Environ. Sci. Technol. 1987, 21, 884-890
Molecular Microbial Ecology of a Naphthalene-Degrading Genotype in Activated Sludge James W. Blackburn" Graduate Program in Ecology and Department of Chemical Engineering, The University of Tennessee, Knoxville, Tennessee 37996
Rakesh
K. Jaln
Department of Microbiology, The University of Tennessee, Knoxville, Tennessee
Gary
37996
S. Sayler
Department of Microbiology and Graduate Program in Ecology, The University of Tennessee, Knoxville, Tennessee
The concentration of cells in an activated sludge system containing a gene known to participate in degradation of naphthalene was experimentally related to the biotransformation and mineralization of naphthalene. The gene probe analysis for the naphthalene catabolic genotype was more sensitive in this system than other naphthalene degrader microbial analysis methods for naphthalene catabolic cells. Other live cells present were 1000-10 000-fold more numerous than the genotype. Naphthalene biotransformation and mineralization rates fell when the mean value of genotype replicates dropped below lo7 genotypically positive cells/mL. The ability to enumerate a critical genotype and relate it to enzymatic activity in a mixed culture suggests an improved capability for system understanding at the ecological level and the potential for process control a t the genotype level.
Introduction All natural and engineered biological treatment systems are complex and contain many types of both procaryotic and eucaryotic microorganisms. In the case of procaryotic cells, which as a group share many physiological and biochemical traits, wide variability can exist in the capacity to carry out specific enzymatic transformations such as the catabolism of xenobiotic and/or pollutant chemicals. These variations in specific catabolic activities in the same or different species can often be traced to the presence or absence of specific catabolic plasmids within the cell that confer the ability to biodegrade one or more organic compounds (1). Plasmids may be acquired or lost from a given species on the basis of environmental conditions and/or stress. As a consequence, the concentration of a plasmid conferring a specific catabolic genotype in an ecosystem a t a given instant, the molecular microbial ecology, may determine the cell's or system's instantaneous catabolic activity (2). The concentration or frequency of a given plasmid-encoded catabolic genotype is regulated by a variety of factors such as competition or predation, which in turn are influenced by environmental factors such as nutrient concentrations, flow rates, chemical contamination, and/or stress (3-5). In addition, the expression of activity can be strongly influenced a t the cellular and molecular levels. Genetic regulation and control of gene expression are common, and subsequent enzyme synthesis may be under the molecular control of chemical substrates (parent compounds), metabolites in a pathway, or other biopolymers *Address correspondence to this author at the Energy, Environment and Resource Center, 327 s. Stadium, The University of Tennessee, Knoxville, T N 37996. 884
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and cellular catabolites (6). Even if a catabolic genotype is ecologicallyfavored, its final activity may vary depending on molecular level control and the biochemical environment. The objective of this study is to determine if the concentration or frequency of a critical catabolic genotype is a reliable measure of the catabolic activity in a biological treatment system operating on an industrial waste water. If so, greater ecological understanding as well as system monitoring and control based on genotype concentration may be possible. The potential importance of subpopulations of specific biodegradative bacteria in biological treatment systems has been studied (7,8). Active degradative subpopulations for toluene, aniline, and phenol in batch, fill-and-draw (sequenching batch), and continuous systems ranged between 2 and 5 orders of magnitude less than the total population of culturable cells, even though biotransformation proceeded rapidly. However, existing selective enrichment microbiological procedures seemed inadequate for determination of small subpopulations capable of degrading volatile and recalcitrant compounds, and therefore, comparison of kinetics for many compounds on a per-degrading-cell basis (second order overall) was not straight-forward. Although it has been assumed in the past that organisms capable of growth on agar medium containing a pollutant or recalcitrant compound as a sole carbon source must be capable of catabolism of that substrate, the utility and reliability of this approach are influenced by the following (5): poor or slow growth of the populations, no growth due to cometabolism of the substrate or auxotrophic nutrient requirements, cross-feeding, and detection of poorly selectable phenotypes or genotypes that are not expressed on specific media. Considering the above, nucleic acid hybridization techniques (9,10) have proven very useful for the detection of specific DNA segments carrying genes of known degradative pathways (5,11,12). Furthermore, it is possible to detect low frequencies of degradative subpopulations or catabolic genes within the complexity of waste water populations. Detection of low gene frequencies in environmental microcosms has also been shown (5). Unambiguous results in biodegradation and fate research mandate careful experimental design. An understanding of kinetic fate processes in the reactor of choice is needed. Equations and protocols for describing chemical fate processes can be rigorously derived, are easily used, and are reported in detail elsewhere (7, 13). The choice of naphthalene as the parent compound was governed by the need for a well-characterized catabolic plasmid (NAH7) and a known catabolic biochemical pathway (14-18). Eunn and Gunsalus (15)suggested that
0013-936X/87/0921-0884$01.50/0
0 1987 American Chemical Society
Table I. Characteriqation of Full-scale Treatment Plant Influent" chemical parameter naphthalene ethylbenzene toluene benzene ethyl acrylate ethanol tetralin 1,2-dichloroethane total organic carbon
influent chemical concn, mg/L mean maximum minimum 0.95 6.00 2.70 12.7 2.00 8.52 0.68 2.40 4600
5.70 31.0 8.40 38.7 5.60 6.10 7.40 8.70
0.38 0.88 0.29 1.3 0.55 0.27 0.03 0.30
"Ref 19.
