JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 95, No. 3,245-251. 2003

Influence of Ethanol Concentration on Biofilm Bacterial Composition from a Denitrifying Submerged Filter

Used for Contaminated Groundwater M I G U E L A N G E L GOMEZ, 1'2. JUAN M I G U E L GALVEZ, 2

E R N E S T O H O N T O R I A , 1'2 AND JESI~IS G O N Z A L E Z - L 6 P E Z 2

Department of Civil Engineering, University of Granada, 18071 Granada, Spain 1 and Environmental Microbiology Group, Institute of Water Research, University of Granada, 18071 Granada, Spain 2

Received 5 August 2002/Accepted 31 October 2002

The influence of the ethanol concentration on the composition and activity of a developed bio- film in a denitrifying submerged unidirectional filter was studied. Process yields (represented as inorganic nitrogen removal), total platable bacteria, denitrifying bacteria, nitrate- and sulphate- reducing bacteria and denitrifying activity (N20 production) were compared at different ethanol concentrations (0 to 46.74 mg/-1). The biofdm exhibited a diverse bacterial composition and higher microbial development at the entrance of the unidirectional biofilter. The number of ,cells per gram of dry weight of biofilm was increased when the ethanol concentration increased, ,with the exception of nitrate reducers, for which the number of cells decreased per gram of biofilm in relation to height. Five different species of denitrifying bacteria were isolated from the biof'dm, all of which were gram-negative rods. All of the species manifested an increase in denitrifying activ- ity when the ethanol concentration was increased. In this sense, the number of denitrifying bac- teria in the biof'dm was positively correlated with the ethanol concentration. Both nitrate- and sul- phate-reducing bacteria were present in the biofilm in the lower and higher part of the column. Nitrate-nitrogen removal in the submerged filter showed a high correlation with the influent eth- anol concentration.

[Key words: biofilm composition, ethanol, nitrate, nitrite, denitrifying bacteria, nitrate reducing bacteria]

Biodenitrification has proved to be one of the more ad- vanced and selective methods for removing nitrate by dis- similatory nitrate reduction, and transforms it into nitrogen gas. This process has been applied to nitrogen removal from wastewater (1) and contaminated groundwater (2), with bio- film technology achieving the highest nitrogen removal rate per reactor volume (3).

Different types of biofilm reactors have been used from the beginning of the 20th century for the biological treatment of water and wastewater. However, the technology based on a submerged filter seems to have better applicability for freshwater biological treatment (4). In this process, the bac- terial film grows on a support through which the water passes. The submerged filter operates as a high rate biologi- cal and mechanical filter in the same reactor and the biofilm is always submerged in water.

To apply submerged filter biodenitrification to contami- nated water such as groundwater, a carbon source dosage is required in addition to pH, temperature and oxygen concen- tration controls (5). Complex and simple carbon compounds have been employed in wastewater and freshwater nitrogen removal (6). In this context, ethanol is considered the most suitable carbon source for nitrogen removal from contami-

* Corresponding author, e-mail: mgomezn@ugr.es phone: +34-58-246153 fax: +34-58-246138

245

nated groundwater (7). Denitrifying biofilms are very complex systems formed

mainly by denitrifying bacteria embedded in a polymer ma- trix structure. However, other physiological groups can de- velop which affect the activity of the biofilm. In this sense, it has been established that the microbial composition of a biofilm is a function of the environmental conditions (8). Nitrate-reducing bacteria, that only reduce nitrate to nitrite, are present in a heterotrophic biofilm (9). The major or mi- nor presence and activity of this physiological group de- pends on several factors such as type of carbon source, con- centration of the carbon source and dissolved oxygen con- centration (10).

Denitrifying bacteria, that reduce nitrate and nitrite to either nitrous oxide and molecular nitrogen, are taxonomi- cally diverse and are considered ubiquitous (11). The deni- trifying bacteria are responsible for most of the conversion of inorganic nitrogen to dinitrogen (N2) in a biofilm. Several authors (12, 13) have reported that the addition of different carbon sources affects the bacterial composition and activity of biofilms, but no study has been done on tl~e influence of the carbon source concentration on the biofilm in a denitri- lying submerged filter.

