Robert F. Hickey

Flexible Community Structure Correlates with Stable Community Function in Methanogenic Bioreactor Communities Perturbed by Glucose

ABSTRACT Methanogenic bioreactor communities were used as model ecosystems to evaluate the relationship between functional stability and community structure. Replicated methanogenic bioreactor communities with two different community structures were established. The effect of a substrate loading shock on population dynamics in each microbial community was examined by using morphological analysis, small-subunit (SSU) rRNA oligonucleotide probes, amplified ribosomal DNA (rDNA) restriction analysis (ARDRA), and partial sequencing of SSU rDNA clones. One set of replicated communities, designated the high-spirochete (HS) set, was characterized by good replicability, a high proportion of spiral and short thin rod morphotypes, a dominance of spirochete-related SSU rDNA genes, and a high percentage ofMethanosarcina-related SSU rRNA. The second set of communities, designated the low-spirochete (LS) set, was characterized by incomplete replicability, higher morphotype diversity dominated by cocci, a predominance of Streptococcus-related and deeply branching Spirochaetales-related SSU rDNA genes, and a high percentage of Methanosaeta-related SSU rRNA. In the HS communities, glucose perturbation caused a dramatic shift in the relative abundance of fermentative bacteria, with temporary displacement of spirochete-related ribotypes byEubacterium-related ribotypes, followed by a return to the preperturbation community structure. The LS communities were less perturbed, with Streptococcus-related organisms remaining prevalent after the glucose shock, although changes in the relative abundance of minor members were detected by morphotype analysis. A companion paper demonstrates that the more stable LS communities were less functionally stable than the HS communities (S. A. Hashsham, A. S. Fernandez, S. L. Dollhopf, F. B. Dazzo, R. F. Hickey, J. M. Tiedje, and C. S. Criddle, Appl. Environ. Microbiol. 66:4050–4057, 2000).

Bacterial community fingerprinting of amplified 16S and 16–23S ribosomal DNA gene sequences and restriction endonuclease analysis(ARDRA)

The 16S and 23S rRNA genes have been utilized for phylogenetic analysis of both prokaryotic and eukaryotic organisms (see Section 3). In addition to direct comparison of the nucleic acid sequences [9], numerous groups have used the rapid method of polymerase chain reaction (PCR) amplification of this gene [6] as well as the complete rRNA locus [3, 4, 8] for a simple method for identification of bacterial genera and species. In these latter procedures, the amplified ribosomal gene (rONA) is subjected to restriction endonuclease digestion; this has been termed ARDRA (Amplified Ribosomal DNA Restriction Analysis [8]). The resulting restriction fragment pattern is then used as a fingerprint for the identification of bacterial genomes. This method is based on the principle that the restriction sites on the RNA operon are conserved according to phylogenetic patterns.

Parallel Processing of Substrate Correlates with Greater Functional Stability in Methanogenic Bioreactor Communities Perturbed by Glucose

ABSTRACT Parallel processing is more stable than serial processing in many areas that employ interconnected activities. This hypothesis was tested for microbial community function using two quadruplicate sets of methanogenic communities, each set having substantially different populations. The two communities were maintained at a mean cell residence time of 16 days and a mean glucose loading rate of 0.34 g/liter-day in variable-volume reactors. To test stability to perturbation, they were subjected to an instantaneous glucose pulse that resulted in a 6.8-g/liter reactor concentration. The pattern of accumulated products in response to the perturbation was analyzed for various measures of functional stability, including resistance, resilience, and reactivity for each product. A new stability parameter, “moment of amplification envelope,” was used to compare the soluble compound stability. These parameters indicated that the communities with predominantly parallel substrate processing were functionally more stable in response to the perturbation than the communities with predominantly serial substrate processing. The data also indicated that there was good replication of function under perturbed conditions; the degrees of replication were 0.79 and 0.83 for the two test communities.

Monitoring of the anaerobic methane fermentation process.

The anaerobic methane fermentation process is being used more frequently for the treatment of liquid wastes. This is primarily due to the newer technologies that are able to realize the advantages of anaerobic treatment while minimizing the detriments. One obstacle remaining that would further increase the usage of these processes involves the need to monitor the process. Traditional indicators are adequate for gradual changes and for detecting process upsets once they are underway. However, the newer technologies are high-rate systems, and for these systems, shock overloads and toxic doses require prompt corrective responses in order to avoid process failure. Thus, along with the newer technologies have come new process indicators for monitoring the anaerobic methane fermentation process. An ideal indicator would be one that is easy to measure, related to the current metabolic status of the system, and perhaps available for on-line measurement. This paper will review the indicators that have been proposed for monitoring the anaerobic processes.

Biological activated carbon in fluidized bed reactors for the treatment of groundwater contaminated with volatile aromatic hydrocarbons

Abstract A comparison of fluidized bed reactor systems with (1) adsorptive removal capacity only using granular activated carbon (GAC) without microbial growth, (2) combined biological and adsorptive removal mechanisms using GAC with microbial growth and (3) biological removal only using nonactivated carbon with microbial growth was performed. These three systems were fed groundwater contaminated with benzene, toluene and xylene (BTX). The breakthrough profiles, steady-state removal of BTX and system responses to step increases in applied organic loading rates were investigated. During start-up, even though the same amount of inoculum was added to the two biological systems, the time required until effective biodegradation commenced in the system employing GAC as a biomass carrier was less than that observed for the system using non-activated carbon (200 vs 500 h). Significantly less BTX was released during this period by the system with combined removal mechanisms and the development of a contiguous biofilm was more rapid. Under constant, steady state, organic loading conditions (3 and 6 kg-COD/m 3 -day), BTX removals were similar for the two biological systems, although the system employing the GAC carrier had lower effluent concentrations at the lower loading rate. More than 90% of the BTX were removed in both systems. During step increases in organic loading, however, the combination of biological and adsorptive removal capacity resulted in enhanced BTX removal and more stable operation. Scanning electron microscopy was used to examine the extent of surface coverage of the GAC and non-activated carbon by the biofilm. Particles from both systems were observed to be completely covered by a contiguous, thick biofilm.

