Biodegradative Processes and Biological Waste Treatment - ACS

Chapter 2

Biodegradative Processes and Biological Waste Treatment Analysis and Control 1

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Gary S. Sayler and James W. Blackburn 1

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Downloaded by UNIV LAVAL on May 4, 2016 | http://pubs.acs.org Publication Date: July 13, 1990 | doi: 10.1021/bk-1990-0433.ch002

Center for Environmental Biotechnology and Department of Chemical Engineering, University of Tennessee, Knoxville, TN 37932

Opportunities exist to dramatically enhance the extent and kinetics of biodegradation of agricultural, industrial and domestic wastes through an integrated systems approach. Such a systems approach combines the strengths of modern molecular biology, and ecological and engineering science to achieve new levels of process understanding and control that can be applied to the development and optimization of efficient biodegradation systems for a variety of wastes. A fundamental research agenda consisting of microbial strain development, bioanalytical monitoring methods and environmental and reactor systems analysis has been identified as a critical framework for the integration of science and engineering disciplines to achieve process optimization. Among the available methods to provide needed quantitative monitoring of critical biodegradative populations, gene probe technology has been shown to be particularly useful. This technology has been successfully integrated with frequency response analytical techniques in developing a new system analysis protocol for biodegradation process control. A new bio-analytical method that utilizes engineered bioluminescent bacteria has also been developed as a remote, on-line sensor of biodegradative activity in waste treatment processes. The coupling of new measurement technology with system analytical methods provides new insight for process design and operation and prediction of optimal regimes of biodegradation performance. 0097-6156/90/0433-0013$06.00/0 © 1990 American Chemical Society

Glass and Swift; Agricultural and Synthetic Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1990.


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AGRICULTURAL AND SYNTHETIC POLYMERS

The abilities of microorganisms to degrade the vast majority of natural and manmade organic substances is well known and is the basis of domestic and industrial waste treatment practice, composting technology, biomass conversion, major carbon cycling components, pesticide degradation and problem soils, and environmental remediation and restoration of contaminated areas (1). Often major problems arise when rates of degradation or conversion occur much slower than accumulation of the organic substrate, such as the case of solid waste accumulation in cattle feedlot operations, or occur too slowly to make commercial fermentations profitable such as biomass to methane. Some of these rate limitations may be due to an inherent biological resistance to degradation, such as hydrolytic depolymerization of polymers, like cellulose, lignin, nylon, (ΖΛ) etc. or the fact that sufficient numbers of active microbes or nutrients cannot be brought into contact rapidly enough to process the organic material (4). Recent advances in genetic engineering may contribute to partial solutions of these problems in terms of developing more versatile and active organisms for degradation processes. However, by itself genetic engineering cannot solve ecological and engineering problems that may ultimately control rate limiting processes. There are major opportunities for integrated biological, engineering and ecological research in order to develop truly effective, predictable and safe applications of biotechnology for chemical control and waste treatment. Collectively, the development, application and control of biological processes for waste treatment is an integral component of environmental biotechnology (£). Operationally, environmental biotechnology for hazardous wastes must proceed as an integrated science and engineering effort in order to achieve successful lab to field scale-up operation. However, such an integrated research strategy is seldom available on the national scene. The resulting limitations for hazardous waste control have been documented by the Office of Technology Assessment (£) and by a recent NSF Environmental Biotechnology Research Planning Workshop (5). From this workshop a consensus research agenda has been formulated that encompasses the major research needs in environmental biotechnology. This agenda is described in Table 1. While research accomplished in any one of the areas in the agenda will contribute to advancing technology for hazardous waste control; major advances are anticipated by interfacing both science and engineering research across the research agenda. This is particularly important because of the rapid advances made in the molecular understanding of the genetic elements and pathways involved in biodégradation, and an unprecedented ability to utilize genetic engineering technology to develop new and improved biodegradative microorganisms (2), which in itself may be an object of federal regulatory oversight (8.9). The objective of this report is to summarize some of the modern molecular approaches for the analysis of microbial populations involved in biodegradative processes and to examine the applications for systems analysis protocols that contribute to better processes control and optimization.


