JOURNAL
OF BIOSCIENCE
AND BIOENGINEERING
Vol. 87, No. 2, 189-193. 1999
Treatment of Aquarium Water by Denitrifying Photosynthetic Bacteria Using Immobilized Polyvinyl Alcohol Beads HISASHI NAGADOMI,’
TAKAKO HIROMITSU,2 KENJI TAKEN0,2 AND KEN SASAK12*
MASANORI
WATANABE,3
Nagao Co. Ltd., 256 M&aura, Okayama 72912121,l Materials and ScienceEngineering, Hiroshima-Denki Institute of Technology, 6-20-I Nakano, Aki-ku, Hiroshima 739-0321 ,2 and Department of Molecular Biotechnology, Graduate School of Engineering, Hiroshima University, 1-4-I Kagamiyama, Higashi-Hiroshima 739-0046,3 Japan
Received5 June 19981Accepted6 November 1998 During the purification of an aquarium for carp breeding, a relatively high level of chemical oxygen demand (COD) was removed by filtration systemspacked with both alginate- and polyvinyl alcohol i.PVA)-immobilized gel beads of Rhodobacter sphaeroides S. Low nitrate accumulation was observed in the alginate gel beads packed system due to denitrihcation, but high levels of nitrate and nitrite accumulation were observed in the PVA gel beads packed system. This phenomenon was caused by the inhibitory effect of PVA on nitrite reductase. The boric acid used for hardening gel beads of PVA slightly inhibited nitrate reductase. On the other hand, during the denitrifying growth experiments for this strain, boric acid inhibited cell growth, but PVA only partially inhibited cell growth. Based on electron equivalent (Y,.), growth yields using various kinds of concentrations of PVA were almost identical. It was suggested that PVA might only limit the growth rate of this strain by the inhibition of nitrite reductase. [Key words: denitrification, polyvinyl alcohol, alginate, immobilization, Rhodobacter sphaeroides]
In closed aqua-environments such as fish breeding ponds and ponds on golf courses, eutrophication has been frequently observed due to the accumulation of phosphorous and nitrogenous compounds. This polluted environment sometimes causes to sudden death of fish or foul odors. Therefore, circulating filtration systems that remove organic materials, phosphorous and nitrogenous compounds have been developed and applied (1). These systems, however, are usually based on the activities of microorganisms that grow naturally in a given environment. Sometimes, the capacity of the systems to remove phosphorous and nitrogenous compounds is not sufficient to prevent eutrophication. In addition, more effective and compact equipment is required to minimize the space needed and maintenance costs (1). The removal of nitrogenous compounds such as ammonia, nitrate and nitrite has been performed in bioreactors using denitrifying bacteria (2-5) and polyvinyl alcohol (PVA)-immobilized photosynthetic bacteria with denitrifying activity (6). The authors noted in a previous study that the purification of an aquarium for breeding fish (carp) could be accomplished using a simple circulating filtration system that was packed with alginate-gelimmobilized photosynthetic bacteria with denitrifying ability (7). The authors then succeeded in effectively removing chemical oxygen demand (COD) and nitrogenous compounds. Alginate gel beads, however, decomposed gradually and the removal effect decreased after 2 weeks. However, the PVA gels were quite stable over a one month period (8, 9). Photosynthetic bacteria can consume various types of organic matter with a relatively high growth rate and can consume phosphorous and nitrogenous compounds simultaneously (10). Therefore, photosynthetic bacteria have been used for water purification of fish breeding ponds in Japan. In addition, photosynthetic bacteria
have also been used as a feed supplement to intensify the color of carp (Nishiki-goi) and to prevent fish disease during breeding (10). This study investigates the characteristics of water purification and the denitrification of PVA-immobilized photosynthetic bacteria by focusing on the long-term purification of fish breeding ponds and the maintenance of the denitrifying ability of the photosynthetic bacterium, Rhodobacter sphaeroides. In addition, the accumulation of nitrite in the water by PVA-immobilized photosynthetic bacteria was also analyzed in order to elucidate the denitrifying activities in PVA gel beads. MATERlALS
AND METHODS
