Physiology and kinetics of autotrophic denitrification by Thiobacillus ...

Appl Microbiol Biotechnol (1985) 22:283--288

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Biotechnology © Springer-Verlag 1985

Physiology and kinetics of autotrophic denitrification by Thiobacillus denitrificans Giinter Claus* and Hans Jiirgen Kutzner Institut fiJr Mikrobiologie, Technische Hochschule Darmstadt, Schnittspahnstrasse 9, D-6100 Darmstadt, Federal Republic of Germany

Summary. A strain of Thiobacillus denitrificans was isolated after enrichment under anaerobic conditions by the continuous culture technique using thiosulfate as energy source and nitrate as electron acceptor and nitrogen source. The isolate was an active denitrifyer, the optimal conditions being 30°C and pH 7.5--8.0. Denitrification was inhibited by sulfate (the reaction product) above 5 g SO4/1, whereas high concentrations of the substrates nitrate and thiosulfate were less harmful; nitrite affected denitrification above 0.2g NO 2/1. During the time course of denitrification in a batch culture growth and substrate consumption slowed down already after only half the substrate was utilized due to product inhibition. The following parameters were determined in continuous culture under nitrate limitation: /-~rnax 0.11 h - 1, Ks = 0.2 mg NO 3/1, maximum denitrification rate=0.78 g N O 3 / g cells.h, YNo~=0.129 g cells/g NO3, Ys2o3=0.085 g cells/ g $20 3. Nitrite did not accumulate during steady state denitrification; the denitrification gas was almost pure N2. The concentrations of N20 and NO were below 1 ppm. =

Introduction Biological denitrification has shown considerable promise as an effective and economic method to remove nitrate nitrogen from waste water (Winkler, 1984). Many heterotrophic bacteria are capable of performing denitrification using organic Present address: Benckiser-Knapsack GmbH, Dr.-AlbertReimann-Strasse 2, D-6802 Ladenburg/Neckar, Federal Republic of Germany Offprint requests to: H. J. Kutzner

*

carbon supplements. Denitrification can also be carried out autotrophically by Thiobaeillus denitrificans using inorganic sulfur compounds as energy sources. The principle of the denitrification process by T. denitrifieans is given in the following equations (Zumft and Cardenas 1979, Schedel and Triiper 1980): 5 S2Oy- +25 H20 ---, 1 0 S O 4 - + 10H + +40 [H] 8NO~ + 8 H + +40 [H] 4 N2 + 24 H20

(2)

5 S 2 0 3 - + 8 N O 3 -t- H 2 0 4N2+ 1 0 S O 4 - + 2 H +

(3)

Equation (1) describes the anaerobic oxidation of thiosulfate as energy source (electron donor); the reducing power is transferred via an electron transport chain to nitrate which is reduced in four steps to nitrogen gas (Eq. 2): this process of nitrate reduction supplies the energy (ATP) to the organism used for biosynthetic purposes. Eq. (3) describes the overall process. Since CO2 is the carbon source of this chemolithoautotrophic bacterium, reducing power is also needed for CO2-reduction. It should be emphasized that the reducing power needed for CO2 assimilation has to be supplied as NADPH2; since the oxidation of thiosulfate is not coupled with the reduction of this coenzyme, NADPH2 has to be formed by the ATP-consuming process of reverse electron flow. Common sewage plants are in most cases efficient enough to remove excess nitrogen from domestic water (20--40 mg/1 as N) by tertiary treatment, i.e. nitrifcation and denitrification. The denitrification of industrial waste water of high nitrate content resembles more a biotechno-

284

G. Claus and H. J. Kutzner: Autotrophic denitrification by Thiobacillus denitrificans

logy process: Since poor in carbon and energy source these have to be added to the waste and thus allow to make the choice between heterotrophic and autotrophic denitrification. Further if the nitrate content is that high as to necessitate dilution before treatment questions arise such as which process might be most appropriate, i.e. continuous culture in a stirred vessel vs. a plugflow reactor, and which parameters are optimal in regard to maximal denitrification rate a n d / o r maximal nitrate removal. In this paper we report on some basic experiments with T. denitrificans which were performed before we employed this organism in a pilot scale process to be described in another paper (Claus and Kutzner 1985).