a self-transmissible plasmid, NAH7, carried genes for naphthalene degradation in a Pseudomonas putida. Yen and Gunsalus (18) showed that there are two clusters of structural genes for the degradation of naphthalene on an approximately 83-kb (kilobase pairs) NAH7 plasmid, which are separated by a 7-kb region containing the regulatory genes. One cluster encodes enzymes mediating the conversion of naphthalene to salicylate while the other contained genes for the catabolism of salicylate via the ortho-cleavage pathway. The upper pathway genes coding for initial naphthalene biotransformation (- 15-kb EcoRI fragment of NAH7) have been cloned into vector pKT230 to create plasmid pDTG113 (D. T. Gibson, personal communication). Plasmid pDTG113 was used in this study to detect the naphthalene catabolic genotype. After the enzymatic biotransformation of naphthalene, the first and all subsequent metabolites of this pathway contain various oxygen-containing moieties. The addition of oxygen-containing groups increases the water solubility of the metabolites, reduces the compound's Henry's law constant (a "water-air equilibrium distribution coefficient"), and leads to metabolites with reduced stripping activity. The practical result is that only the parent compound is volatile (relative to the metabolites) and the presence of naphthalene in the off-gas (naphthalene stripped) provides an inverse measure of the magnitude of the biotransformation of naphthalene.
Experimental Section Reactor Feed and Inoculum. Waste water for reactor feed and biomass were obtained from a large southern petrochemical and chemical production facility in which full-scale fate sampling and analysis for specific organic compounds has occurred (19). For several compounds including naphthalene, the compound fates have been studied, and the transient concentrations in the influent and at many locations have been statistically quantified. Table I presents a summary of the characterization of the influent to the full-scale plant. Table I1 shows an analytical characterization of the waste water fed to our reactor, while Table I11 presents a biological characterization of the initial biomass obtained from the plant and used to inoculate our reactor. It is noted that the naphthalene concentrations of the reactor feed (Table 11) and the plant influent (Table I) are very similar even though wide variations in the plant influent concentrations are experienced. Equipment and Operation. The experimental equipment used here is shown schematically in Figure 1. The feed was maintained at 4 "C in a refrigerator and in a 9-L Tedlar bag to minimize volatile losses and composition changes. It was fed to the system with a stainless
Table 11. Feed Waste Water Characterization" parameter
concn, mg/L 11000 2 400
chemical oxygen demand dissolved organic carbon total nitrogen total phosphorus naphthalene PH metals exceeding 1 mg/L ca1cium iron magnesium si1icon sodium
50-54 38-43 0.98 4.8 (pH units) 9.4 1.6 1.8 3.5 1200
Other metals detected (less than 1mg/L): aluminum, barium, boron, chromium, copper, manganese, strontium, titanium, vanadium, and zinc. Other metals below detection limits: antimony, arsenic, beryllium, cadmium, cobalt, lead, lithium, molybdenum, nickel, selenium, silver, thallium, and tin. Table 111. Characterization of Biomass Inocula concentrations sample 2 sample 1 Sample as Received suspended solids" 6040 volatile suspended solids" 5070 Solids after Centrifugation suspended solids" 16 700 volatile suspended solidsa 15 800 microbial population 2.8 X 10'O total cellsb culturable cellsc 4.3 x 109 2.5 X lo6 NAH7 genotyped 1.6 X lo6 cells culturable on naphthalenee
6280 5080 15 700 14 500 2.0 X IO" 1.5 x 1010 1.7 X lo7 1.4 X lo6
Solids after Freezing and Thawing suspended solids" 15 500 15 800 volatile suspended solids" 14 400 14 600 microbial population total cellsb 1.2 X 10'O 2.3 X loxo 1.2 x 109 3.2 x 109 culturable cellsC NAH7 genotyped 3.7 x i o 6 1.2 x 107 1.2 x 106 7.0 x 105 cells culturable on naphthalenee "Units of mg/L. bAODC enumeration (cfu/g of wet solids), ref 22. 'YEPG enumeration (cfu/g of wet solids), ref 20. dColony
hybridization gene probe method (cfu/g of wet solids), ref 5 and 10. e Growth on media with naphthalene vapor as the sole carbon source (cfu/g of wet solids), ref 15.