Microorganism and culture medium A denitrifying photosynthetic bacterium, R. sphaeroides S (7, 8) was used in this study. Stock culture was maintained as described previously (11). Glutamate-malate (GM) medium (11) which contained 5 g/l of KN03 was used for denitrifying growth of this strain. GM medium without KN03 was used for precultivation medium. Cultivations Precultivation was performed in a 300-ml conical flask (with a silicon stopper, 200ml medium) for approximately 3 d at 30°C under static-light conditions using two tungsten bulbs (5 klx, 20 W/m2, on the surface of a culture vessel) as described previously (11). The main cultivation (denitrifying culture) was performed at 30°C for 5 d under dark anaerobic conditions in a 1.5-l Roux bottle (with a silicon stopper) containing GM medium with 5 g/f of KN03. After inoculation with the preculture broth (2%, v/v), the medium was sparged with sterile nitrogen gas (99.9%) in order to make it anaerobic. Culture bottles were mixed vigorously by hand at 12-h intervals in order to maintain their homogeneity. After cultivation, cells were harvested by centrifuga-
* Correspondingauthor. 189
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tion (10,000 xg, 20min) and washed twice with deionized water, then used immediately for cell immobilization. Cell immobilization Cell immobilization was performed according to the conventional immobilization procedure using Na-alginate (Wako Chemical Co. Ltd., Osaka) and polyvinyl alcohol (PVA, Kuraray PVA-HC, Kuraray Co, Ltd., Osaka) (9). The cell suspension in deionized water (ODGa=5.0, approximately 2.5 g/c) was mixed completely with 4% (w/w) autoclaved alginate or PVA solution and titrated into CaC12 (2%, w/w) or saturated boric acid solution, respectively to form the gel beads. The gel beads with immobilized cells (3-4mm in diameter) were then soaked in respective solution for 20 h at 4-7°C in order to harden the gel beads. These gel beads with immobilized cells were used to purify aquarium water and in denitrifying experiments. The gel beads were washed several times with large amounts of dechlorinated tap water (approximately 25-30°C) before use in order to remove the extra alginate or PVA on the surface of the gel beads. Purification of fish breeding water Fish (carp) breeding was performed as described previously (7) using 25 1 of dechlorinated tap water in an acryl aquarium equipped with an airlift circulated filter vessel with a diameter of 10 cm, a height of 7 cm and an air supply of 1 Urnin. In the filter tank, 30 or 60 g (wet base) of gel beads as packed to the glass wool in the filter vessel. Five carp of approximately 5 cm in length (Nishiki-goi, one year old, approximately 10 g) were bred in this aquarium and were fed with 1.0 g/25 1 of pellet feed (Swimy mini, Japan Pet Feed Co. Ltd., Tokyo) per day in each aquarium for 15 to 20d. Three aquariums of the same type were prepared: one was as a control (no gel beads) and the other 2 were packed with 30 and 60g respective gel beads. Denitrification by gel beads In order to analyze the denitrifying characteristics of the gel beads, 0-30g of gel beads as put into a 1.5-l Roux bottle with 1 1 of denitrifying medium (10) and denitrification was carried out for 20 d at 30°C under dark anaerobic conditions (the medium was sparged with nitogen gas for several minuts during each sampling period). The medium with gel beads was gently agitated with a magnetic stirrer (approximately 100rpm) in order to maintain its homogeneity. The composition of the denitrifying medium was as follows (g/l): glucose 1.0, KN03 1.08, KH2P04 0.11, (NH&SO4 1.O, yeast extract 0.1, thiamine-HCl 1 x 10 3, nicotinic acid 1 x lop3 and biotin 1 x low5 (10). Denitrification by resting cells Resting cells were established in a 300-ml conical flask (with 200 ml liquid medium with a silicon stopper) using freshly prepared suspended cells in denitrifying medium so as to separately study the effects of PVA and boric acid on denitrification. The cell concentration was set at ODm=2.0 (1 .Og cells/l denitrifying medium) and 0, 5 and 10% PVA was added to each of three conical flasks (300m1, liquid volume 200 ml). These 3 flasks were incubated for 20d at 30°C under dark anaerobic conditions. Flasks were shaken vigorously by hand at 12-h intervals in order to maintain homogeneity. For the boric acid tests, 0, 3, 6 g/l of boric acid was added to cells suspended in a denitrifying medium (OD6m =2.0) in a 300-ml conical flask (with 200ml liquid volume) and the cultures were maintained at 4-7’C for about 20 h in a refrigerator. The suspended cells were then collected by centrifugation (lO,OOOxg, 20min) and
.I. BI~SCI.