We report the effects of different ethanol concentrations on the biofilm bacterial composition to elucidate the effects of the carbon source concentration on the biological activi-

246 GOMEZ ET AL. J. B~oscI. B~OENG.,

ties of a denitrifying submerged filter.

MATERIALS AND METHODS

Pilot-plant submerged filter The reactor used in this study consisted of a methacrylate cylindrical (3.0 m high and 0.3 m di- ameter, Fig. 1) column of the anoxic submerged biofilter-type, op- erating with an upward flow of groundwater and an upward flow of rinsing water and air for filter cleaning. Residual clayey schists from a brick factory (Cerfimicas Sties, Granada, Spain) were used as a support medium for biofilm growth, packing the column up to a height of 2.0 m. The average diameter of the particles was 2- 4 mm with a density of 1.75 g cm 3. To maintain the biofilm sup- port medium completely submerged in water, a communicating- vessels system was employed for operation. A parallel tube (3.5 m high and 0.05 m in diameter, Fig. 1) was connected to the meth- acrylate cylindrical column. The parallel tube operating with a downfiow and the packed column with an upflow function as com- municating vessels. The reactor was fitted with a sampling port set at different heights, to sample the water (0, 10, 66, 123 and 200 cm) and the biofilm (16, 64 and 123 cm). The submerged biofilter was cleaned using an upward flow of air (70 mh -1) for 1 min, rinsed with water (50 mh -1) and an upward flow of air (70 mh -l) for 10 min. A flow of water was applied for 5 rain to eliminate the re- maining biofilm (14).

Experimental procedure The water to be treated was groundwater from La Vega aquifer (Granada, Spain). The follow- ing characteristics of the water were determined daily for one month, according to the Standard Methods (15): NO3-, 50-70 mgt~l; NO2, 0.0~).01 mg/-l; PO43-, 0.4-0.8mg/-1; $04 ~, 180- 210mg/-l; 02, 2.0--4.5 mg/-1; and pH 7.0-7.5. The groundwater was pumped at a flow rate of 0 . 5 / m i n 1 using a piston pump. Ni- trate was supplemented by the addition of an appropriate volume of a concentrated stock solution of NaNO 3 giving a final concen- tration of 100 mg/-1 according to Gomez et al. (7). The system was operated under continuous ethanol addition in the concentration range of 0.0 to 46.47 mg/-1, maintaining different ethanol-carbon vs nitrate-nitrogen ratios (0.0 to 1.08 C/N). Each ethanol concen- tration was assayed over a period of 7 d. A concentrated stock solution of ethanol was stored in a tank from which it was pumped to the influent pipe. A stoichiometric quantity of sodium sulphite (8.0 mg Na2SO 3 per liter) was added to eliminate dissolved oxy-

6

FIG. 1. The pilot scale plant. 1, Submerged filter (V=0.21 m3); 2, influent tank (V= 1.7 m3); 3, carbon source tank (V=0.03 m3); 4, piston pump; 5, effluent tank (V=0.5 m3); 6, rinsing pump; 7, air compressor; 8, safety valve; 9, U-bend.

gen. The submerged biofilm support was inoculated with an acti- vated sludge supplemented with nitrate (1 g/-') and ethanol (0.5 g/-1), which was recirculated for 7 d, after which the influent water was pumped in. The water temperature in the system was in the range of 20_+5°C.

Sampling procedure Every 24 h, water samples (200 ml) were collected from the inlet and the outlet of the column obtain- ing five replicates for each C/N ratio assayed. For the given C/N ratio of 1.08 (46.74 mg ~ ethanol vs. 100 mg t -1 nitrate), water samples were collected through different sampling ports located along the column height. Nitrate and nitrite were routinely moni- tored in all samples.

For 0.0, 19.04, 32.02 and 46.74mg1-1 ethanol concentration assays (C/N ration of 0.0, 0.44, 0.74 and 1.08, respectively), the inert support was removed from the reactor through a sampling port located along the column, using a cylindrical sampler. Sam- ples (1 g) of the inert substrate were taken every week for a month, from three different heights (16, 64 and 123 cm) and were thor- oughly mixed prior to analysis.

