Final Report: Integrating Molecular and Biochemical Techniques to Characterize Adaptation Mechanisms of Anaerobic Microbial Communities Exposed to Chlorinated Organics.EPA Grant Number: R823351
Title: Integrating Molecular and Biochemical Techniques to Characterize Adaptation Mechanisms of Anaerobic Microbial Communities Exposed to Chlorinated Organics.
Investigators: Stahl, David A. , Rittmann, Bruce E.
Institution: Northwestern University
EPA Project Officer: Hahn, Intaek
Project Period: October 1, 1995 through September 30, 1998
Project Amount: $406,372
RFA: Exploratory Research - Environmental Biology (1995) RFA Text | Recipients Lists
Research Category: Biology/Life Sciences , Health , Ecosystems
Objective:Natural microbial communities often develop the ability to transform initially recalcitrant organic compounds. However, this process, referred to as adaptation, may be lengthy, requiring months or years to occur. Therefore, the persistence of some compounds in natural or engineered environments may be controlled by the adaptation period, as well as by the rate of biodegradation. Adaptation to recalcitrant substrates is currently difficult to predict or accelerate because the biological and chemical events contributing to this process in complex systems are not well understood.
The adaptation of native anaerobic microbial populations to chlorinated aromatic compounds exemplifies our lack of understanding of community adaptation mechanisms and the difficulties encountered in trying to elucidate them. Anaerobic biotransformation of a number of chlorinated aromatics in undefined enrichments has been observed, and several dehalogenating isolates have been obtained from a variety of anaerobic environments. However, biotransformation of aryl chlorides in complex samples is typically preceded by lengthy adaptation periods. Six isolates that are able to mediate aryl reductive dehalogenation in pure culture have been obtained, but the significance of these organisms in natural and engineered systems that are contaminated with chloroaromatic compounds is not known. Furthermore, in environmental systems, these organisms depend on syntrophic relationships with other community members that provide them with nonchlorinated electron donors, carbon sources, and growth factors. Thus, sustained mineralization of a chlorinated aromatic compound as the sole growth substrate requires the involvement of multiple organisms. Therefore, the adaptation of anaerobic microbial communities to chlorinated aromatic compounds is a particularly complex research topic that has many important applications in natural and engineered systems.
The overall goal of this research was to take the study of microbial community adaptation to pollutants beyond the "black box" approach, which has been used with limited success in the past. The approach taken in this research is to retain the microbial complexity of environmental systems, and at the same time, track and link changes in the concentrations of key populations and their functions by applying emerging molecular tools to overcome the limitations of culture-based techniques. Thus, there are three primary objectives of this research: (1) to develop innovative experimental approaches, which make it possible to link changes in community structure and function, for studying how microbial communities adapt to and transform pollutants; (2) to improve our understanding of the mineralization of 3-CB and 2-CP by complex anaerobic microbial communities; and (3) to advance our knowledge of how anaerobic microbial communities adapt to 3-CB and 2-CP. The significant progress made toward satisfying these three objectives is summarized below.
Summary/Accomplishments (Outputs/Outcomes):Two innovative experimental approaches were developed and applied in this research. A key feature of these experimental techniques is that they make it possible to link changes in community structure and function in complex microbial systems. The first innovative approach involved the following steps: 1) enrichment of populations involved in the mineralization of a chlorinated substrate, (2) identification of active populations using DNA-based evaluations (PCR and DGGE), and (3) rRNA-based quantification of the identified populations during the periods of adaptation and biotransformation. Enrichment of populations involved in the mineralization of the chlorinated substrates was accomplished either by repeatedly supplying the chlorinated substrate to a microbial community or by perturbing the community with a high concentration of a metabolite of the chlorinated substrate. For example, repeated additions of 3-CB for over 300 days and perturbations with high concentrations of benzoate resulted in the augmentation of Syntrophus-like populations (as inferred by 16S rRNA sequence relationship) in 3-CB-degrading sediment and digester sludge communities, respectively. The potential importance of the Syntrophus-like populations was recognized when they were detected using PCR amplification and DGGE. The use of rRNA-based evaluations revealed that changes in the concentrations of Syntrophus-like populations and 3-CB transformation in the digester sludge and sediment communities were indeed correlated. Thus, although these organisms have not been isolated, their phylogenetic affiliation and increased abundance associated with adaptation, suggest they are functionally similar to characterized proton reducing syntrophs.
The second innovative experimental approach involved applying integrated molecular evaluations and chemical measurements to microbial communities that were exposed to chlorinated substrates, during the periods of adaptation and biotransformation. Chemical measurements of the chlorinated substrates, metabolites, and products provided information about changes in the microbial community function and environmental conditions. The molecular evaluations, which included rRNA-based hybridizations, PCR amplification, and DGGE, made it possible to identify differences in the structures of communities that did and did not develop the ability to transform chlorinated substrates, track changes in the structure and diversity of a community over time, and link changes in community structure and function.