Materials and methods Isolation of Thiobacillus denitrificans To obtain a strain of T. denitrificans continuous culture was employed for the enrichment of this organism. A 500 ml fermenter (Biotec Fe 007) served as culture vessel, and the selective nutrient solution of Baldensperger and Garcia (1975) was used (g/l): Na2SzO~-5H20, 6.0; KNO3, 3.0; NaHCO3, 1.5; Na2HPO4, 1.5; KH2PO4, 0.3; MgSO4.TH20, 0.5; pH 7.5; one liter was supplemented with 1 ml of the following trace element solution (g/l): Na2MoO4.2 H20, 1.0; FeSO4.7 HzO, 38.0; CaCO3, 2.0; ZnSO4-7 H20, 1.5; MnClz.4HzO, 1.0; C H S O a . 5 H 2 0 , 0.25; COC12.6H20, 0.25; NiC12.6H20, 0.25; H3BO3, 0.5; HCI (32%), 50.0. The medium was inoculated with garden soil and incubated under anaerobic conditions at 28 ° C. After commencement of growth the continuous culture was started, using a dilution rate of D=0.06 h -1. After 5 days a pure culture was isolated using the same medium with agar (anaerobic conditions). The agar plates yielded tiny white colonies of Gram-negative rods, typical of 2: denitrificans (Kuenen and Tuovinen 1981).

Culture conditions A. Experiments to determine the optimal temperature and pH for denitrification were performed in 10 ml screw cap test tubes. The same medium as used for the isolation was employed. The inoculum consisted of cells of a four days old culture, i.e. centrifuged from the same volume and medium. The extent of denitrification was determined after 48 hours incubation. -Testing the influence of ions related to the process such as NO 7, NO 2, $20~--, and S O 4 - the same test tubes and inoculum were used. The following base medium was supplemented with various concentrations of the ions mentioned above. Base medium (g/l): NazS203.5H20, 3.0; KNO3, 1.5; NaHCO3, 1.0; NazHPO4, 1.5; KHzPO4, 0.3; MgSO4-7H20, 0.5; trace element solution (see above), 1 ml; pH 7.5. The extent of denitrification in the base medium after 48 hours incubation was set 100%. B. The time course of denitrification in batch culture was determined in a 10 1 laboratory fermenter (Biostat V, B. Braun Melsungen) at 30°C. The same medium as for the isolation

was used; however the content of nitrate and thiosulfate was increased to 10.0 g Na2S203.5 H20 and 4.8 g KNO3 per liter. Before inoculation with a four day old culture (500 ml, i.e. 5%) the nutrient solution was gassed with nitrogen to exhaust oxygen. The pH of 7.5 was maintained by automatic addition of 1 tool/1 KOH by a Metrohm pH-stat-unit, Dosimat 655 with recorder. C. The kinetic parameters, e.g. growth rate and denitrification rate, were determined in a chemostat: The 101 laboratory fermenter was inoculated like the batch culture, and after a distinct turbidity became apparent the continuous flow was started. Each dilution rate was kept constant until a steady state had been reached as it could be recognized by continuous analysis of nitrate removal and formation of dinitrogen gas.

