Simona Ciiinska, Vit Mate@, Jakub KrejCi, TomaS Janoch & Eva Kyslikova. Institute of Microbiology, 142 20 Prague 4, Videiiska 1083, Czechoslovakia.
J . Chem. Tech. Biotechnol. 1992, 55, 33-38
Water Denitrification by an Immobilized Biocatalyst Simona Ciiinska, Vit Mate@, Jakub KrejCi, TomaS Janoch & Eva Kyslikova Institute of Microbiology, 142 20 Prague 4,Videiiska 1083, Czechoslovakia (Received 7 February 1992; accepted 28 April 1992)
Abstract: The continuous denitrification of drinking water was performed by microorganisms from activated sludge chemically immobilized in a water-soluble polymer. The influence of temperature, pH, input nitrate and dissolved oxygen concentrations on the process rate was studied. Nitrates were reduced on average from 25% to 6.8 mg dm-$ NOS-N over a 100-day operation. Key words : denitrification, nitrate, drinking water, activated sludge, immobilized microorganisms.
NOTATION Ethanol concentration (mg dm-3) Oxygen concentration as a percentage of coz saturation (YO) COD Chemical oxygen demand (mg dm-7 m Dry mass of the biocatalyst (g) MLSS Mixed liquor suspended solids of activated sludge (g dm-$) NO;-N Concentration of nitrite nitrogen (mg dm-') NO;-N Concentration of nitrate nitrogen (mg dm-7 Q Flow rate (cm3 h-'; dm3 h-' ) SDR-N Specific denitrifying rate expressed in nitrogen (mg g-' h-l) Time when half of initial nitrate and nitrite to5 concentrations are reduced (h) ce
Subscripts in res
Initial Residual
1 INTRODUCTION Nitrate contamination of the environment is becoming an increasing problem. Nitrate concentrations in groundwater reached serious levels some years ago and are still rising.'-4 There are several methods of removing nitrates from groundwater supplies, only three of which show limited potential for full-scale a p p l i ~ a t i o n . ~These processes are ion exchange,' reverse osmosis7 and J . Chem. Tech. Biotechnol.
biological denitrifi~ation'.~or a combination of ion exchange and biological denitrification.'". The biological reduction of nitrates to gaseous nitrogen appears to be the most effective approach to water denitrification.l* Most of the methods studied make use of immobilized biosystems, especially physical or physico-chemical adsorption of microorganisms to the surface of insoluble carriers such as sand, plastic or ceramic particles. Various processes have been developed using pure microbial c ~ l t u r e s l ~but - ' ~ the use of mixed cultures may be more promising."." Adsorbed microorganisms, immobilized by weak hydrogen bonds or by electrostatic interactions with the carrier (fixed-film processes), are used for the denitrification of drinking water in certain full-scale processes.''-'s These weakly-bonded microbes might be easily washed out from the support into the treated water, causing subsequent microbial pollution. Other, more innovative processes, using microorganisms entrapped into alginate beads, have also been evaluated. l9 All these processes have their disadvantages ; surplus biomass has to be removed from the system and the denitrification activity is highly sensitive to temperature changes of water during the year. Another way to immobilize the microorganisms is by chemical bonding. Strong covalent bonds are formed between the microbes and the carrier during the chemical immobilization. The immobilization of a mixed microbial population of activated sludge into a water-soluble polymer gives rise to biomass aggregates. The aggregates exhibit high
33 0268-2575/92/$05.00 0 1992 SCI. Printed in Great Britain 3-2
S &insku
34
et al.
FEED WATER
D WATER
STORAGEKNI%
OF ETHANOL AND
DENlTRl FlCATION COLUMN SAND FILTER
NITRATE SOLUTION Fig. 1. Diagramatic representation of pilot plant system employed for the study of denitrification by an immobilized biocatalyst.
denitrification activity and possess suitable mechanical and hydrodynamic properties." Preparation of the biocatalyst consists of mutual chemical immobilization of the microorganisms by a reactive, water-soluble polymer and subsequent hardening of the particles using various procedures.'" The polymer may be formed by the reaction of branched polyethyleneimine and glutaraldehyde.21 A covalent linkage is thus formed between free functional groups of surface cell structures and reactive groups of the polymer.
