water for Springfield, MA. Cores from the filter were used to quantify with depth acriflavine direct cell counts (AFDC) using epifluorescence microscopy, and ...
Wat. Sci. Tech. Vol. 20, No. 11/12, pp. 293-299, Printed in Great Britain. All rights reserved.
0273-1223/88 $0·00+·50 1989 IAWPRC
1988.
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MICROBIAL POPULATION DISTRIBUTIONS AND BENZOATE MINERALIZATION KINETICS IN A MUNICIPAL SLOW SAND FILTER T. Taylor Eighmy, Michael R. Collins, Stergios K. Spanos and James Fenstermacher Department of Civil Engineering, University of New Hampshire, Durham, NH 03824, U.S.A.
ABSTRACT The microbial populations within the media of a slow sand filter were evaluated with regard to distribution and activity. The filter that was investigated was one of ten filters providing water for Springfield, MA. Cores from the filter were used to quantify with depth acriflavine direct cell counts (AFDC) using epifluorescence microscopy, and nutritionally specific heterotrophs (R2A), aquatic organic matter (AOM) utilizers, benzoate utilizers, catechol utilizers, heterotrophic iron precipitators, and manganese oxidizers using various spread plate techniques. Extractable Fe and Mn, as well as Folin-reactive material (FRM) were 1 quantified with depth. The ability of schmutzdecke populations to mineralize [U_ 4C] benzoate was also examined. AFDC counts revealed that the schmutzdecke contained the highest counts (109 to 1010/g dry wt) with counts declining by one to two orders of magnitude with depth in the filter media. All nutritionally specific plate counts exhibited similar trends with depth; R2A plate counts were generally less than 1% of the AFDC. A significant percentage of the colonies on the R2A media were pigmented. AOM, benzoate, and catechol utilizers were more prevalent in the schmutzdecke (as a percentage of the AFDC) than with depth in the cores. Small, opaque-white colonies were observed on the AOM, benzoate, and catechol media; suggesting the same bacterium (small flagellated rod) was responsible for growth on the three media. FRM correlated well with the AFDC with depth; the data suggests that the Folin reagent complexed with bacterial aromatic amino acids and was thus a good measure of microbial biomass. Extractable Fe and Mn correlated with AFDC, FRM, and with heterotrophic Fe precipitator and Mn oxidizer plate counts. The data strongly suggest that extractable Fe and Mn is complexed to the bacterial biomass in the filter. All schmutzdecke samples were able to Typically, over 50% of the label rapidly mineralize benzoate in the 1.0 to 1000 ng/ml range. was converted to l4C02 after 20 h of incubation. Mineralization was usually biphasic at 100 and 1000 ng/ml. Typical first order rate constants were 3 to 8/d. Non-purgeable dissolved organic carbon (NPDOC) removal in the filter was only 15%. Based on NPDOC and UV absorbance removals, the AOM fraction that was removed was hydrophobic and aromatic. The data suggests that while NPDOC removals are low, the microbial populations present in the filter are capable of promoting bioadsorption and biodegradation of certain fractions of the AOM in the raw water during winter operation.
KEYWORDS Slow sand filters, microbial populations, benzoate mineralization, microbial enumeration, iron, manganese, biomass.