steel positive displacement pump (3.2-mm piston). Feed was pumped continuously to the reactor but was intermittently directed through a volume-displacement flow meter for flow rate measurement. All wetted parts in the feed system were stainless steel, glass, poly(tetrafluoroethylene) (TFE), or Tedlar. Buffer (per liter: NaN03, 4.0 g; KH2P0,, 1.5 g; Na2HP04,0.5 g; FeCl3-6H2O,0.0005 g; MgS04.7H20,0.2 g; CaC12.2H20,0.01 g; pH 7.0) was introduced to the reactor from a glass reservoir through silicone tubing with a peristaltic pump controlled by an electronic timer. The reactor system consisted of a New Brunswick Scientific BiOFLO Model C30 chemostat modified with a solid virgin TFE top and TFE and silicone tubing. The reactor liquid volume was 350 mL. Temperature, agitation, and airflow controls were integrated into the system. An external glass clarifier (150 mL) received the reactor overflow, and the system effluent flowed from the clarifier to an effluent receiver. Settled biomass was returned to Environ. Sci. Technol., Vol. 21, No. 9, 1987
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TO OFFGAS CONTROL SYSTEM
d
POSITIVE DISPLACEMENT PUMP
PERISTALTIC PUMP
EFFLUENT
(ON ELECTRONIC TIMER) RECEIVER Flgure 1. Equipment used in this study.
the reactor with a timed peristaltic pump. Airflow into the reactor was continuously metered and humidified to minimize water evaporative losses in the reactor. The general experimental protocol included a series of preliminary operations leading to continuous reactor operation and data collection. Inoculum (after arrival from overnight shipment on ice) was centrifuged, resuspended as 1-2-g samples in glycerol-water (50% v/v), and fast frozen in liquid nitrogen with storage a t 183 K. The acclimation process (a fill-and-draw or sequencing batch mode) began with charging a flask with thawed inoculum and adding the buffer solution. On each of 5 days, liquid in the flask corresponding to 4,8,12,16, and 20 vol % was replaced with actual waste. Culturable cell counts on yeast extract peptone glucose or YEPG (20) media were performed and showed a 10-fold increase during acclimation. Waste water feed was prepared by pressure filtration through a glass fiber filter to remove solids and emulsions. Naphthalene was lost during this process. Consequently, the feed was supplemented with naphthalene to approximately 1 mg/L naphthalene. Radiolabeled [I4C]naphthalene was added in an n-butanol solution and mixed; feed radiolabel activity was 2200 dpm/mL. Typical system operating conditions are shown in Table IV. Biomass that attached as film on the reactor or clarifier surfaces was manually resuspended daily, and the mean cell residence time in the reactor system was controlled at about 5 days by daily measurement of both the biomass present and lost to the effluent and subsequent daily wastage of ' I 6of the biomass. The waste sludge was separated from reactor liquid by centrifugation of a calculated volume and split between two tubes to form two equal biomass pellets. These centrifuge pellets (reactor biomass) were used as samples for both analysis for 14C in the biomass and for extraction and analysis for sorbed naphthalene. One pellet was digested with a commercial 886
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Table IV. Typical Reactor Operating Conditions
dissolved organic loading, mg/day hydraulic residence time, days mean cell residence time, days temperature, "C system operating pressure, mmH,O relative PH dissolved oxygen, % saturation airflow rate, L/min agitation power input, hp/1000 gal reactor volume, L clarifier, L
0.25 3.5 5 20