2. SAYLER AND BLACKBURN


Table 1.

Biological Waste Treatment

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Fundamental components for a research agenda leading to the development of effective Environmental Biotechnology for hazardous wastes

Agent (Strain) Development * Source and Selection * Characterization * Modification and Improvement * Model Systems (mixed and pure) * Applications * Evolutionary Relationships/Diversity * Stress Responses * Collections and Libraries Process and System Analytical Tools * Quantitative Analytical Techniques (chemical/physical measurements) * Bio-analytical Methods * Molecular Analysis Methods * Monitoring Applications * Remote Sensing * Biomonitors * Reporter-Signal Analysis (structure and function) Environmental System Analyses * Ecological Interactions * Environmental Fate and Abiotic Processes * Population Dynamics (organisms and genes) * Environmental Stability * Determination of Kinetic Parameters * Micro-habitats (niche invasions) * Organismal or Genetic Mobility * Controllability and Environmental Modification * Stress-induced Effects Reactor System Analysis * Reactor Design * Transient Outcomes and Perturbations * Dynamic Analysis * Ecological Interactions * System Stability and Component Stability * Online Analysis and Control * Kinetic Parameters Analysis Science/Economic Policy Analysis * Regulatory and Other Constraints to Application * Costs and Benefits of Competitive Cleanup Technologies * Potential Risks and Liabilities



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Molecular Tools. The application of biodegradative processes in control of environmental contaminants is ultimately determined by understanding and controlling microbial community structure and activity. Since microbial communities are heterogenous and composed of both biodegradative and non-degradative populations, specific and quantitative methods are needed to predict the genetic potential of a community to degrade specific contaminants, and to evaluate controlled interventions designed to establish or enhance biodegradative activity. Successful application of biological processes for bioremediation and control of hazardous environmental contaminants is dependent on the understanding and control of the structure and activity of biodegradative microbial communities. Because these communities are complex mixtures of microbes involved in degradation of specific toxicants (7), as well as microbes involved in biogeochemical transformations of non- anthropogenic materials, there are major needs to develop and apply specific and quantitative monitoring technology to discriminate those organisms whose specific role is the removal of contaminants of environmental concern (7.8). This apparent need is further reinforced by the development of improved biodegradative microorganisms (10), by classical methods or genetic engineering techniques, that are intended for bioaugmentation purposes and process development. In addition, the whole area of biostimulation presupposes that naturally occurring biodegradative organisms and processes can be selectively enhanced to achieve more efficient rate of biological transformation under environmental contaminants. Occurring simultaneously with increases in the molecular understanding of biodegradative processes has been the development of new molecular analytical monitoring techniques that permit both sensitive and specific quantitation of microbial populations involved in biodégradation (Table 2). Many of these techniques offer advantages over more conventional measures of microbial population abundance and activity. In general, they are best applied in combination with conventional enumeration methods to develop new information on the fate and dynamics of individual populations. The concern over recombinant DNA (rDNA)-containing or genetically engineered microbes (GEM) in the environment has driven the search for gene-or DNA-specific molecular monitoring methods to provide information on the fate, persistence, amplification, and transfer of genes or rDNA sequences in the environment^). This is precisely the same information needed to monitor and control genes involved in biodégradation processes relative to long term system optimization. Over the past decade, a variety of new techniques have emerged in the field of molecular biology that have demonstrated great utility in developing more sophisticated and specific approaches for the analysis of microbial communities(Table 2). These approaches include new immunological and protein analysis techniques, as well as methods to directly analyze the genetic structure and information of microbial systems. Plasmid and nucleic acid recovery and analysis methods when integrated with genotype specific, nucleic acid hybridization techniques have shown great utility in enhancing our understanding of the biodegradative potential of microbial communities and the response of these communities to engineering practice designed to promote in situ or reactor



Table 2.