INTRODUCTION The dissolved aquatic organic matter (AOM) in most natural waters is composed of aquatic humic substances (fulvic and humic acids). These constituents typically comprise up to 50 percent of the AOM in water; amino acids, carboxylic acids, proteins, and carbohydrates cons titute the remaining fraction (Amy et al., 1987). During chlorine disinfection of natural waters, the
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aquatic organic matter serves as precursor material for the formation of chlorination by products such as trihalomethanes (THMs). THMs are carcinogenic and are a regulated primary drinking water standard. Concerns about THMs have generated interest in water treatment processes which effectively remove the precursor material and reduce the THM formation potential (THMFP). Slow sand filtration of surface waters is an older but reliable treatment process known for its ability to remove turbidity, coliforms, viruses, and Giardia cysts from surface water supplies (Bellamy et al., 1985; Fox et al., 1984; McConnell et al., 1984). There has not been a lot of research on the microbiology of slow sand filters (Huisman and Wood, 1974); however, data suggests that the filters, particularly the schmutzdecke on the media surface, are microbiologically very active. The schmutzdecke has been hypothesized to promote removal of dissolved ADM by adsorption and subsequent biodegradation of the absorbed material. Surprisingly, non-purgeable dissolved organic carbon (NPDOC) removal in slow sand filters has been poor; typical percent removals are only 15 to 20% (Fox et al., 1984). Fulvic and humic acids are believed to be the metabolic end products from microbial biodegradation of lignins (Christman and Oglesby, 1971). Consequently, they are considered to be quite recalcitrant (Alexander, 1965). Recent evidence suggests that these ADM constituents are capable of supporting some microbiological growth (Bott et al., 1984; Geller, 1985; Meyer, et al., 1987; Tranvik and Hofle, 1987). The large size and aromaticity of fulvic and humic acids presents a problem to bacteria that require these substances as a carbon and energy source. Bacteria must be able to break down the larger structures to smaller molecular weight subunits. Those aromatic subunits require ring cleavage before mineralization can occur. Aerobic utilization of benzene-like aromatics requires enzymatic modification to catechol, protocatechuate, or gentisate before ring cleavage can occur (Alexander, 1977). Some research has shown that benzoate stimulates biodegradation of fulvic acid (Hann, 1977; Rifai and Bertru, 1980). Other work has shown that fulvic acid induces the transcription and translation of a ring cleaving dioxygenase (catechol-2,3-dioxygenase) that is essential in the utilization of benzoate and other similar monoaromatic substrates (Hann, 1976; 1983). Cometabolism of ADM with monoaromatic substrates has also been observed (Hann, 1983; Tranvik and Hofle, 1987). Conversely, Shimp and Pfaender (1982) found that biodegradation of monosubstituted phenols decreased in the presence of humic acids. We hypothesize that the ability of slow sand filter bacteria to ring cleave and mineralize benzoate is an important indicator of their ability to biodegrade ADM. Benzoate is found in humic and fulvic acids (Bollag, 1983; Ishiwatari and Machihara, 1983) and its utilization is clearly linked to fulvic acid biodegradation (Hann, 1976; 1977; 1983; Rifai and Bertru, 1980). The results presented here describe preliminary work on the characterization of municipal slow The sand filter microbial populations from filters which serve the City of Springfield, MA. studies were conducted to understand population distributions and metabolic activity in fully operational facilities. The data indicates that the municipal filters contain extensive microbial populations that are nutritionally diverse, capable of mineralizing benzoate, and are probably responsible for removing small molecular weight hydrophobic aromatic substances from the raw water during winter operation.
METHODS
Municipal Plant Description Three operational municipal slow sand filters have been visited in 1987 as part of our research effort on slow sand filters. Results from the January trip to the Westfield, MA filters are reported here. The West Parish filters, located in Westfield, MA, serve nearby Springfield, MA. There are ten filters in operation which cover 6.5 acres. They provide 10 MGD of finished water. The filters are operated at 1.15 ftfh. The sand media is 12 to 42 inches deep with an effective size of 0.3 mm and a uniformity coefficient of 2.3. The schmutzdecke is manually removed from the media surface by raking and shoveling when production falls below 0.5 MGD/acre. Hoyt (1986) provides a more detailed description of the facility.
Coring and Sample Preservation A 2.0 inch I.D. clear PVC tube (36" long) was used to core the filters. The filters were drained almost to the media surface before sampling. The coring device was driven into the
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filter media until 15 to 24" of media was cored. The tube was then manually excavated. Some compression of the core usually occurred (1" per 12"). Samples were collected with depth from the extruded core and stored in Whirl-Pak bags at 4'C until analysis. Influent and effluent raw water samples were also collected and appropriately stored for subsequent analyses.