Several advances in our understanding of the mineralization of 3-CB and 2-CP by complex anaerobic microbial communities were achieved. DNA-based evaluations of 3-CB-degrading sediment communities revealed that the structures of these communities continued to change, even after being enriched on 3-CB for hundreds of days. In addition, these evaluations suggested that communities that have a common inoculum, but independently develop the ability to transform a chlorinated substrate, retain unique overall structures, yet share some common populations. Thus, the presence of the common populations may be required in order for a community to adapt to a specific chlorinated substrate.
Using a combination of DNA- and rRNA-based evaluations and chemical measurements, several populations that appear to be involved in the biotransformation of 3-CB and 2-CP in anaerobic sediment and sludge communities were identified. For example, rRNA-based evaluations and chemical measurements identified a correlation between Desulfovibrionaceae abundance and 2-CP transformation in the sediment community; however, the exact nature of this relationship was not elucidated. Two populations in the 2-CP transforming communities were also detected that are phylogenetically related to members of a co-culture reportedly responsible for the anaerobic transformation of phenol to benzoate. This co-culture consists of a Desulfotomaculum-type and a Clostridium-type species. Syntrophus- and/or Syntrophomonas-like populations also appeared to play an important role in the transformation of 2-CP in the sediment community, and, as previously mentioned, in the 3-CB-degrading digester sludge and sediment communities. These populations were most likely mediating fermentation of benzoate, which is a metabolite of 3-CB and 2-CP dehalogenation under anaerobic conditions. Molecular perturbation experiments and DNA-based evaluations identified populations closely related to the dehalogenating species Desulfomonile tiedjei (97-99% 16S rRNA sequence similarity) in both the sediment and digester sludge 3-CB-transforming communities. Chemical measurements and rRNA-based analyses clearly revealed that archaeal populations benefited from the biotransformation of 3-CB and 2-CP in the sediment community, and metabolic perturbation experiments suggested that methanogenic populations enhanced 3-CB transformation in the sediment community by providing a hydrogen sink.
These results suggested that key populations in the 3-CB-degrading digester sludge and sediment communities have the same general functions and interactions as the three members of a defined consortium that grows on 3-CB as it sole growth substrate (Dolfing and Tiedje, 1986). Briefly, in this consortium, D. tiedjei mediates reductive dehalogenation of 3-CB to benzoate, which is fermented to H2, CO2, and acetate by a syntrophic population. This reaction is thermodynamically feasible because a hydrogenotrophic methanogen and D. tiedjei maintain low H2 levels in the consortium.
Two other interesting observations regarding the mineralization of 3-CB and 2-CP in the sediment community were made. Chemical measurements and rRNA-based evaluations revealed that perturbations of the sediment community with 2-CP or 3-CB additions caused some populations to increase transiently. This suggests that some populations overshot, and subsequently corrected, their activity levels, in response to the chlorinated substrate additions. In addition, a previously unreported biotransformation pathway, the transformation of 2-CP to 3-CB, and the subsequent accumulation of 3-CB, was sometimes observed in the sediment community. This biotransformation is analogous to the production of benzoate from phenol, which presumably also occurred in the sediment community. The populations responsible for catalyzing the transformation of phenol to benzoate in the sediment community may also have mediated cometabolic transformation of 2-CP to 3-CB. The accumulation of 3-CB in 2-CP-transforming communities has important implications for the bioremediation of 2-CP-contaminated waste streams or environmental sites, under anaerobic conditions.
Several advances in our understanding of how anaerobic microbial communities adapt to 3-CB and 2-CP also were made in this research. The adaptation of the digester sludge and sediment communities to 3-CB and of the sediment community to 2-CP was highly reproducible. Therefore, it is unlikely that rare genetic changes contributed to the adaptation process in these systems. DNA-based evaluations suggested that selective enrichment of a D. tiedjei-like population played a role in the adaptation of the sludge community to 3-CB. However, rRNA-based evaluations failed to reveal a correlation between selective enrichment of a D. tiedjei-like population, or any other targeted populations, and adaptation to 3-CB or 2-CP. On the other hand, the rRNA-based evaluations suggested that selective enrichment of Syntrophus populations contributed to the adaptation of the sediment community to benzoate that was produced from the biotransformation of 2-CP. Finally, chemical measurements and metabolic perturbation experiments provided strong evidence that depletion of preferential organic substrates contributed to the adaptation of the digester sludge and sediment communities to 3-CB.