Analytical methods The cell mass was determined routinely by measuring the optical density of the culture broth at 560 nm. A calibration chart, i.e. cell dry weight vs. turbidity, was prepared by filtering the broth through pre-weighed membran filter discs (0.45 p~m) and drying at 105°C for 12h. Nitrate was determined potentiometrically using the ion sensitive nitrate electrode Orion 93-07, the reference electrode Orion 90-02 and the Orion Ionalyzer 901. The ion strength was adjusted with 2% of a 1 tool/1 KHzPO4 solution. Nitrite was determined photometrically at 546 nm after formation of the diazonium salt of sulfanilic acid and its coupling with N(1)-naphthylethylenediamine hydrochloride (Freier 1964). Thiosulfate was determined as iron complex of thiocyanate photometrically at 460 nm (S6rbo 1957). Sulfate was titrated with Pb(C104)2 under potentiometric indication with an ion selective lead electrode (Ingold). The denitrification gas was passed through granular sodium lime to absorb CO2 and then determined with a gas meter (Brand, Wertheim, FRG). Occurrence of N20 and NO in the denitrification gas was determined by gas chromatography equipped with an electron capture detector (for NzO) and by a chemiluminescence detector (for NO) respectively. (These analysis were carried out by Dr. W. Seller, MPI ffir Chemie Mainz, Abt. Chemic der Atmosph~ire). All photometric measurements were made on a Zeiss PMQ 2 spectrophotometer (Zeiss, Oberkochen, FRG). All chemicals were of analytical grade and purchased from E. Merck (Darmstadt, FRG).

Results and discussion

Nitrogen source Since a medium with nitrate as electron acceptor as well as nitrogen source was employed for enrichment and isolation of T. denitrificans, a strain was obtained which did not need ammonium. Thus our isolate is capable of assimilatory as well as of dissimilatory nitrate reduction -- similar to the strain " R T " described by Baldensperger and Garcia (1975). Other strains of T. denitrificans described in the literature, however, required am-

G . Claus and H. J. Kutzner: Autotrophic denitrification by Thiobacillus denitrificans

285

Denitrification (%)

Denitrification (%) 10o-

f ° ~

°

8o6o-

60-

4o-

40-

zo-

20o

o1o

2'o

~0

4'0

0

6

~

6 pH

Temperature ('C)

Fig, 1. Influence of temperature on denitrification activity. (Maximal denitrification set to 100%)

Fig. 2. Influence of pH of medium (initial) on denitrification activity. (Maximal denitrification set 100%)

monium as nitrogen source (Baalsrud and Baalsrud 1954; Hutchinson et al. 1967).

the denitrification of high concentrated nitrate waste water would be the amount of the sulfate produced. -- Nitrite as an intermediate of denitrification exhibited a strong inhibition at rather low concentrations (Fig. 4). Since all compounds were used as sodium salts, sodium chloride was also tested to exclude an inhibitory effect of sodium or of osmotic pressure. At the concentrations used for these compounds sodium chloride has no effect on denitrification; only 30 g/1 (0.52 mol/1) and more are inhibitory to the process (Fig. 3).

Effect of temperature and pH on denitrification These experiments were carried out in test tubes. After 48 h of incubation the extent of denitrification was evaluated by the determination of residual nitrate. As shown in Figs. 1 and 2 the optimal conditions for denitrification with our isolate of T. denitrificans were 30 ° C and an initial p H 7.5 to 8.0. Under these conditions about 80% of the nitrate supplied were reduced. The same optima for temperature and p H were found for the strain of T. denitrificans employed by Steinmtiller and Kutzner (1981) for denitrification. These isolates thus prefer a rather high p H as compared with the strain of Baalsrud and Baalsrud (1954) whose optimum was between p H 6.2 and 7.0. Most authors employ media for T. denitrificans with a p H of 7.0 (Taylor et al. 1971; Justin and Kelly 1978).

