSTRACT Cellulose degradation, fermentation, sulfate reduction, and methanogenesis are microbial processes that coexist in a variety of natural and engineered anaerobic environments. Compared to the study of 16S rRNA genes, the study of the genes encoding the enzymes responsible for these phylogenetically diverse functions is advantageous because it provides direct functional information. However, no methods are available for the broad quantification of these genes from uncultured microbes characteristic of complex environments. In this study, consensus degenerate hybrid oligonucleotide primers were designed and validated to amplify both sequenced and unsequenced glycoside hydrolase genes of cellulose-degrading bacteria, hydA genes of fermentative bacteria, dsrA genes of sulfate-reducing bacteria, and mcrA genes of methanogenic archaea. Specificity was verified in silico and by cloning and sequencing of PCR products obtained from an environmental sample characterized by the target functions. The primer pairs were further adapted to quantitative PCR (Q-PCR), and the method was demonstrated on samples obtained from two sulfate-reducing bioreactors treating mine drainage, one lignocellulose based and the other ethanol fed. As expected, the Q-PCR analysis revealed that the lignocellulose-based bioreactor contained higher numbers of cellulose degraders, fermenters, and methanogens, while the ethanol-fed bioreactor was enriched in sulfate reducers. The suite of primers developed represents a significant advance over prior work, which, for the most part, has targeted only pure cultures or has suffered from low specificity. Furthermore, ensuring the suitability of the primers for Q-PCR provided broad quantitative access to genes that drive critical anaerobic catalytic processes.

Identification of stress-related proteins in Escherichia coli using the pollutant cis-dichloroethylene

Aims:  To complement our proteome study, whole‐transcriptome analyses were utilized here to identify proteins related to degrading cis‐1,2‐dichloroethylene (cis‐DCE).

Comparison of microbial community composition and activity in sulfate‐reducing batch systems remediating mine drainage

Five microbial inocula were evaluated in batch tests for the ability to remediate mine drainage (MD). Dairy manure (DM), anaerobic digester sludge, substrate from the Luttrell (LUTR) and Peerless Jenny King (PJK) sulfate‐reducing permeable reactive zones (SR‐PRZs) and material from an MD‐treatment column that had been inoculated with material from a previous MD‐treatment column were compared in terms of sulfate and metal removal and pH neutralization. The microbial communities were characterized at 0, 2, 4, 9, and 14 weeks using denaturing gradient gel electrophoresis and quantitative polymerase chain reaction to quantify all bacteria and the sulfate‐reducing bacteria of the genus Desulfovibrio. The cultures inoculated with the LUTR, PJK, and DM materials demonstrated significantly higher rates of sulfate and metal removal, and contained all the microorganisms associated with the desired functions of SR‐PRZs (i.e., polysaccharide degradation, fermentation, and sulfate reduction) as well as a relatively high proportion of Desulfovibrio spp. These results demonstrate that inoculum influences performance and also provide insights into key aspects of inoculum composition that impact performance. This is the first systematic biomolecular examination of the relationship between microbial community composition and MD remediation capabilities. Biotechnol. Bioeng. 2008;101: 702–713.

High frequency dielectrophoretic response of microalgae over time.

The high frequency dielectrophoresis (>20 MHz) response of microalgae cells with different lipid content was monitored over time. Chlamydomonas reinhardtii was cultured in regular medium and under nitrogen‐depleted conditions in order to produce populations of cells with low and high lipid content, respectively. The electrical conductivity of the culture media was also monitored over the same time. The upper crossover frequency decreased for high‐lipid cells over time. The single‐shell model predicts that the upper crossover frequency is dictated primarily by the dielectric properties of the cytoplasm. The high frequency DEP response of the high‐lipid cells’ cytoplasm was changed by lipid accumulation. DEP response of the low‐lipid cells also varied with the conductivity of the culture media due to nutrient consumption. Relative lipid content was estimated with BODIPY 505/515 dye by calculating the area‐weighted intensity average of fluorescent images. Finally, microalgae cells were successfully separated based on lipid content at 41 MHz and DEP media conductivity 106 ± 1 μS/cm.

Molecular assessment of the sensitivity of sulfate-reducing microbial communities remediating mine drainage to aerobic stress.