Stimulating the anaerobic biodegradation of explosives by the addition of hydrogen or electron donors that produce hydrogen

The anaerobic biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), and 2,4,6-trinitrotoluene (TNT) by a methanogenic mixed culture was investigated. Microcosms containing a basal medium and the mixed culture were amended with ethanol, propylene glycol (PG), butyrate or hydrogen gas as the electron donor and a mixture of TNT (50 microM), RDX (25 microM), and HMX (8 microM). After 29 days TNT and RDX were completely transformed to unidentified endproducts in the bottles amended with ethanol, hydrogen, or PG, while 53%, 40%, and 22% of the HMX was transformed, respectively. There was no loss of RDX or HMX in the electron donor unamended control bottles. The ethanol and PG were transformed to near stoichiometric amounts of acetate and propionate, suggesting the immediate electron donor supporting the transformation of the explosives was the H2 evolved during the metabolism of the parent substrate. Our findings suggest that the addition of H2 or electron donors that produce H2 may be a useful strategy for enhancing the anaerobic biodegradation of explosives in contaminated groundwater and soils.

Anaerobic treatment of pinkwater in a fluidized bed reactor containing GAC

Pinkwater is generated during the handling and demilitarization of conventional explosives. This listed hazardous waste contains dissolved trinitrotoluene (TNT) and cyclo trimethylene trinitramine (RDX), as well as some by-products. It represents the largest quantity of hazardous waste generated by the operations support command, and its treatment produces a by-product hazardous waste--spent granular activated carbon (GAC). Anaerobic treatment in a fluidized bed reactor (FBR) containing GAC is an emerging technology for organic compounds resistant to aerobic biological treatment. Bench scale batch studies using an anaerobic consortium of bacteria fed ethanol as the sole electron donor demonstrated the transformation of TNT to triaminotoluene (TAT), which then degrades to undetectable end products. RDX is sequentially degraded to nitroso-, dinitroso-, trinitroso- and hydroxylaminodinitroso-RDX before the triazine ring is presumably cleaved, forming methanol and formaldehyde as major end products. The bacterial members of the anaerobic consortia are typically found in sludge digesters at municipal or industrial wastewater treatment plants. The results of a pilot scale evaluation of this process that was conducted at McAlester Army Ammunition Plant (MCAAP, OK) over a 1 year period are reported in this paper. The pilot test experienced wide fluctuations in influent concentrations, representative of true field conditions. The FBR was a 20 in. (51 cm) diameter column with an overall height of 15 ft (4.9 m) and a bed of GAC occupying 11 ft (3.4m). Water was recirculated through the column continuously at 30 gpm (114 l/min) to keep the GAC fluidized, and pinkwater for treatment was pumped into the recirculation line. Several flowrates were evaluated to determine the proper mass loading rate (mass of TNT and RDX per reactor volume per time, kg/m(3) per day) which the reactor could handle while meeting the discharge limitations. Based on the tests performed, a 1 gpm (3.785 l/min) rate in the 188 gal (710 l) volume of the fluidized GAC bed was determined to consistently meet the discharge requirements. This information was used to develop a cost estimate for a system capable of treating the total effluent currently produced at MCAAP. The cost of installing and operating this system was compared to the cost of GAC adsorption for MCAAP at current pinkwater generation rates. The GAC-FBR system had an annual operating cost of approximately US

Effects of a long-term periodic substrate perturbation on an anaerobic community

19K, compared to US

Changes in bacterial community structure correlate with initial operating conditions of a field-scale denitrifying fluidized bed reactor.

71 K annually for GAC adsorption. When including the amortization of the capital equipment required for the GAC-FBR, the payback period for installation of this new process was estimated at 3.7 years.

Long-term evaluation of adsorption capacity in a biological activated carbon fluidized bed reactor system

The response of a glucose-fed anaerobic community to a long-term (>400 days) periodic substrate perturbation was examined. A completely mixed “mother” reactor operated at steady state with a 10-day hydraulic retention time (HRT) and constant 8 g/liter glucose feed served as the source of organisms for a “daughter” reactor. The daughter reactor was initially operated in the same manner as the mother reactor, i.e. at a 10-day HRT and a constant 8 g/liter glucose feed. After reaching steady state, the daughter reactor was subjected to a periodic organic loading pattern in which the influent glucose concentration was alternately varied from 16 to 0 g/liter (mineral media only) on a 6-day cycle. A rapid accumulation in glucose fermentation intermediates occurred immediately after initiating the perturbation. Effluent chemical oxygen demand (COD) increased from ca 400 to a peak level of 6000 mg/liter, methane content of the biogas decreased from 50 to less than 20% and the pH decreased from 7.0 to 6.2. This was followed by a 230-day metastable “steady state” during which the effluent COD concentration fluctuated about a new median value of 5000 mg/liter and the acetate and butyrate concentrations varied in a cyclic, inverse pattern. At the end of the metastable period, volatile fatty acid concentrations decreased rapidly. Within ca 40 days, a new steady state was established. These observations indicate that the anaerobic system was able to adapt to the periodic substrate perturbation through a long-term change in microbial community.