Time course of denitrification in batch culture This experiment was carried out in a 10 1 fermenter with stirring; the result is shown in Fig. 5" Denitrification (%) 100- - - O

~

Q

80-

Effects high concentrations of thiosulfate, nitrate, nitrite, and sulfate (see Eq. 3)

60-

40-

As it can be seen, T. denitrificans is most sensitive to sulfate, the endproduct of the process (Fig. 3). The inhibition began at concentrations above 5 g / 1 (0.05 mol/l); at 20 g/1 no denitrification activity could be observed. 5 g S 0 4 / 1 correspond to the oxidation of 2.9 g $20 3/1 and the concommitant reduction of 2.0 g NO 3/1. The substrates of the process, nitrate and thiosulfate, showed inhibition between 10 and 20g/1 (ca. 0.2 mol/1 (Fig. 3)). These results indicate that the limiting factor for

20-

0

0

j 10

20

30

40

50

Concentration (g/I)

Fig. 3. Influence of some ions on denitrification activity. Resuits of low concentrations not yet inhibitory are not shown, i.e. curves of the respective salts start at the highest concentration without negative effect. © - - © Sulfate; []--EJ Thiosulfate; A - - A Nitrate; 0 - - 0 NaC1

286

G. Claus and H. J. Kutzner: Autotrophic denitrification by Thiobacillus denitrificans

Denitrification (%)

tion of sulfate during denitrification (Eq. 1). Nitrite was produced only in small amounts after 60 hours and disappeared later on.

100-O-o

Continuous culture experiments

6040-

For the determination of some kinetic and stoichiometric parameters of the denitrification process a nitrate-limiting chemostat culture was employed. The following equations for the calculation of the maximal specific growth rate ~tmax and the saturation constant Ks were used (Veldkamp 1976):

~

200

I 0.5

0.0

i 1.0

i 1.5

2.0

Nitrite (g/l)

Fig. 4. Effect of nitrite on denitrification activity. The extent of denitrification without nitrite was set 100%

After a lag period of about 10 hours growth and denitrification commenced; the reduction of nitrate was parallel to the consumption of thiosulfate; also the production of nitrogen gas and protons was stoichiometrically associated with nitrate reduction. As to be expected growth was parallel to the denitrification process. After 70 hours of cultivation a drop in the optical density occurred, due to the clumping of cells at this time. Remarkably was the observation that the denitrification rate and also the growth rate already decreased after only just half of the nitrate and thiosulfate were consumed. Due to the stoichiometric relation between thiosulfate disappearance and sulfate occurrence it is assumed that this was due to the inhibitory effect of the increasing concentra-

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NO3-'$203~NO2gll II mgll 160]

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~ z x

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%

.~"

0.6 6

120

-150

Ks + SR

(4)

]•max = Dc - SR

(5) Do = critical dilution rate SR = incoming substrate concentration = substrate concentration in the culture vessel at dilution rate D Figure 6 shows the steady state relationships observed at various dilution rates: The cells were washed out of the system when the dilution rate reached the critical value D e = 0.11 h-1. If we assume that the saturation constant (Ks) is very low as compared with the incoming substrate concentration (SR) ~max becomes equal to Dc. The critical dilution rate found here is in accordance with those described by other authors,

0

A []

H+ N2 mmol mg I.h I'h 25. o

0B-(16.t

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!

02 2

~ m 0 - • - I t ~

6

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30-r~--~O

,

60

~

70

,

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-- O0 0

~00

Time(h)

Fig. 5. Time course of denitrification by T. denitrificans in batch culture: (©) Nitrate concentration, (/x) thiosulfate concentration, ( n ) nitrite concentration, (B) cell mass, optical density at 560 nm, (O) N2 evolution, (A) H+-ions evolution

0

•

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Cetts NOk ~03" (g/O (g/t) (g/t)

0.04

0.06

0.C)8

0.10

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0 ~-0

Dilution rate (h "1)

Fig. 6. Steady state results from the nitrate-limiting chemostat: (D) cell concentration, (A) nitrate concentration, (O) thiosulfate concentration, (/',) rate of N2 evolution, (©) rate of H +ions evolution. Incoming substrate concentrations: 1.23 g NO3/1; 1.90 g $203/1