Analytical determinations Water samples were filtered through 0.22-1xm membrane flters (HAWP; Millipore, Bedford, MA, USA). Nitrate and nitrite were measured by an ion-chro- matography system using conductivity detection (Dionex® DX- 300; Dionex, Sunnyvale, TX, USA). Separation and elution of the anions were carried out on an anion analytical column (Ionpac® AS14; Dionex) using a carbonate/bicarbonate eluent and a sup phuric regenerant. Before measuring, the filtered samples were diluted to achieve nitrate and nitrite concentrations lower than 10 mg/-1. The pH and dissolved oxygen levels were measured con- tinuously in the effluent using a pH meter and an OXI 921 elec- trode (Crison®; Crison, Madrid, Spain) respectively. Ethanol con- centrations were measured in the influent and effluent by gaseous phase chromatography (Perkin-Elmer® Autosystem GC; Perkin Elmer, Wellesley, MA, USA).

Biofilm production was estimated by dry weight according to Gomez et al. (7). For these studies, 1 g of clayey schists was added to sterile glass bottles containing 100 ml of sterile saline solution (NaC1 0.9%). The biofilm was separated from the inert substrate by sonication (1 min) and the suspended solids obtained were de- termined by vacuum filtration of the 100ml of saline solution through a pre-weighted fibber glass filter (0.45 p~m), then dried for 24 h at 105°C.

Total biofdm bacteria (total platable counts) Biofilm total bacteria were counted by the dilution-plate technique, using trypti- case soy agar (TSA; Difco, Franklin Lakes, NJ, USA). The biofihn was separated from the inert substrate as previously described for biomass production estimation and homogenized using a magnetic stirrer at maximum speed for 1 h. The inoculated agar plates (three replicates) were incubated at 22+ 1°C for 2 d for aerobic bacteria and for 5 d for anaerobic bacteria (Anaerogen system; Oxoid, Hampshire, UK) before the colonies were counted.

Denitrifying bacteria Denitrifying bacteria were determined by plating on nitrate-sucrose-agar (NSA) medium with the fol- lowing composition (per liter of distilled water): NaNO 3 2.0 g, K2HPO 4 1.0 g, MgSO4.7H20 0.5 g, KC1 0.5 g, FeSO4.7H20 0.01 g, yeast extract 1.0 g, sucrose 30.0 g and agar 20.0 g, at pH 7.2. The inoculated agar plates (three replicates) were incubated an- aerobically (Anaerogen system; Oxoid) at 30-1°C for 2 weeks before the colonies were counted. Approximately 5 randomly se- lected bacterial colonies were subcultured from each NSA plate. Single colonies were restreaked on NSA for purification.

Pure isolates from NSA medium were identified using Bergey's Manual criteria (16) and those of Jeter and Ingraham (11). The fol- lowing tests were used: cell morphology, Gram staining, motility, positivity or negativity for catalase, oxidase, urease and [3-galac- tosidase, nitrate reduction to nitrite, esculin hydrolysis, poly-~-

VOL. 95, 2003 INFLUENCE OF ETHANOL ON BIOFILM BACTERIAL COMPOSITION 247

hidroxybutyrate (PHB) accumulation, growth at 40°C, gowth in the presence of 2% NaC1, starch hydrolysis, PHB hydrolysis, pro- duction of fluorescent pigments, presence or absence of arginine dihydrolase and growth on sucrose, glucose, L-sorbose, D-xylose, maltose, saccharate, mannitol, ethylene glycol, 2,3-butylene gly- col, geraniol, azelate, levulinate, glycolate, L-serine, L-arginine, L-histidine, betaine and sarcosine. Denitrification activity (N20 production), was determined by the acetylene inhibition method (17). The culture medium for denitrification was nitrate sucrose broth (NSB). After 100% (v/v) of the atmosphere had been re- placed by He, a hermetically closed vial containing 5 ml of NSB was inoculated with each isolated strain and then incubated at 30°C, and 0.25-ml gas samples were assayed for NzO after 1, 2, and 3 d by injection into a Varian Start 3400 GC (Varian, Palo Alto, CA, USA) equipped with a thermal conductivity detector.

Nitrate reducing bacteria Nitrate reducing bacteria were estimated by conventional bacterial dilution on NSA plates as de- scribed by Rodina (18). To determine the ability to reduce NO 3 to NOz-, the isolated strains were inoculated (three replicates) into bacto-nitrate broth (Difco) and were incubated at 30+ 1°C for 48 h. After incubation, Griess reagent (sulphanilic acid plus c~-naphthyl- amine) was added. Positivity was indicated by a pink or red color.

