The Effect of Different Carbon Sources on Respiratory Denitrification Nautska et al

JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 82, No. 3, 277-285. 1996

The Effect of Different Carbon Sources on Respiratory Denitrification in Biological Wastewater Treatment NATUSCKA M. LEE* AND THOMAS WELANDER,*

Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, S-221 00 Lund, l and Anox AB, Ideon Research Park, S-223 70 Lund,2 Sweden

Received 12 December 1995/Accepted 30 June 1996

The respiratory denitrification activity of activated sludge bacteria with diierent carbon sources (acetic acid, crude syrup, hydrolyzed starch, methanol-with and without a small amount of yeast extract) was studied in long-term continuous cultivations and batch tests. Mass balance calculations showed that the main product in long-term cultivations with all carbon sources was molecular nitrogen. However, the type of carbon source had a significant influence on the denitrification rate, denitrification yield, sludge yield and the composition of the microflora. With acetate and methanol higher denitrification yields, lower sludge yields and more true (end product N& denitrifying bacteria were obtained than with crude syrup and hydrolyzed starch. Furthermore, with acetate a higher growth rate and a higher denitrification rate was obtained than with methanol.

[Key words: biological wastewater treatment, nitrogen removal, respiratory denitrification, acetic acid, hydrolyzed starch, methanol]

Respiratory denitrification is one of the main microbiological processes for nutrient removal in biological wastewater treatment. Preceded by ammonification and nitrification, which reduce organic nitrogen into oxidized inorganic nitrogen compounds such as nitrate, denitrification is the last step in the nitrogen cycle where the nitrate is transformed through several intermediary products (NO*-, NO, N20) to molecular nitrogen. It is an anaerobic process where the oxidized nitrogen compound serves as an electron acceptor, while the electron donor may be of either inorganic or organic nature (1). In municipal wastewater treatment, the respiratory denitrification relies upon a carbon source as an electron donor. However, the concentration of available carbon source in the wastewater may not always be sufficient, so that an external carbon source may be needed (2). Some of the main requirements for a suitable external carbon source, apart from low costs, are a non toxic/non dan- gerous nature, a low sludge yield and the ability to stimu- late a complete denitrification without the need for ad- aptation of the microflora, so that environmentally detrimental, intermediary products such as nitrite and nitrogenous oxides can be avoided.

Several external carbon sources have been tested and found to have various effects on the respiratory denitrification in terms of the denitrification rate, the denitrification yield and the sludge production (3). However, these studies have mostly been performed on unadapted wastewater or activated sludge in batch reactors for kinetic determinations, whereas only a few have paid attention to the long-term influence of a carbon source on the denitrifying microflora (4-6). Some of these studies have focused on the effect of methanol and have shown that appendaged bacteria, mainly Hyphomicrobium, seem to be favoured (7, 8). In other microbiological investigations in different types of denitrification systems with external carbon sources, in- cluding methanol, both Hyphomicrobium as well as other bacterial genera have been observed (9-13; Dr.

* Corresponding author.

Hilde Lemmer, personal communication, Bavarian State Agency, D-8000 Munich 22, Germany).

The aim of this study was to determine the denitrification activity of bacteria selectively enriched from activated sludge with different carbon sources. Both long-term cultivations in chemostats as well as batch tests were performed in order to determine the denitrification rate, the denitrification yield, the sludge yield and the microbial composition. Four carbon sources were selected: acetate and methanol, which are commonly used either in full scale or in laboratory scale (3), and two carbohydrate products, crude syrup and hydrolyzed starch, which have been used to some extent as external carbon sources.

MATERIALS AND METHODS

Chemicals All chemicals were of pro analysis quality, except for crude syrup and hydrolyzed starch. Crude syrup, a by product from sugar refining, contains 800g sucrose/l and 50-lOOg/l unspecified organic matter, and the COD content is 9OOg/l. Hydrolyzed starch consists of 60% lower (mono-, di-, tri-saccharides) and 40% higher sugar compounds, and the COD content is 860 g/l.

Analyses Nitrate, nitrite and ammonium were analysed according to (14). Chemical Oxygen Demand (COD) was measured as described in (15). Acetic acid and methanol were measured by the gas chromatographic method as described in (16). Starch and sucrose/D- glucose/Dfructose were analysed enzymatically by kits from Boehringer Mannheim, no. 207748 and no. 716260, respectively. Total Suspended Solids (TSS) were determined according to (17), using 0.45 pm membrane filters (Schleicher & Schuell). Nitrogen gas, dinitrogen oxide, nitrogen oxide and carbon dioxide were analysed by gas chromatography as described in (18). The oxidation-reduction potential (ORP) was monitored in the reactors throughout the experiment as described in (19). Microscopic examinations by means of phase contrast and interference microscopy (Nikon Optiphot-2) of the

277

278 LEE AND WELANDER J. FERMENT. BIOENG..

microflora in the chemostats were carried out throughout the experiment.