287

G. Claus and H. J. Kutzner: Autotrophic denitrification by Thiobacillus denitrificans

e.g. Justin and Kelly (1978): Dc=0.08 h -1, Baldensperger and Garcia (1975): #m~x=0.14h -1 (calculated from the generation time of 5 h for strain " R T " under aerobic and anaerobic conditions). Nitrate (which is supplied in a concentration of 1.2 g/l) could not be detected over a wide range of dilution rates: until D = 0 . 0 8 6 h -~ its concentration was below the detection limit of 0.5 mg N O 3 / I . Therefore Ks could not be read from the graph at 1~2De as it is usually done but had to be calculated according to Eq. (5) at higher dilution rates. At D = 0 . 0 9 3 h -~ the nitrate concentration Y in the culture vessel was 1 mg N O 3 / 1 , i.e. K s = 0 . 2 mg N O 3 / 1 . A similar value, 0.13 mg NO 3/1, has been found for T. denitrificans by Batchelor and Lawrence (1978). Thus the saturation constants are in about the same range as those obtained for heterotrophic mixed populations which range between 0.27 to 0.5 mg NO3-/1 (Christensen and Harrem6es 1977; Engberg and Schroeder 1975). The maximum rate of N2 evolution as measured at D = 0 . 1 0 h -1 was 24.5 mg N2/1.h (165 mg N2/g cells- h); it corresponds to the rate of nitrate consumption of 0.78 g NO 3 / g cells.h. A similar value, i.e. 160 mg N2/g cells.h, was reported by Shimizu et al. (1978) for the heterotrophic bacterium Paracoccus denitrificans with glucose as energy source. An analysis of the denitrification gas at the dilution rate of 0.07 h - ~ showed that it consisted of almost pure dinitrogen; the concentrations of N20 and NO were below 1 ppm. Data in the literature stem all from batch culture experiments and vary greatly: Carlson and Ingraham (1983) found up to 50% N 2 0 in the gaseous products with Pseudomonas aeruginosa and Paracoccus denitrificans, whereas Pseudomonas stutzeri produced very little N20. Similar results were obtained by Burth and Ottow (1983): much N 2 0 w a s produced with Acinetobacter spec. and Pseudomonas aeruginosa, little with Bacillus licheniformis and Moraxella spec. Nitrite did not accumulate during steady state operation. Cell mass and thiosulfate concentration were nearly constant between D =0.06 and 0.1 h -1. -- From the data obtained several parameters were estimated (Table 1). The values for YNO~ and Ys2or are somewhat lower than those reported by Justin and Kelly (1978): these authors found Ys2o7 = 11.37 g cells/ mol $ 2 0 ~ (corrected for maintenance), i.e. equivalent to YNo~-=8.55 g cells/mol N O ~ , if $203--/NO3- (molar) is 0.75 as given by these authors. In contrast, we obtained for $ 2 0 3 / N O 3

Table 1. Kinetic constants and stoichiometric coefficients

from the nitrate-limiting chemostat culture Maximum specific growth rate

0.11 h -1

O~r,,x) Maximum specific rate of N2 evolution Maximum specific rate of NO 3 reduction Saturation constant Ks Yield coefficient YNO7 Yield coefficient Ys2o3 820 7 / N O ~ (molar) H + / N O ;- (molar) N 2 / N O ;- (molar) SO 7 / N O ;- (molar)

165 mg Nz/g cells.h 0.78 g NO3/g cells-h 0.2 mg NO3/1 0.129 g cells/g NO ;(7.98 g cells/mol NO 7) 0.085 g cells/g SzO 3 (9.52 g cells/mol S20 3) 0.81 0.32 0.47 1.62

(molar) the value of 0.81. The difference could be partly due to the fact, that in our strain assimilative nitrate reduction took place in addition to the dissimilatory process. In chemostat experiments with higher concentrations of nitrate (above 2.0 g NO3/1) and stoichiometric amounts of thiosulfate no stable steady state could be reached and nitrite accumulated. This was obviously also an effect of the formation of the inhibiting concentrations of sulfate. Acknowledgements. This work was supported by a grant from the Bundesministerium for Forschung und Technologie, 02 WA 119. We thank Dr. W. Seiler of the Max Planck Institut fiir Chemie, Mainz, for the analysis of the denitrification gas for N~O and NO.