Sulphate reducing bacteria The population of sulphate-re- ducing bacteria were estimated by conventional bacterial dilution on NSA plates as described by Rodina (18). The production of H2S by bacteria was detected in hermetically closed test tubes contain- ing 5 ml of Sturm medium (18) with suspended indicator paper soaked in a saturated solution of lead acetate. Prior to inoculation, the atmosphere of the vial was substituted with helium. Inoculated test tubes (three replicates) were incubated at 30+1°C for one week.

Scanning electron microscopy Cells of the biofilm were immediately fixed with 3% glutaraldehyde for 2 h, then rinsed and treated with 1% osmium oxide for 3 h. Subsequent dehydration in- cluded rinsing and retention in a graded ethanol series (30%, 50%, 70%, 90% and 100%). Finally, the samples were dried to the criti- cal point and mounted on support stubs. The samples were viewed by a Hitachi scanning electron microscope without gold coating.

Statistical considerations The arithmetic mean_+ standard error (S.E.) was used for nitrate and nitrite effluent concentration selection after each C/N ratio assay. Values above the mean plus S.E. and below the mean minus S.E. were discarded.

Data obtained through this study were analyzed by computer assisted statistics, using Statgraphics Plus for Widows 3.0 (Statical Graphics Corp., 1997). The least significant differences test (LSD- Test) was used to measure the difference between the C/N ratio and denitrifying bacterial activity. An analysis of variance (ANOVA) test was used to assess the homogeneity of variance with a significance level of 1% (P<0.01).

RESULTS

Development of an active biofilm from groundwater can be a very slow process due to its low bacterial and nutri- tional loads. Thus, bioreactor inoculation is required and in our case we used activated sludge from an urban wastewater treatment plant, due to its high bacterial load. Once the inoculation stage was finished (7 d), the presence of the biofilm was confirmed by electronic microscopic surface screening (Fig. 2a, b). These observations confirmed the suitability of both the support material and inoculum for creating a biofilm in the bioreactor. However, the numbers of cells (predominantly composed of rod-shaped bacteria) and structure o f the biofilm were modified during the opera- tion time of the bioreactor.

The inorganic nitrogen removal rate of the submerged fil- ter showed a high positive correlation with the ethanol con- centration in the influent (r=0.93). Once the biofilm was formed, polluted groundwater (100 mg NO 3- l -~) was treated resulting in 2% inorganic nitrogen removal without the ad- dition o f ethanol. The total inorganic nitrogen concentration in the effluent decreased immediately when ethanol was added reaching 99% removal with a C/N ratio in the influ- ent o f 1.08 (46.74 mg 1-1 ethanol). The nitrate concentration decreased immediately when the ethanol concentration in- creased (Fig. 3), but, the nitrite concentration initially in- creased, but was not detectable later as the ethanol concen- tration rose. In all tests, the residual ethanol concentration in the treated water was analyzed (Fig. 3). The highest carbon source concentrations were detected in treated water during

(a) (b)

FIG. 2. Scanning electron micrograph of the biofilm after the inoculation stage (a) and under running conditions (b).

248 GOMEZ ET AL, J. BIOSCL BIOENG.,

TABLE 1. Effect of ethanol concentration on number ofheterotrophic aerobic and anaerobic bacteria, nitrate- and sulphate-reducing bacteria and denitrifying bacteria in a denitrifying submerged filter for nitrate contaminated groundwater treatmenP

Bacteria Colony forming units/mg of biofilm x 10 s

0.0 b 19.04 b 32.02 b 46.748

Heterotrophic aerobic bacteria Heterotrophic anaerobic bacteria Nitrate reducing bacteria Denitrifying bacteria Sulphate-reducing bacteria

13.4_+ 1.4 27.0_+4.6 21.0_+ 1.4 24.5 _+2.5 290.1-+19.0 180.0_+10.5 170.0_+7.8 180.2_+9.6 34.1-+6.0 30.7_+ 1.7 30.9_+0.68 45.1_+3.23 37.2___5.8 77.5_+3.3 72.4_+7.05 115.1 _+4.9 2.81 _+0.73 2.9_+0.5 2.4_+0.16 5.7_+0.28

Values are means of five replicates_+S.E. 100 mg l < of nitrate.

b Influent ethanol concentration.