Extraction and Enumeration Bacteria were extracted from the sand media using sodium pyrophosphate (Balkwill and Ghiorse, 1985). Approximately 3 g wet weight of sand was added to 25 ml of a 0.1% (w/v) Na4P207 • 10 H20 solution (pH 7.0, sterile). The sand was well mixed with a vortex mixer for two 30s bursts. Serial dilutions were conducted on the extraction supernatant. Sterile 0.1% sodium pyrophosphate (pH 7.0) was used as the diluent. All enumerations are reported per g dry weight of sand. Dry weight/wet weight determinations on appropriate duplicate core samples were done after drying at 70'C for 72 h. Acriflavine direct counts (AFDC) were used to enumerate total counts in the appropriate extraction dilution. The method outlined by Bergstrom et al. (1986) was used. A 1.0 mM acriflavine (AF) solution in 5 mM citrate-HCl (pH 4.0) was used to stain the bacterial DNA. A 5.0 min stain time was used. Two mls of the appropriate extraction dilution was fixed with 40 �L of 10% (w/v) glutaraldehyde, filtered through black 0.22 �m polycarbonate nucleopore filters, stained with 1.0 ml of AF, filtered after the 5.0 min exposure, and then examined under oil with a Nikon Biophot microscope. A variety of spread plate techniques were used to enumerate nutritionally-specific populations in the extractions. Incubations were conducted at 20'C. The R2A media developed by Reasoner and Geldrich (1985) was used to enumerate low carbon-requiring heterotrophs. An aquatic organic matter (AOM) media was made from locally obtained AOM (0.003 giL ) and a base nutrient solution developed by Hann (1974). A benzoate media and a catechol media were made by substituting the AOM with either benzoate (0.003 giL) or catechol (0.003 giL). A media used to enumerate heterotrophic iron precipitators was also employed (Clark et al., 1967). Manganese oxidizers were enumerated using Ghiorse's recommended media (Ghiorse, 1984).
Iron. Manganese, and Folin-Reactive Material Analyses Sand samples within a core were subjected to a nitric-acid digestion (Lyons et al., 1983). The digestate was analyzed for Fe and Mn using a Perkin-Elmer 2000 flame atomic absorption spectrophotometer. The method of standard additions was used to determine the concentration of nitric acid-recoverable Fe and Mn. The Lowry method (Lowry et al., 1951) was used to determine the concentration of Folin-reactive materials (FRM) with depth in each core. The first sodium pyrophosphate extract was used for analysis. Bovine serum albumen was used as the standard material.
Benzoate Mineralization The methods of Subba-Rao et al. (1982) and Simkins and Alexander (1984) were adopted for use in our mineralization studies. The mineralization assays were conducted with sodium pyrophosphate extractions of schmutzdecke samples from appropriate cores. Final cell concentrations in the incubation solutions were approximately 5 x 106 cells/mI. Uniformly labeled benzoate was used in the assay (Amersham, 120 mCi/mmole). Cold benzoate was used to augment the labeled benzoate at higher benzoate concentrations. Initial DPMs in all incubations were approximately 10,OOO/ml. Four benzoate concentrations were evaluated (1.0, 10, 100, and 1000 ng/ml). During the course of the incubations (20'C), 1 ml aliquots were An ethanolamine trap was collected, acidified, and placed under vacuum to drive off l4C02' The counts remaining in initially used to verify the conversion of l4C-benzoate to l4C02' solution represent free, bound, or incorporated benzoate and its metabolic byproducts. Mineralization rates were determined by looking at DPM losses over time.
Organic and Inorganic Water Analyses Influent and effluent water samples to the filters were analyzed for turbidity, color, pH, alkalinity, hardness, and dissolved oxygen using Standard Methods (APHA, 1985). NPDOC determinations and UV absorbance values (@ 254 nm, pH 7.0) were determined on the raw and treated waters. NPDOC was determined with a Dohrmann 80 organic. carbon analyzer. UV absorbance was measured with a Bausch and Lomb 2000 spectrophotometer with a 1 cm pathlength. THMFP was determined according to Collins et al. (1985). The incubations took place at 20'C, pH 7, for 168 h with a C12/NPDOC mass ratio of 5.0. A Perkin-Elmer Sigma 2000 Gas
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Chromatograph with an EC detector was used to quantify THMs after a liquid/liquid extraction into pesticide grade pentane.
RESULTS AND DISCUSSION As shown in Figure 1, the schmutzdecke always had the highest were usually 109/g dry wt. Similar levels have been observed (GAC) media treating seawater (Shimp and Pfaender, 1982), and Albright, 1986). AFDC always declined one order of magnitude and thereafter gradually declined with depth.