BASF, Germany) and glutaraldehyde (Merck, Germany). The particles were hardened by the further addition of Sedipur. The biocatalyst was separated from the reaction mixture by filtration, dehydrated by ice-cold acetone and dried for 20 h at room temperature."' The size of aggregated particles varied between 0.1 5 and 4.60 mm. The immobilized particles were firm and rough with good sedimentation properties. The biocatalyst was stored as a suspension in 1 OO/ (w/v) glucose at 5-12°C. The dry mass content of the biocatalyst was 30.5% (w/w).
2.3 Analytical methods 2 MATERIALS AND METHODS 2.1
Microbial source material
A mixed population of activated sludge from a wastewater treatment plant, with specific denitrifying rates of 6.2 and 5.0 mg g-' h-' NO;-N, was used for laboratory and pilot plant tests, respectively. The biomass was concentrated by means of biological denitrification flotation and subsequently dewatered in a decantation centrifuge (Alfa Laval AVNX 309 B; Alfa Laval, Sweden) without the use of organic flocculants." A cell paste of 12 OO/ dry mass was produced.
2.2 Immobilization of microorganisms The concentrated activated sludge was immobilized by a water-soluble polymer, formed by the reaction of Sedipur (cationic flocculant containing polyethyleneimine;
Standard methods were used for the analytical determination of indices of water quality.23 Ethanol analysis was performed by gas chromatography using direct injection of the sample without any pretreatment. Particle size was measured by a computerized system from Leitz T.A.S. (Germany).
2.4 Apparatus and process conditions Laboratory continuous tests were performed in a plexiglass column (inner diameter, 26 mm ; height, 250 mm). Feed water passed up the column at a rate of 150cm3h-l. The column was packed with l o g of the biocatalyst. The column was fed with drinking water in which the nitrate concentration was set to 22.6 mg dm-3 NO;-N by the appropriate addition of potassium nitrate. This feed was used for the majority of laboratory tests. The only exceptions were experiments which served to investigate the influence of input nitrate
35
Water denitrijcation by an immobilized hiocatalyst
concentration on the specific denitrifying rate (SDR-N). Ethanol was used as an electron donor in all experiments; the amount added was 2.06 mg mg-' NO,-N, i.e. 1.5 multiple of stoichiometry. The SDR-N of the immoilized microorganisms was calculated as follows : SDR-N = [NOi-Ni, - (NO,-N m
+ NOi-N)rcs]Q (mg g-' h-')
The SDR-N of unbound biomass was measured using Ehrlenmeyer flasks with a working volume of 500 cm". Potassium nitrate was added to the sludge suspension at time zero to attain a concentration approximately 22.5 mg dm-3 NO;-N. The residual concentrations of nitrates and nitrites in sludge water were measured at selected time intervals. The SDR-N of activated sludge was calculated as follows : SDR-N =
(NO,-N + NO,-N),, (mg g-' h-l) 2 MLSS f0+
Pilot plant experiments were performed in a glass column (inner diameter, 110 mm; height, 1000 mm). Treated water overflowed from the column head. The column was packed with 2 kg of the biocatalyst. The rising water stream retained biocatalyst particles of 3.0 to 4.5 mm at the bottom of the column, particles of 1.0 to 3.0mm in the middle and, in the upper part, 0.2 to 1.0 mm particles. The porosity of the expanded biocatalyst bed was 0.6. A sand filter was positioned after the column (inner diameter, 300 mm; height of the sand layer, 600 mm; water level above sand layer, 500 mm). Drinking water with nitrate concentrations from 20.3 to 34.0 mg dm-3 NO,-N was used as a feed. The flow rate in the column was in the range of 16.0 to 26.0 dm-3 h-' and the upflow rate ranged from 1-7 to 2.8 m h-l. Ethanol, chosen as an electron donor since this is preferred to methanol in Czechoslovakia, was used in an amount equal to a multiple of stoichiometry in the range from 1.2 to 1.6. A diagram of the pilot plant system is presented in Fig. I .