Some example molecular methods offering improved specificity and sensitivity for quantifying biodegradative organisms or genes in environmental samples

Currently Available Technology Downloaded by UNIV LAVAL on May 4, 2016 | http://pubs.acs.org Publication Date: July 13, 1990 | doi: 10.1021/bk-1990-0433.ch002

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Immunological Techniques Fluorescent Antibodies Enzyme-linked immunosorbant assays Nucleic Acid Analysis Techniques rRNA sequence analysis DNA/RNA probe technology Plasmid analysis Restriction digestion RFLP analysis Genetic Reporter Strain Techniques Bioluminescent strain analysis Selectable phenotypic markers Analytical Chemical Techniques Signature fatty acid analysis Pyrolysis GC/MS analysis

(See Ref. 9 for specific examples.)



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level biodégradation. Nucleic acid hybridization (gene probe) detection technology can also be used successfully to rapidly screen microbial populations for organisms with degradative genes and the eventual recovery of organisms with new or enhanced biodegradative activity. Such organisms represent new source material for bioaugmentation and strain improvement using classical selection or genetic engineering technology. Technologies that focus on the specific nucleic acids responsible for the degradative capacity of a biological process hold promise for predicting the genetic potential of a biodegradative system. One such example may be plasmid fingerprinting. Plasmids, which are extra chromosomal genetic elements, are often associated with specific biodegradative pathways (7.11). Many of these plasmid can be readily isolated from bacteria involved in biodégradation and similar plasmids can be diagnosed by digesting the plasmid DNA with specific restriction endonucleases (DNA cutting enzymes that recognize particular nucleic acid base pair sequences) and examining the molecular weight distribution of the resulting DNAfragmentsusing electrophoresis techniques. Such an example is presented in Figure 1 for a plasmid associated with 4-chlorobiphenyl degradation. Such information can be used to determine if a unique plasmid is present and is stably maintained in microorganisms promoting a particular degradative process. While such techniques are good for molecular characterization and ecological monitoring for genetic elements in biodegradative processes they can be difficult to apply to real time, quantitative process monitoring. As a group, nucleic acid hybridization techniques and sequence analysis provide molecular monitoring for dynamic populations and/or genes involved in biodégradation (12.13.14.15V Potentially hybridization technology can be applied to real time, quantitative process monitoring. The strategy of nucleic acid hybridization as a monitoring technology is described in Figure 2 (9). Fundamentally, target DNA (unknown) from bacterial colonies or DNA extracted from environmental samples (16.17) is denatured from a double stranded to a single stranded form and conveniently bound to a solid support such as a nylon membrane. At an appropriate buffer and temperature condition, a known probe DNA (also in single stranded form) is added to the reaction mixture. Under the proper conditions, the single stranded probe DNA forms base pair hydrogen bonds with homologous complementary base sequences of the target DNA; if these sequences are present in the unknown target DNA. This process of hybridization or reassociation of the single stranded probe DNA and single stranded complementary target DNA results in the restoration of a double stranded hybrid DNA molecule. In order to detect and discriminate these positive hybrid molecules, it is necessary to have a detectable tag or label on the probe DNA. This is accomplished by cross-linking various fluorescent, chromogenic, enzymatic or immunological reagents to the probe DNA or by biochemical synthesis of radio-isotopically labeled DNA. While the non-isotopic detection methods offer many advantages and are rapidly progressing, isotopically labeled DNA probes (most often labeled with ^P), are the most sensitive and generally used. Detection of the positive hybrids, indicating the presence specific DNA




SAYLER AND BLACKBURN

Figure 1. Restrictionfragmentfingerprint for the 4-chlorobiphenyl catabolic plasmid pSS50 determined by agarose gel electrophoresis. (Lanes:A, k Hind III standard; B, undigested; C-H enzymatic digests of pSS50, C, Esq RI; D, Bam HI; E , Hind III; F, EÇQ RI and l a m HI; G , EÇQ RI and Hind III; H,flifldΙΠ and £am HI.) Glass and Swift; Agricultural and Synthetic Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1990.