LOG AFDC OR CFU/GRAM DRY WT 3 4 5 6 7 8 9 10 II
I'g FRM/g dry 500
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0-0 Mn OXIDIZING PLATE COUNTS 0-0 AQUATIC ORGANIC MATTER UTILIZER S A---tI. BENZOATE UTILIZERS +-+ CATECHOL UTILIZERS
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concentration of AFDC; levels on granular activated carbon in marine sediments (Velji and directly below the schmutzdecke,
39
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COUNTS
Fig. 1 - Population distributions with depth. % RSDs were usually 20 to 40% for plate counts, 10% for AFDC
65
Fig. 2 -
FRM , Fe, and Mn distributions with depth. % RSDs were usually 3 to 8%
The Lowry method for determination of cell protein was used to estimate microbial biomass with depth in each filter core. The colorimetric reaction occurs with aromatic amino acids. The method has also been used to quantify phenolics in humic and fulvic acid fractions (Sharma and Krishnan, 1966). The levels of FRM , using bovine serum albumin as a standard, closely correlated with AFDC with depth in each of the filter cores (Figure 2). Dividing typical protein concentrations by the AFDC results in values of 0.8 to 2.0 x 10-12 g protein/cell. The data presented by Herbert (1961) suggests that 0.2 to 0. 8 x 10-12 g protein/cell is a typical protein level. Thus, the Folin-reaction appears to be quantifying mostly cell protein and not fulvic and humic phenolics, indicating that the assay is an adequate measure of cell biomass in the filter core samples. Nutritionally specific plate counts exhibited similar trends with depth. Invariably, plate counts were always 2-3 orders of magnitude lower than the AFDC. Highest plate counts were always seen with the low carbon heterotrophic R2A media. Usually, 5 to 10% of the colonies were pigmented (yellow, orange, purple). Colony pigmentation is usually the result of growth of aerobic, gram-negative rods (Reasoner and Geldreich, 1985). Other studies have also observed colony pigmentation on the R2A and other similar low carbon media (Reasoner and Geldreich, 1985; Maki et al., 1986). The ability of microbial populations to mineralize benzoate to C02 has been directly related to their ability to biodegrade fulvic acids (Hann, 1974, 1977, 1981, 1983). The enzymatic conversion of benzoate to catechol is an important step in the ring cleavage process and subsequent mineralization of 3-C byproducts to C02' Consequently, the ability of microbial There were no filter populations to grow up on AOM, benzoate, and catechol was investigated.
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distinct trends in the numbers of each nutritionally specific category that were observed (Fig. 1). Each category comprised a more significant proportion of the AFDC in the schmutzdecke samples, suggesting that these bacteria were preferentially located in the schmutzdecke. A small «1.0 mm diameter) whitish opaque colony was observed to be the predominant colony on all three media. Preliminary investigations indicate that the bacterium was a small flagellated rod. Heterotrophic iron precipitators and autotrophic manganese oxidizers were also abundant in the filters (Fig. 2). Iron and manganese are both secondary water quality standards. Microbial activity plays an important role in governing the biogeochemical fate of iron and manganese (Ghiorse, 1984). Some data suggest that slow sand filter microbial populations affect the fate of Fe and Mn during the slow filtration process (Huisman and Wood, 1974). Both heterotrophic iron precipitators and autotrophic manganese oxidizers would tend to promote oxidation of reduced species and/or extracellular deposition of oxidized forms of both metals. Strong correlations were found to exist between extractable Fe and Mn and AFDC, and with heterotrophic iron precipitators and autotrophic manganese oxidizers. Fe and Mn also strongly correlated with FRM; suggesting that Fe and Mn were associated with microbial biomass in the filter cores. Benzoate mineralization kinetics and rates are shown in Figures 3 and 4, respectively. Mineralization kinetics obtained in this study were similar to ones found by Subba-Rao et al. (1984) and Simkins et al. (1986) for lake water and wastewater populations and for a Pseudomonas isolate. The cell concentrations during the incubations in this study were similar to the ones used in the above mentioned studies.
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