3 RESULTS AND DISCUSSION
The denitrification activity of the biocatalyst depends on many physico-chemical and technological parameters and maximum efficiency of the process may be achieved by optimization of these parameters. The influence of temperature, pH of feed water, input NO,-N concentration and oxygen saturation on SDR-N was studied in continuous laboratory tests and the
0,5 U
Z
Z
I
rn 0
I
-
I
0
10
I
2 0 3 0 T L'Cl
I
4 0 5 0
-
LL
n
Fig. 2. Influence of temperature on the specific denitrifying rate (SDR-N) of native (0) and immobilized ( 0 )biomass at pH 7.0.
influence of temperature and pH of feed water on SDRN compared for both native and immobilized biomass. The optimum temperature range, from 35 to 5OoC,was very similar for both suspended native biomass and the immobilized biocatalyst. The SDR-N of native biomass reached a maximum at 48°C. The maximum SDR-N of immobilized biomass was achieved at 40°C (Fig. 2). The shift of the optimum temperature between the native and immobilized forms was probably caused by the immobilization technique used, which is in agreement with data reported earlier.24However, the denitrifying rate of immobilized biomass was less influenced by water temperature changes. This property of the immobilized form is valuable from the point of view of process control in the lower temperature range. The lower sensitivity of immobilized biomass to temperature changes was perhaps due to the protective role of the polymer. The enzymatic activity of immobilized, whole microorganisms has been previously shown to be more stable to environmental effects than either free whole microorganisms or native enzymes.*j.26 According to the authors, the reason for the increased stability was the physical structure of the matrix. The highest SDR-N of immobilized biomass was attained in the pH range from 7.5 to 8.5 and the optimum pH was shifted towards a lower pH compared to suspended free microorganisms. Figure 3 shows the response of SDR-N to pH for the free and immobilized biomass. Immobilization did not prevent a lowering of activity in the range below pH 7.5, as found previously with immobilized pure cultures of denitrifying bacteria.27s28 The optimum temperature and pH ranges found in the studies reported here are in agreement with data reported p r e v i o ~ s l y30. ~ ~ ~ Enzyme reaction rate may depend on substrate concentration and the influence of nitrate concentration in feed water on SDR-N was tested accordingly. Higher
36
"
"
~
4
5
6
7
8
reaction concept of biological denitrification"' and other published data.3z A higher nitrate concentration, however, decreased nitrite reduction. Nitrite concentration in treated water increased simultaneously with increasing nitrate concentration in the feed (Fig. 4). The purification process must be controlled such that the lowest possible nitrite concentration is ensured or, alternatively, nitrite must be subsequently removed in the following stages of water treatment. In dissimilatory nitrate reduction, microorganisms utilize nitrates as a terminal respiratory electron acceptor instead of molecular oxygen.33The potential inhibitory influence of dissolved oxygen was therefore examined in the continuous denitrification of feed water by immobilized biomass together with ethanol consumption (Table 1). The concentration of dissolved oxygen was varied either by compressed air saturation (100 YO)or by adding sodium sulphite (0 YO). After changing from aerobic to anoxic conditions, the immobilized biomass gave an increased SDR-N by only about 14%. Immobilized cells significantly differ in this property from suspended free cells, whose nitrate reduction was completely inhibited by the presence of dissolved oxygen. This feature of the biocatalyst may be explained as follows. During immobilization, the integrity of the cytoplasmic membrane may be impaired. The membrane potential may therefore change and the intensity of electron flux towards nitrite reductase increased. Nitrogen-containing intermediates could thus be formed at such a concentration which might inhibit terminal o ~ i d a s e s . ~The * ability of immobilized cells to denitrify under aerobic conditions is technologically feasible. However, the presence of dissolved oxygen caused an increased ethanol consumption due to the presence of aerobic microorganisms in water to be treated ; nitrite production may therefore be e n h a n ~ e d . ' ~ The results of pilot plant tests are summarized in Table 2 as mean values over 10-day periods. The inlet water temperature ranged from 13 to 1 9 O C , the average pH value was 6.9 and dissolved oxygen saturation of water was 80 YO.Nitrate concentrations in treated water ranged from 0.3 to 17.3 mg dm-3 NO,-N according to the feed
9
PH Fig. 3. Influence of pH in the specific denitrifying rate (SDR-N) of native (0) and immobilized ( 0 )biomass at 20°C.