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resuspended in fresh denitrifying medium in a conical flask (300ml , liquid 200ml) without boric acid so that the cell concentration was OD660=2.0. The same procedure was conducted for the PVA test with the flasks incubated for 20 d. In denitrification by resting cells, KN03 (1.08 g/l) or KNOz (1.0 g/l) was used as the terminal electron acceptor for denitrification (see Results and Discussion). Denitrifying growth experiment A 300-ml conical flask (200 ml of denitrifying medium) with a silicon stopper was used in the experiment. After the addition of 0 (control), 2 or 5% PVA and 3 or 5 g/l of boric acid to the medium, the flasks were autoclaved (lOlb, lOmin), then the preculture broth was added (lo%, v/v). The flasks were incubated for 3 d at 30°C under dark anaerobic conditions after sparging with pure nitrogen gas. Flasks were shaken by hand at 12-h intervals to maintain homogeneity. Analyses Cell concentration, COD, residual nitrate (as mg/l of NOi--N), nitrite (as mg/l of NOT-N), and glucose concentrations were analyzed as described previously (11). Pack-test (Kyouritsu Rikagaku Co. Ltd., Tokyo) was used for semiquantitative nitrite analysis for carp breeding experiments. RESULTS AND DISCUSSION Carp breeding and water purification by gel beads The water quality profiles from aquariums for carp breeding using different gel beads in the water circulation filter were compared. As shown in Fig. la, the COD increase in the aquarium with alginate gel beads packed in the filter vessel was slightly suppressed during the 2week carp breeding period compared with that of the water quality of the control experiment (no gel beads).
?
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FIG. 1. Purification of an aquarium for fish (carp) cultivation using (a) alginateand 0~) aolvvinvl alcohol (PVA)-immobilized gel beads bf R.-sphaeroide~ s backed in a circ&ting kdter vessel. Syk bols: 0, control (no gel beads); 0, 30 g (wet base) of gel beads; q , 60 g (wet base) of gel beads.