Sulfate-reducing permeable reactive zones (SR-PRZs) are microbially-driven anaerobic systems designed for the removal of heavy metals and sulfate in mine drainage. Environmental perturbations, such as oxygen exposure, may adversely affect system stability and long-term performance. The objective of this study was to examine the effect of two successive aerobic stress events on the performance and microbial community composition of duplicate laboratory-scale lignocellulosic SR-PRZs operated using the following microbial community management strategies: biostimulation with ethanol or carboxymethylcellulose; bioaugmentation with sulfate-reducing or cellulose-degrading enrichments; inoculation with dairy manure only; and no inoculation. A functional gene-based approach employing terminal restriction fragment length polymorphism and quantitative polymerase chain reaction targeting genes of sulfate-reducing (dsrA), cellulose-degrading (cel5, cel48), fermentative (hydA), and methanogenic (mcrA) microbes was applied. In terms of performance (i.e., sulfate removal), biostimulation with ethanol was the only strategy that clearly had an effect (positive) following exposure to oxygen. In terms of microbial community composition, significant shifts were observed over the course of the experiment. Results suggest that exposure to oxygen more strongly influenced microbial community shifts than the different microbial community management strategies. Sensitivity to oxygen exposure varied among different populations and was particularly pronounced for fermentative bacteria. Although the community structure remained altered after exposure, system performance recovered, indicating that SR-PRZ microbial communities were functionally redundant. Results suggest that pre-exposure to oxygen might be a more effective strategy to improve the resilience of SR-PRZ microbial communities relative to bioaugmentation or biostimulation.

MICROBIOLOGY OF SULFATE-REDUCING PASSIVE TREATMENT SYSTEMS 1

Little is known about the microbiology of passive mine drainage treatment systems, such as sulfate-reducing permeable reactive zones (SR-PRZs). We have recently developed a suite of molecular biology tools in our laboratory for characterizing the microbial communities present in SR-PRZs. In this study our suite of tools is used to characterize two different field bioreactors: Peerless Jenny King and Luttrell. Both bioreactors are located near the Ten Mile Creek Basin near Helena, MT, and both employ a compost-based substrate to promote the growth of sulfate-reducing bacteria (SRB) for production of sulfides and precipitation of metals. In summer, 2005, the reactors were sampled at multiple locations and with depth. DNA was extracted from the compost material and followed by cloning of polymerase chain reaction (PCR) amplified 16S rRNA genes, restriction digest screening, and DNA sequencing to provide insight into the overall composition of the microbial communities. To directly examine the SRB populations, a gene specific to SRB, apsA, was PCR-amplified, cloned, and sequenced. This revealed that Desulfovibrio spp. were prevalent in both Luttrell and Peerless Jenny King. At Peerless Jenny King, one Desulfovibrio spp. found was noted to be particularly aerotolerant. This analysis also revealed that Thiobacillus denitrificans were common at Peerless Jenny King. This is an organism that oxidizes sulfides in the presence of nitrate, which is undesirable for biozone function. In order to quantify SRB, quantitative real-time PCR (Q-PCR) was used targeting two specific groups of SRB, Desulfovibrio and Desulfobacteria. These results indicated that these two SRB groups, which have distinct substrate requirements, vary in distribution between the two bioreactors and with depth. It is hoped that an improved understanding of the microbiology of these systems will help to improve design and operation of passive treatment systems employing sulfate reduction.

Active community profiling via capillary electrophoresis single-strand conformation polymorphism analysis of amplified 16S rRNA and 16S rRNA genes

Here, we report the validation and advancement of a high-throughput method for fingerprinting the active members of a microbial community. This method, termed active community profiling (ACP), provides information about both the composition and the activity of mixed microbial cultures via comparative measurements of amplified 16S rRNA (RNA) and 16S rRNA genes (DNA). Capillary electrophoresis is used to resolve single-strand conformation polymorphisms of polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR) products, producing electropherograms representative of the community structure. Active members of the community are distinguished by elevated RNA:DNA peak area ratios. Chemostat experiments with defined populations were conducted to validate the ACP approach. Using a pure culture of Escherichia coli, a direct correlation was found between the growth rate and the RNA:DNA peak ratio. In a second validation experiment, a binary culture of E. coli and Pseudomonas putida was subjected to a controlled environmental change consisting of a shift to anaerobic conditions. ACP revealed the expected cessation of growth of P. putida, an obligate aerobe, while the corresponding DNA-only analysis indicated no change in the culture. Finally, ACP was applied to a complex microbial community, and a novel binning approach was demonstrated for integrating the RNA and DNA electropherograms. ACP thus represents a significant advance from traditional DNA-based profiling techniques, which do not distinguish active from inactive or dead cells, and is well suited for high-throughput community analysis.