- 2 c : 1
E
I
Z
' 1
K
n
v)
0
I
I
I
LO
80 N G ~- N [mg. 1-1I
120
Fig. 4. Influence of input nitrate concentrations on the specific denitrifying rate (SDR-N) of immobilized biomass (0) and residual nitrite content ( 0 )at 20°C and pH 7.0.
nitrate concentrations in the feed were found to give a higher SDR-N for the immobilized biomass (Fig. 4). This relationship is in agreement with the first-order
TABLE 1 Effect of Dissolved Oxygen Concentration on the Specific Denitrifying Rate (SDR-N) and Ethanol Consumption in the Continuous System used for Denitrification
~
Time
c,,?
(17)
( "/.)
Feed water
Treated water
SDR-N
Q
NO,-N
ce
NO,-N
NO,-N
c,
(cm3 h-1)
(mg dm-3)
(mg dm-3)
(mg dm-3)
(mg dm-3)
(rng dm-3)
100
144
100 0 100
270 262 141 142
30.0 1 22.08 25.05 25.19 28.40
52.0 55.0 54.0 53.5 53.0
1.17 5.64 8.48 2.76 4.65
2.35 2.54 0.03 1.50 0.04
0 28.0 30.0
(mg g-' k ' )
C,,NO;-N (mg mg-')
1.25 1.23 1.42 0.97 1.10
1.90 1.86 I .46 1.81 1.33
~~~
24 48 72 96 120
0
14.5
21.0
37
Water denitrijication by an immobilized biocatalyst TABLE 2 Pilot Plant Continuous Denitrification of Drinking Water During a 100-day Operational Period Time (days)
Feed water
Q
NO,-N
(dm3kl) (mg dm-3)
10 20 30 40 50 60 70 80 90 100
16 20 21 25 26 21 20 22 21 22
20.33 22.65 22.75 22.55 34.00 34.00 3254 23.85 22.50 23.00
Denitrified water
NO;-N
NO,-N
(mg dm-3)
(mg dm-3)
0.26 2 51 3.8 1 7.35 17.27 12.87 9.79 5.37 3.57 5.78
1.01
water temperature and to the column load. Nitrite concentrations in treated water increased up to 1.84mg dm-3 NO;-N during the first 20 days of operation. After this period, nitrite concentrations gradually decreased. If the sand filter was employed, a further reduction of nitrate and nitrite concentrations occurred (Table 2). Denitrification by the biocatalyst did not influence ammonium ions, iron, manganese, chloride and sulphate concentrations. The content of phosphate decreased from 0.06 to 0.02 mg dm-3. The total hardness of water increased from 2.30 to 2.35 mmol dm-3 and the acid-consuming capacity changed from 1.40 to 2.10 mmol dm-3. The initial COD of 1.05 mg dm-3 increased to a maximum of 8.4 mg dm-3 in the effluent from the column but the sand filter decreased this value to 2.1 mg dm-3. Microbiological quality of the treated water did not meet current drinking water standards because of a higher count of psychrophilic bacteria and occurrence of coliforms in some samples. These results may be caused by the conditions of biocatalyst storage and unsterile running of denitrification in the presence of organic substrate, which enables growth of contaminating microorganisms. The immobilization method, however, prevents immobilized cells from growth and multiplication. To ensure the quality of drinking water, disinfection must be employed. According to the results of pilot plant tests, the volumetric load of 10 dm3 water per 1 kg of biocatalyst (dry mass 30.5% (w/w)) per hour is the optimum to ensure reduction of a nitrate concentration from 22.5 mg dm-3 to less than 5.0 mg dm-3 NO;-N. The SDR-N did not change significantly over a 100-day operation. Thus the operational half-life of the biocatalyst may be estimated at several months. Bed exchange will be necessary rather for the reason of mechanical properties of the biocatalyst than the loss
1.84 0.93 0.32 0.78 0.5 1 0.48 0.18 0.05 0.03
Sand jltered water
NO,-N (mg dm-3)
NO,-N (mg dm-7
0.32 1.49 0.94 1.25 9.06 8.27 4.25 2.18 0.65 0.57
0.90 0.24 0.04 0.0 1 0.02 0.0 1 0.0 1 0 0 0
of denitrifying activity. After a 100-day operation, the average particle diameter decreased by 0.05 to 2.09 mm.
ACKNOWLEDGEMENT The authors acknowledge the kind help of Mrs Kristina Krejeova with the preparation of illustrations.
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