TABLE 2. Effect of ethanol concentration on number of denitrifying bacteria isolated from biofilms developed in a denitrifying submerged filter for nitrate contaminated groundwater treatment"

Denitrifying bacteria Colony forming units/mg of biofilm x 10 s

0.0 b 19.04 b 32.02 b 46.74 b

Pseudomonas alcaligenes ° Moraxella d Pseudomonas pickettii ~ Alcaligenes denitrificans e Agrobacterium radiobacter d

6.75+2.8 28.8+3.1 4.8+0.5 7.6+1.15 11.8 __+2.7 0.0085 _+ 0 . 0 0 1 9 0.006_+0.0005 0.8 -+ 0.3 18.6_+6.1 16.9-+0.2 14.1_+ 1.2 42.09_+ 1.2

0.012_+0.0009 19.07_+2.1 39.9_+3.4 44.5-+6.2 0.011_+0.0001 12.5_+1.6 13.4_+2.7 19.9__+2.7

Values are means of five replicates_+ S.E. 100 mg/-~ of nitrate.

b Influent ethanol concentration. ~,d., Bacteria with a common letter are not statistically significant different (1°<0.0l).

the maximum yield phases, not reaching 1165 rng/I. The presence of different microorganisms in the biofilm

was studied using several ethanol concentrations (Table 1). The bacteria which accounted for the greatest proportion of the isolates consisted of heterotrophs grown under anaero- bic conditions. Only denitrifying bacteria showed a high correlation with the ethanol concentration in the treatment system (Fig. 4).

The number of denitrifying bacteria vs nitrate reducers as deduced from Table 1, showed a substantial increase (1.13 _+ 0.33 to 2.52 + 0.06) when transferring from a system without the addition of carbonaceous compounds to one with a C/N ratio of 0.44 (19 .04mg/q ethanol), due to the rapid growth of the denitrifiers.

The effect of the ethanol concentration on denitrifying bacteria was studied in detail (Table 2). Pseudomonas pick- ettii and Alcaligenes denitrificans were numerically the

most predominant in the biofilm examined, showing statisti- cally significant differences, with respect to their numbers, from the rest of the denitrifiers isolated (P<0.001) which was presumably related to the increase in ethanol concentra- tion.

The mean denitrifying activity of the different strains in- creased in accordance with the increase in the influent etha- nol concentration (Table 3). For all but the highest ethanol concentration evaluated, statistically significant differences in activity were observed (P<0.01). In addition, statistically significant differences in activity were observed (P<0.01) between the isolated. P. picket t i i and A. denitr~'cans ac- tivities were unaltered by the ethanol concentration. The highest mean activity was achieved by Pseudomonas alcali- genes and Agrobaeterium radiobacter especially when a high concentration of ethanol was used.

Nitrate (100 mg/- i ) in the groundwater was removed in

120

o • =~ loo

.~ ~ 6o

2 ~ 4O

20

z 0~- o 0.2 0.4 0.6 0.8 1

C/N ratio of the influent

2

.5 =

g

0.5

0 ~ 1.2

FIG. 3. Residual nitrate (asterisks), nitrite (circles) and ethanol (squares) concentrations in contaminated groundwater (100 mg NO3-/-~) treated in a denitrifying submerged filter with different ethanol con- centrations in influent (rag/-1).

14

~= 12 o

~ 1 0

6

i~ •

2

o 0

, i , , r , I , , , , I , , , , I , , , , r

10 20 30 40 50 Influent ethanol concentration (mg/-1)

FIG. 4. Linear regression between number ofdenitrifying bacteria in the biofilm and influent ethanol concentration (rag ?~).

VOL. 95, 2003

TABLE 3.