References Baalsrud K, Baalsrud KS (1954) Studies on Thiobacillus den# trificans. Arch Microbiol 20:34--62 Baldensperger I, Garcia IL (1975) Reduction of oxidized inorganic nitrogen compounds by a new strain of Thiobacillus denitrificans. Arch Microbiol 103:31--36 Batchelor B, Lawrence AW (1978) Autotrophic denitrification using elemental sulfur. J Water Poll Contr Fed 50:1986--2001 Burth I, Ottow JCG (1983) Influence of pH on the production of N20 and N2 by different denitrifying bacteria and Fusarium solani. Environ Biogeochem 35:207--215 Carlson CA, Ingraham JL (1983) Comparison of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans. Appl Environ Microbiol 45 : 1247-- 1253 Christensen HM, Harrem6es P (1977) Biological denitrification of sewage: a literature review. Pro Water Technol 8 : 509--555 Claus G, Kutzner HJ (1985) Autotrophic denitrification by Thiobacillus denitrificans in a packed bed reactor. Appl Microbiol Biotechnol 22:289--296

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G. Claus and H. J. Kutzner: Autotrophic denitrification by Thiobacillus denitrifieans

Engberg D J, Schroeder ED (1975) Kinetics and stoichiometry of bacterial denitrification as a function of cell residence time. Water Res 9:1051--1054 Freier RK (1974) Wasseranalyse. Walter de Gruyter, Berlin Hutchinson M, Johnstone KI, White D (1967) Taxonomy of anaerobic thiobacilli. J Gen Microbiol 47:17--23 Justin P, Kelly DP (1978) Growth kinetics of Thiobaeillus denitrifieans in anaerobic and aerobic chemostat culture. J Gen Microbiol 107:123--130 Kuenen JG, Tuovinen OH (1981) The genera Thiobaeillus and Thiomierospira. In: Starr MP, Stolp H, Trtiper HG, Balows A, Schlegel HG (eds) The Prokaryotes. Springer, Berlin Heidelberg New York, pp 1023-- 1036 Schedel M, TAper HG (1980) Anaerobic oxidation of thiosulfate and elemental sulfur in Thiobacillus denitrificans. Arch Microbiol 124:205--210 Shimizu T, Furuki T, Waki T, Ichikawa K (1978) Metabolic characteristics of denitrification by Paraeoeeus denitrificans. J Ferment Technol 56:207--213 SOrbo B (1957) A colorimetric method for the determination of thiosulfate. Biochim Biophys Acta 23:412--416

Steinmtiller W, Kutzner HJ (1981) Nitrat-Eliminierung aus vorwiegend anorganisch belasteten Industrie-Abw~issern dutch autotrophe Denitrifikation. Landwirtsch Forsch Sonderheft 37: 527--540 Taylor BF, Hoare DS, Hoare SL (1971) Thiobacillus denitrificans as an obligate chemolithothroph. -- Isolation and growth studies. Arch Microbiol 78:193--204 Veldkamp H (1976) Continuous culture in microbiol physiology and ecology. Meadowfield Press Ltd, Durham, England Winkler M (1984) Biological control of nitrogenous pollution in wastewater. In: Wiseman A (ed) Topics in enzyme and fermentation biotechnology, vol 8. Ellis Horwood, Chichester, 31--124 Zumft WG, Cardenas J (1979) The inorganic biochemistry of nitrogen bioenergetic processes. Naturwissenschaften 66:81--88

Received November 29, 1984/Revised February 26, 1985