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experiments In addition,nitratewasnot accumulated in the water ed in the waterof PVA gel bead-packed when the alginate gel beads were packed, whereas the opposite was observed in the control experiment. This indicates that denitrification is active in the alginate gel beads as has been noted previously (7). Around the gel beads, organic matter such as feces of carp and microorganisms in the water was accumulated like sediment mud. This organic matter was decomposed to volatile fatty acids or other organic acids by digestion under microaerobic conditions. Strain S probably utilized these organic acids as substrates (electron donor). Gel beads, however, began to decompose after 2 weeks and COD removal activity began to decrease. For the PVA gel beads (Fig. lb), COD reduction was active as similarly observed in the aquarium with alginate gel beads, however, nitrate accumulation was observed in all cases. It was suggested that denitrification was not so active in PVA gel beads compared with in alginate gel beads. In addition, 2 carp died in the aquarium with 60 g of PVA gel beads which did not occure in aquariums with alginate gel beads. In that aquarium, approximately 1.Omg/l of NOT-N accumulation was detect(a) 2
0
5
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15
20
Time(d)
(b)
(the analysis was semi-quantitative using a test kit). Generally, nitrite was relatively toxic for the carp (7). Thus, it was observed that in aquariums with PVA gel beads denitrification was suppressed and nitrite sometimes accumulated. However, COD reduction with the PVA gel beads was almost the same as with the alginate gel beads. In addition, the mechanical stability of the PVA gel beads was very high compared with the alginate gel beads (9). A detailed analysis of the characteristics of denitrification with PVA gel beads was performed. Denitrifying ability of gel beads To determine the denitrification by gel beads in more detail, a denitrifying medium was used for the denitrification by strain S because the authors have already analyzed in detail the characteristics of the denitrifying growth of this strain using denitrifying medium (11). As shown in Fig. 2a, COD and nitrate were reduced when 30g alginate gel beads were added to the denitrifying medium. In particular, the relatively rapid reduction of nitrate was observed and approximately 50 mg/l of nitrite accumulation was observed after 10 d, but this nitrite was reduced again after 20d. This phenomenon was in good agreement with the denitrification of R. sphaeroides S in which nitrate was reduced as follows: NOj+NOz+NO+N20+N2 (11). In general, NO was not observed as an intermediate product of this step in the denitrification of bacteria (12). In addition, N20 could not also be detected in the denitrification by the photosynthetic bacteria, strain S (7, 13). From the results in Fig. 2a, denitrifying activity including cell growth was still active in the alginate gel beads. On the other hand, in the case of PVA gel beads, COD and nitrate reductions were low as compared with those in the alginate gel beads. In particular, a large amount of nitrite accumulation (maximum 85 mg/l) was observed but reconsumption was not noted when 30g of PVA gel beads were used. It appears that PVA immobilization of cells seemed to suppress denitrification. In addition, the death of carp in the aquarium with 60g of PVA gel beads may have been caused by the accumulation of nitrite (approximately 1.O mg/l of NOT-N) in the water. Therefore, in order to elucidate the suppression of denitrification by PVA, the effects of PVA and boric acid on denitrification were analyzed separately using resting cells. Effects of PVA
0
5
10
15
Time(d)
FIG. 2. Denitrification with (a) alginate- and (b) polyvynil alcohol (PVA)-immobilized gel beads of R. sphueroides S. Denitrification was performed in a denitrifying medium (1 f) with 10 and 30 g (wet base) gel beads under dark anaerobic conditions. Symbols: 0, control (no gel beads); 0, 10 g gel beads was mixed into the broth; q , 30 g gel beads was mixed into the broth.
and boric
acid on denitrification
The effects of boric acid and PVA on denitrification with resting cells are shown in Figs. 3 and 4. In Fig. 3a, nitrate reduction was suppressed with the increase in the boric acid concentration. The resting cells were soaked for ca. 20 h in a boric acid-containing denitrifying medium and then they were incubated for 15 d in a boric acid-free denitrifying medium. A small amount of nitrite accumulation was observed in the control experiment only (Fig. 3a). When nitrite was used as an electron acceptor in place of nitrate (Fig. 3b), the level of nitrite consumption was almost the same as that in the control experiment. These results suggest that boric acid seems to act as an inhibitor for nitrate reductase during the 20-h soaking period to harden the PVA gel beads. On the other hand, when PVA was added without soaking in boric acid, the nitrate comsumption in the PVA-added experiments was comparable to that in the
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(a)
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FIG. 3. Effects of boric acid on denitrilication in the resting cell system of R. sphaeroidesS. Cells were suspended in 0,3 or 6 g/l boric acid in a denitrifying medium within a 300-ml conical flask (containing 200 ml of liquid) and stored at 4-7°C for 20 h. After that, cellswere collected by centrifugation and washed twice using a boric acid-free denitrifying medium. Cells were resuspended in fresh denitrifying medium without boric acid. Cells were incubated for 15 d at 3O“C under dark anaerobic conditions. Nitrate (a) and nitrite (b) were used as the electron acceptor in denitrifying medium. Symbols: 0, control (no boric acid was added); 0,3 g/l of boric acid was added; 0 ,6 g/l of boric acid was added.