Organismal sample

Isolated DNA

Lyse organisms and release DNA

Probe hybridizes to complementary DNA under controlled conditions

Figure 2. The general use of DNA probes to detect biodegradative genes in target bacterial colonies or DNA extracts. (Reprinted with permission from Ref. 9. Copyright 1988 CRC.)

Add labeled Nucleic Acid probe

Prehybridize to reduce interference and non-specific binding

Wash away excess probe and detect positive hybrids

Denature to separate the strands and fix to membrane

c l i O f S >>4 Yi. Λ

Transfer organisms on hybridization membrane

Cultivate or

Directly deposit DNA


id Ο r




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sequence in the unknown target DNA mixture, is easily quantitated by liquid scintillation spectrometry or by radioautography when employing ^ labeled DNA probes (2). TTiis technology has found wide applications for detection of bacterial populations and individual species in the environment or in waste treatment systems that contain specific genes for catabolism of environmental contaminants (12.14.15). The technology is easily integrated with conventional microbiological cultivation, plating and enumeration procedures to both enumerate bacterial colonies containing degradative genes of interest (12.14.16) and recovery of pure cultures of degradative organismsQS). A typical example of detecting the genes for naphthalene degradation in bacterial colonies from a mixed culture is given in Figure 3. In this example ^P-labeled probe DNAfroma Pseudomonas putida naphthalene catabolic plasmid was used to demonstrate that Bacteria capable of naphthalene degradation predominated in the inoculum of a continuous stirred soil slurry biotreatment reactor. Another example application of this technology is to estimating the genetic potential of bacterial populations in subsurface soils at a Manufactured Gas Plant (MGP) site to degrade aromatic hydrocarbons. Again DNA specific for naphthalene degradation was used as a ^-labeled probe to determine which bacterial colonies contained gene sequences for naphthalene degradation and to determine relative concentration of these bacteria at different depths in contaminated MGP soils. Table 3 indicates the occurrence and differences naphthalene degradative cell densities in subsurface-contaminated MGP soils as determined by plate count enumeration and DNA colony hybridization. Thus far, these examples have demonstrated the use of DNA probe technology to detect catabolic genes in bacterial colonies that have been cultured by conventional microbiological methods. Since the technology is a molecular technology, there is no requirement that colonies of organisms must be used as the source of target DNA. In fact, it is now possible to directly extract total DNA directlyfroman environmental sample or waste treatment population and to use this total DNA as a target to directly determine the frequency or abundance of catabolic genes in a given environment (14.16.17). In this case aliquots of DNA from an environmental sample are bound in single-stranded form to the hybridization membrane as a blot of DNA. This DNA target is probed with an appropriately labeled catabolic DNA probe. Figure 4 is an example of this approach where DNA has been extracted directly from chemically contaminated reservoir sediments and probed directly for the qualitative abundance of genes associated with 4-chlorobiphenyl degradation (plasmid pSS50). Recently, this technology has been advanced in detection sensitivity using polymerase chain reaction (PCR) amplification of specific DNA sequences recovered in environmental extracts (12). PCR is an enzymatic in vitro method for exponentially copying specific sequences in a complex DNA sample. Theoretically 1 gene or DNA fragment represented as single




Figure 3. Autoradiographic detection of naphthalene degradative bacterial colonies from MGP soil enrichments used as inoculum for continuous stirred soil slurry bioreactors.

Figure 4. Comparative detection of 4-chlorobiphenyl catabolic gene abundance by blot hybridization of pSS50 plasmid DNA probe to target DNA extracted from sediments. A & Β spatially separated sediments; C control self hybridizations. (Units A & B, ug total DNA; C ng of probe DNA.)



Table 3.


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DNA probe detection and distribution of naphthalene biodegradative bacteria in Manufactured Gas Plant soils


Culturable Microorganisms

Sample Depth (ft)

YEPGA

Total

7

Biodegradative Processes and Biological Waste Treatment - ACS

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