INFLUENCE OF ETHANOL ON BIOFILM BACTERIAL COMPOSITION

Effect of ethanol concentration on denitrifying activity of denitrifying bacteria isolated from biofilms developed in a denitrifying submerged filter for nitrate contaminated groundwater treatment ~

249

Denitrifying bacteria Denitrifying activity (nmol ?t N20/cfu )

0.0 b 19.04 b 32.02 b,~ 46.74b. ¢

Pseudomonas alcaligenes Moraxella Pseudomonas pickettii Alcaligenes denitrificans Agrobacterium radiobacter

1.25.+0.0718 1.9.+0.12 109.5.+0.045 115.77-+11.2 0.127.+0.015 1.26.+0.15 30.1 .+1.12 30.4.+0.816

5.14.+0.578 5.78.+0.236 45.8.+0.2 47.1 .+ 1.36 5.31 .+0.22 47.7.+1.14 45.5.+0.81 47.5.+0.73 1.15 _+ 0.0855 47.4 .+ 0.43 60.9 .+ 1.06 120.8 .+ 8.56

Values are means of five replicates_+S.E. 100 mg k ~ of nitrate.

b Influent ethanol concentration. C/N ratios with a common letter are not significantly different (P< 0.01).

the lower part (123 cm) of the bioreactor, with no inorganic nitrogen removal capacity in the last 80 cm of the cylinder (Fig. 5). Therefore, 50% of the total amount o f nitrate in the influent was removed in the first 30 cm of the reactor.

A large concentration of denitrifying bacteria and hetero- trophic anaerobic bacteria was observed in the first 66 cm (lower part) o f the reactor (Fig. 6), but the rest of the micro- organisms studied were not present in large numbers. These results showed a decrease in the ratio o f denitrifiers/nitrate reducers, from a mean of 2.35-+0.01 at 16 cm to 1.18_+0.04 at 123 cm.

The pH of the influent and effluent was analyzed with no detection of significant statistical differences between the two. The mean pH value of the influent was 7.25 +0.25 and that of the effluent was 7.6+0.8.

DISCUSSION

Biofilms form in every environment so long as a surface, nutrient and water are available. When forming biofilms, the growth of microorganisms is a function o f different fac- tors, such as the substrate and inhibitory agent concentra- tions, temperature, pH and hydrodynamic conditions. At least three conceptual models o f biofilm structure exist: the heterogeneous mosaic model, the water-channel model and the dense biofilm model, according to substrate concentra- tion (19). In this study, the biofilm structure and its bacterial composition varied during the bioreactor operation (Fig. 2a, b), displaying heterogenic channelled forms (the water-chan- nel model) as described by Beer and Stoodley (20). Low nutrient concentration may be the cause o f this channelled

120~

soL\ ~ ~ 6 0 I k ~ , , , ,

8 40 ~['k.=

0 , , , ~ , " ~ 0 50 100 150 200

Height (era)

FIG. 5. Residual ethanol (squares), nitrate (asterisks) and nitrite (circles) concentrations in treated contaminated groundwater (100 mgNO 3 1 -~) in relation to column height.

structure, due to minor cellular proliferation, as previously reported by Wimpenny and Calasauti (19). Biofilm develop- ment was directly related to the presence of nutrients, and inorganic nitrogen removal.

Disassimilatory nitrate reduction using ethanol, obeys the following reaction (21).

12NO3-+5CzHsOH ---> 6Nz+9H20+10HCO3--+2OH-

This reaction shows that the appropriate C/N ratio to re- move all o f the nitrate is 0.72. However, a higher C/N ratio (1.08; 0.46kg of ethanol per kg of nitrate removed) was necessary in our experiment for the complete elimination o f inorganic nitrogen (100 m g N O 3 U). This consumption of ethanol could be caused by non-denitrifying microorga- nisms in the submerged filter and also by the assimilation o f carbon by the biofilm which is used for cellular growth (22).

When the C/N ratio of the influent was inc, reased (higher ethanol concentrations) the number o f denitrifying bacteria was clearly increased. This result suggests that the higher ethanol concentrations in the media facilitate a better bac- terial energy yield and therefore greater cel]kular prolifera- tion, which consumes carbon and nitrogen by assimilation. In addition, our results show a better adaptation o f the de- nitrifying bacteria to the working conditions set in the sub- merged filter, compared with other microorganisms.

Denitrifying bacteria as well as nitrate reducers are re- sponsible for the production of nitrite, an anion which is

16 66 Height (em)

123

FIG. 6. Number of microorganisms (bacterial cells) in the biofilm at different heights. Crossed bars, Heterotrophic aerobic bacteria; re- ticulated bars, heterotrophic anaerobic bacteria; open bars, denitrifying bacteria; dotted bars, nitrate-reducing bacteria and closed bars, sul- phate-reducing bacteria.