control experiment, however, nitrite was accumulated in the PVA-added (5 and 10%) experiments (Fig. 4a). In addition, when nitrite was used as an electron acceptor in place of nitrate (Fig. 4b), the nitrite consumption in the PVA-added experiment was reduced as compared with the control experiment. These results indicate that PVA mainly inhibits nitrite reductase. Based on the above results, it was suggested that boric acid slightly inhibits nitrate reductase and PVA inhibits nitrite reductase relatively strongly. As a result, a large amount of nitrite accumulation was observed during the denitrification with PVA gel beads. Shen and Hirayama established a bioreactor system for denitrification, in which the photosynthetic bacterium, R. sphaeroides, was immobilized using the PVAboric acid method (6). However, they did not report on nitrite accumulation levels. In addition, the purification of wastewater by PVA-immobilized activated sludge has also been noted, but there is no report about nitrite accumulation to date (8, 9). Effects of PVA on bacterial growth under the deniPVA inhibits nitrite reductase, trifying condition
0
4
a
12
16
Time(d)
FIG. 4. Effects of polyvinyl alcohol (PVA) on denitrification in the resting cells of R. sphaeroidesS. Cells were suspended in 2 or 5% PVA in denitrifying medium and incubated for 16 d at 30°C under dark anaerobic conditions. Nitrate (a) and nitrite @) were used as the electron acceptor. Symbols: 0, control (no PVA was added); 0, 5% PVA was added; q , 10% PVA was added.
therefore, growth experiments with PVA in the medium were performed. A quantitative analysis was also carried out to clarify the quantitative relationship between glucose and nitrate consumption in a resting cell system. As shown in Table 1, cell growth, residual nitrate and nitrite, and glucose are shown after 3 d culture when the glucose was almost consumed in the control experiment (no addition of PVA and boric acid). In the PVA added experiments, a similar level of nitrite accumulation (36.9-67.0mg/l) was observed as in the resting cells, however, no growth was observed in the boric acidadded experiments due to the long-term contact of cells with boric acid. In the denitrifying growth of R. sphaeroides S, the evaluation of growth yield based on electron equivalent (Y,,,) has already been analyzed in order to evaluate the efficiency of energy conversion (11, 12). Y,,. can be expressed as Eq. 1 (11, 12), Ye,.= AX/ - (Epq03AN03 -E~ozAN0i ) (1) where ENo3 and ENoz are the electron equivalents to the reduction of nitrate and nitrite to NZ, respectively. ENo3 and EN02 are -5 and -3 equivalents/mol, respectively (11). On the other hand, the growth yield for glucose, Yx,s can be expressed as,
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1. Quantitative analyses of glucose and NOT-N consumption and NOT-N accumulation after 3-d culture of R. sphueroides S under denitrifying condition
Glucose NOT-N NOT-N AGlucose ANOF-N AN02 -N (g7$.) (g/O (w/O (g/l) (w/l) @g/l) Ow/O 3.23 0.05 13.0 35.3 0.95 16.9 2.54 0.25 None 0.98 248 0 0 0 0 no growth Boric 3 g/l 0 no growth acid 5 g/l 0.98 250 0 0 0 3.56 0.45 81.9 67.0 0.55 12.1 4.78 0.14 PVA 5% 0.54 9.78 2.64 0.14 3.42 10% 0.46 113 36.9 Initial concentration of elucose. NOT-N and NOT-N in the denitrifving medium were 1.OO,0.25, and 0 g/l, respectively. Indicates the amount of change of each value after 3 d culture. a Estimated by Eq. 3 using Yx,s=O.26 g cell/g glucose. Addition