COMPARISON OF INOCULA APPLIED IN THE REMEDIATION OF ACID MINE DRAINAGE BY SULFATE REDUCTION 1

Sulfate-reducing permeable reactive zones (PRZs), such as anaerobic wetlands, sulfate-reducing bioreactors, and permeable reactive barriers, are an attractive means of passively treating mining influenced waters contaminated with heavy metals. While the low cost and maintenance requirements are significant advantages of PRZs, the lack of clear design criteria is a disadvantage. It is not known why some systems will function for long periods of time without need for intervention, while others fail or do not recover well when exposed to stresses such as winter weather or other changes in conditions. This study explores the role of microorganisms in PRZs and the potential to use selected inocula to improve performance with respect to start-up time, sulfate-reducing activity level, and activity retention time. We have compared these attributes using various inocula, including: dairy manure, anaerobic digester sludge, acclimated column inoculum, and inoculum collected from two sulfate-reducing bioreactors operated in the field (Luttrell and Peerless Jenny King). Our results demonstrate that there are clear differences between the inocula and that the Luttrell bioreactor inoculum performs the best in terms of start-up time and overall activity. Sulfate concentrations, metal concentrations, and pH were measured in the aqueous phase to evaluate the ability of the different inocula to remediate acid mine drainage (AMD). In subsequent studies, DNA-based methods that profile the microbial community will be used to determine what kinds of microorganisms are present and to quantify key functional groups, including sulfate reducers, methanogens, and cellulose degraders. The ultimate goal will be to transfer these results to the field by developing the capability to intelligently design inocula for site-specific concerns.

Investigation of the Effect of Growth From Low to High Biomass Concentration Inside a Photobioreactor on Hydrodynamic Properties of Scenedesmus obliquus

Most of the current production cost in algae biodiesel plants utilizing photobioreactors comes from the high energy required for pumping, CO2 transfer, mixing, and harvesting. Since pumping affects the mixing and CO2 transfer, which are the main factors in algae productivities, solutions to reduce the required energy for pumps can significantly make algae biodiesel production more economically feasible. An investigation on the effect of Scenedesmus obliquus’s growth from low to high biomass concentration inside a horizontal tubular photobioreactor to determine the impact that it has on hydrodynamic performances, which will affect cost and production efficiency, was performed. As the biomass concentration increased, the algal culture was found to remain Newtonian. Additionally, the biomass concentration (expressed in cell density) was found to have lower viscosity even at the highest concentrations evaluated at 2.48 × 108 cell/ml (1.372 × 10−3 ± 1.32 × 10−4 Pa s) compared to the Modified Bold’s 3N medium (1.408 × 10−3 ± 9.41 × 10−5 Pa s). Furthermore, the total energy consumption does not appear to depend on the S. obliquus biomass concentrations, but rather on the medium the algae grows in. The rheological properties of autotrophic algae will not have significant impact on energy requirements until technology improves so that the concentrations reach those of heterotrophic algae.

EFFECT OF ORGANIC SUBSTRATE COMPOSITION ON MICROBIAL COMMUNITY STRUCTURE OF PILOT-SCALE BIOCHEMICAL REACTORS TREATING MINING INFLUENCED WATER 1

Mining-influenced water (MIW) is acidic, metal rich water formed when sulfide minerals react with oxygen and water. There are various options for the treatment of MIW; however, passive biological systems such as biochemical reactors (BCRs) have shown promise because of their low cost and maintenance requirements. The purpose of this study was to explore the effect of organic substrate on microbial communities present in pilot-scale BCRs treating MIW in order to understand how substrate-microbe interactions drive performance. Three organic substrates were evaluated: ethanol (ETOH); and two lignocellulose-based mixtures: hay and wood chips (HYWD), and corn stover and wood chips (CSWD). The microbial community compositions were characterized by cloning of 16S rRNA genes and apsA genes associated with sulfate reduction. Quantitative polymerase chain reaction (Q-PCR) was applied to quantify Desulfovibrio-Desulfomicrobium spp. and methanogens. Results revealed distinct differences in microbial compositions and relative quantities of total and sulfate- reducing bacteria (SRB) among the BCRs. In particular, the greatest proportion of SRBs were observed in the ETOH BCRs, but the total number of bacteria was low. The HYWD and CSWD BCRs had highly similar bacterial communities, which were complex in composition in comparison to the ETOH BCRs. Methanogens were found to be present in all BCRs at low levels and were the highest in the lignocellulose-based BCRs. This study demonstrates that substrate influences microbial community composition and diversity, which may play an important role in performance and reliability.