250 GOMEZ ET AL. J. BIOSCI. BIOENG.,

also employed by denitrifiers as an electron acceptor (22). The decrease in the bacterial population in relation to reac- tor height was substantial for the denitrifying bacteria and heterotrophic anaerobic bacteria, but not for the nitrate re- ducers, causing a decrease in the number of denitrifying bacteria vs nitrate reducers. The same results were obtained when low carbon source concentrations were employed. In this sense, decreases in this ratio (denitrifying bacteria vs nitrate reducers) could give rise to increases in the nitrite concentration in the treated water, a highly toxic compound, the maximum permitted concentration in drinking water of which is 0.1 mg 1-1. This anion is easily oxidized by mole- cules used in the chemical disinfection of drinking water (23), although its high concentration will make these com- plementary treatments more complicated.

Neutral to alkaline conditions are optimal for denitrifica- tion. As the pH increases above 9.0, the accumulation of nitrite increases significantly (24). Thus, control of the pH of groundwater during biodenitrification is important be- cause microbial activity can result in pH changes and hetero- trophic denitrification in particular may increase the pH. Several methods, such as reagent addition and use of encap- sulated buffer (25), have been used for pH regulation. How- ever, in our case the inner support behaved as a pH regula- tor, due to its clayey nature. No variation in pH was detected during the process, so a negative influence on microorga- nism growth was not expected.

Biofilm porosity and structure influence the biomass ac- tivity as does the diffusion of nutrients and biofilms can be active or inactive (26). Clogging problems negatively affect the flow in fixed bed reactors (27). Thus, in this study the bacterial distribution in upflow system generated system clogging in the inlet area, slowing the water flow and re- quiring the filter to be washed. This is the main energy cost in the running operation of the plant. Clogging mainly oc- curred in the first few centimeters of the column, while the rest of the filter had no blockage problems. To lengthen the intervals between washes it is necessary to study other fluxes such as downflow and alternative flow.

Sulphate-reducing bacteria were also isolated in the bio- film under our experimental conditions. The population of these microorganisms increased in the biofilm when the in- fluent ethanol concentration was increased. However, the effect on the chemical characteristics of the outlet water was small. This bacterial group u s e 8042- as a terminal electron aceeptor to generate H2S. The concentration of this anion, which may be present in high or low concentrations in groundwater, can increase substantially in the total absence of oxygen and nitrate, elements from which bacteria obtain more energy (28). Under these circumstances, proliferation of this physiological group, which increases the H2S con- centration in the treated water, will affect the organoleptic characteristics of the treated water. The infiuent sulphate concentration oscillated between 180-210 mg ~ , and was increased by sodium sulphite addition (influent dissolved oxygen control). Thus, it is important to control the carbon source concentration, because when it is increased it gives rise to a disassimilatory reduction of sulphate.

The denitrifying bacteria isolated from the biofilm be- longed to five different species, all of which were gram-

negative rods. Some of them exhibited growth correlated with the ethanol concentration, while others did not seem to be adapted to the system, decreasing in number after the in- oculation stage. The presence of different denitrifying bac- teria may affect the result of the process, due to their differ- ent inorganic nitrogen removal abilities. While species such as A. radiobacter developed a high nitrogen removal capac- ity, others did not even reach one fourth of their potential. Denitrifying activity varied not only in relation to the bac- terial strain but also to the carbon concentration. All of the species in the biofilm manifested an increase in denitrifying activity when the carbon concentration was increased, but this increase was higher or lower depending on the bacterial species.

In conclusion, our data suggest that the use of bacterial inocula with high denitrifying activity in association with submerged filters could be of importance to increase the in- organic nitrogen removal capacity ofbiofilms. However, the degree to which our experiments can be extrapolated to other conditions must be considered more attention because a number of factors (temperature, composition of the micro- bial community, etc.) also influence this biological activity. Further development of this process is continuing.

A C K N O W L E D G M E N T S

This study was supported by a grant from the Comisi6n Inter- ministerial de Ciencia y Tecnologia (AMB95-0621) and it was conducted in the Institute of Water Research and Department of Civil Engineering, University of Granada.

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