”
I_
Yx,s = AX/ AS
(2)
where AX is the amount of cell growth (g/l) and AS is the amount of glucose consumed by cells (g/l). From Eqs. 1 and 2 (3) Y,. = Yx,sAS/-(ENO~ANO~ -&o~AN01) The growth yield during denitrifying growth in glucose minimal medium (denitrifying medium) was 0.26 g cell/g glucose. This value was the same as the data in a previous study by us (11). Therefore, using Yx,s and Eq. 3, Y,,. was calculated for each culture and is shown in Table 1. In the control experiment, the Y,,. value was in agreement with that of Pseudomonas denitrificans during denitrifying growth (3.03 g cell/eq.) (12). In addition, the Y,,, values in the PVA-added experiments were not changed. This indicates that PVA did not affect the assimilatory reaction in cells such as the TCA cycle and protein synthesis. PVA mainly affected the rate of nitrate respiration due to the inhibition of nitrite reductase. ACKNOWLEDGMENT This research was supported in part by a Grant-in-Aid (No. 08650953) for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. REFERENCES 1. Kikuta, T. and Fukada, A.: Water quality control of aquarium water for admired fishes, p. 11-17. In Murakami, M. (ed.), Examples of purification of the waters for environmental use. Power-Sha, Tokyo (1996). 2. Nilson, I. and Ohlson, S.: Immobilized cells in microbial nitrate reduction. Appl. Biochem. Biotechnol., 7, 39-41 (1982).
3. Wiffels, R. H., Shukking, G. C., and Tramper, J.: Characterization of a denitrifying bacterium immobilized in K-carrageenan. Appl. Microbial. Biotechnol., 34, 399-403 (1990). 4. Nilson, I., Oldson, S., Haggstrom, L., MolIin, N., and Mosbath, K.: Denitrification of water using immobilized Pseudomonas denitrificans. Eur. J. Appl. Microiol. Biotechnol., 10, 261274 (1980). 5. Klapwik, A., van der Hoeven, J. C. M., and Lettinga, G.: Biological denitrification in an upflow sludge blanket reactor. Water Res., 15, l-6 (1981). 6. Shen, J. and Hirayama, 0.: Denitrification of PVA-immobilized denitrifying photosynthetic bacterium, Rhodobacter sphaeroides. J. Ferment. Bioeng., 75, 43-47 (1993). 7. Sasaki, K., Hashimoto, G., Lin, T., Takeno, K., and Suzuki, K.: Removal of nitrate ion by denitrification and the purification of fishing pond with immobilized cells. Mizushyori-Gijyutsu, 32, 29-35 (1991). (in Japanese) 8. Hashimoto, S. and Furukawa, K.: Immobilization of activated sludge by the PVA-boric acid method. Biotechnol. Bioeng., 30, 52-59 (1987). 9. Hashimoto, S.: Wastewater purification by immobilized microorganism. Yousui to Haisui, 29, 725-734 (1987). 10. Sasaki, K., Tanaka, T., and Nagai, S.: Use of photosynthetic bacteria for the production of SCP and chemicals from organic wastes, p. 247-290. In Martin, A.M. (ed.). Bioconversion of waste materials to industrial products, 2nd ed. Blackie Academic & Professional (Chapman & Hall), London, New York, Tokyo (1998). 11. Sasaki, K., Morii, H., Nishizawa, Y., and Nagai, S.: Denitrifying and photoheterotrophic growth of Rhodobucter sphueroides S under anaerobic-dark and -light conditions. J. Ferment. Technol., 66, 27-32 (1988). 12. Koike, I. and Hattori, A.: Growth yield of a denitrifying bacterium, Pseudomonas denitrificnns, under aerobic and denitrifying conditions. J. Gen. Microiol., 88, l-10 (1975). 13. Satoh, T., Hoshino, Y., and Kitamura, H.: Rhodopseudomonas sphaeroides forma sp. denitrr$cans, a denitrifying strain as a subspecies of Rhodopseudomonas sphaeroides. Arch. Microbiol., 108, 265-269 (1976).