Chemostat experiments The effect of a sole carbon source on the denitrification activity was studied in four 5OOml chemostats operated in parallel on the same basic sterile medium, but with different carbon sources: acetic acid, crude syrup, hydrolyzed starch and methanol. The basic medium was comprised of 3.25 g/l KN03, 190 mg/f NH&l, 520 mg/l K2HP04. 3Hz0 and all necessary trace metals as described in (19). The carbon sources were added to respective media at a concentration corresponding to 1.40 g/l Chemical Oxygen Demand (COD). From day 77 of the experiment, 200mg/l Difco yeast extract (195 mg/l COD) was added to all media in order to see if the denitrification activity could be further improved by adding growth factors. The temperature in the chemostats was kept at 15C. The pH was maintained at 7.0 by automatic titration with 0.5 M HCl. The reactor effluent was led through a gas trap for the determination of the gas production rate and the gas composition. Ini- tially, each of the chemostats was inoculated with 10ml of activated sludge from the anoxic zone at the Malm(i sewage works, where no external carbon source is added. A continuous flow of medium was started, giving a hydraulic retention time (HRT) of 24 h in the chemostats. This HRT was maintained throughout the

0 IO 20 30 40 50 fla 70 80 90 100110 120 0 10 20 30 40 50 60 70 80 90 100110120

Time (d) Time (d)

800

700

600

500 8

400

300

ux)

100

0

ol I I I , I I 1-u I I I I 1oo 0 10 20 30 40 50 60 70 80 90 100110120

Time (d)

experiment, with the exception of the chemostat fed methanol medium, for which the HRT was prolonged to 36 h on day 108 of the experiment. Nitrate, nitrite, ammonium, COD and dry matter in the reactor effluent as well as nitrogen, dinitrogen oxide, nitrogen oxide and carbon dioxide in the produced gas were measured regu- larly during a period of 120 d. At the end of the experiment (10d for the reactors on acetic acid, crude syrup and hydrolyzed starch, 7 d for the methanol reactor), a nitrogen mass balance was performed around the reactor, in order to see if all nitrogen in the influent could be accounted for all nitrogen species measured in the gas and the reactor effluent.

Batch studies The maximum denitrification rate of the enrichments in the chemostats was determined at the end of the experiment by stopping the feed to the chemostats, adding 0.72 g/f KN03 and respective carbon source in an amount corresponding to 0.50 g/l COD and then following the nitrate, nitrite and dinitrogen oxide concentrations for a couple of hours. The maximum denitrification rate was compared with the apparent denitrification rate, which was calculated from the values obtained on nitrate removal, TSS production and the HRT in the chemostat experiments.

Isolation and characterization of deuitrifying bacteria Bacteria were isolated from the enrichments at the end

0 IO 20 30 40 50 60 70 80 90 100110120

Time (d)

FIG. 1. Concentrations of nitrate and COD in the effluent from the denitrifying enrichments on different carbon sources versus time. (a) Acetic acid; (b) crude syrup; (c) hydrolyzed starch; (d) methanol. Symbols: l , nitrate; 0, COD. The concentrations of nitrate and COD in the influent were 450mg N/I and 1,400 mg/l, respectively. From day 77 the COD in the influent was increased to 1,595 mg/l by adding growth factors in form of yeast extract (y.e.). The HRT in all reactors was 24 h, except for in the methanol enrichment in which the HRT was increased on day 108 to 36 h (Fig. Id). A summary of all results is given in Table 1.

VOL. 82, 1996 DENITRIFICATION WITH DIFFERENT CARBON SOURCES 279

of the chemostat experiments (on day 90 for all carbon sources, except for methanol, which was on day 120). Two agar media were used: the basic medium used in the chemostat experiments diluted 10 times with 1 g/l of respective carbon source and 15 g/l agar (Bacto, Difco, Mich., USA), and Tryptone glucose extract agar (TGEA, Difco). Samples were withdrawn from the reactors and diluted in a sterile solution containing 8 g/l sodi- um chloride, 1 g/l peptone (Difco) and 1 g/l Tween 80 (Sigma, MO, USA). The diluted samples were spread on agar plates which were incubated aerobically for 7 d at 25C. In addition, plates with the basic medium were also incubated anaerobically (BBL GasPak Systems) for 14 d. For each sample, 20 colonies were picked randomly from plates with 30-300 colony forming units. The colonies were restreaked several times for purity.

The isolates were characterized by morphology, gram-, cytochrome c oxidase-, catalase- and urease-reactions, oxidative and fermentative acidification of glucose, hydrolysis of starch and gelatin and production of indole and hydrogen sulfide, as described in (20). The isolates were further characterized by the oxidation pattern of 95 carbon sources at 30C within 24-48 h according to the identification system of Biolog (Biolog Inc., California, USA). Identifications were considered as acceptable when the similarity index of the isolate with the Biolog Strain Library was above 0.5, and in consistent with the results from the phenotypical characterizations. The denitrifying ability was first tested by a simplified test based on anaerobic cultivation in broth medium with 0.1% KN03 and 0.17% agar (20). Isolates showing gas production were further tested in liquid medium cultivations for true respiratory denitrifying ability. The cultures were transferred to serum bottles containing the same medium as used in the chemostat experiments but diluted four times. The gas phase in the bottles was replaced by sterile oxygen-free nitrogen gas and the bottles were then incubated at 25C for 24-48 h, after

which nitrate, nitrite, dinitrogen oxide, COD and total suspended solids were analysed.

RESULTS

Denitrification with acetic acid After 50 d of operation, the concentrations of nitrate and COD in the reactor stabilized (Fig. la, Table 1). No acetic acid could be detected in the effluent, which indicated that the denitrification process had become carbon substrate limit- ed. After the addition of yeast extract to the feed from day 77, the average nitrate concentration decreased from 127 to 48 mg N/1, although it showed some variation during the rest of the experiment. The COD concentration in the effluent increased somewhat from 50 to 88 mg/l, but no acetic acid could be detected. Initially (days 14- 39), some nitrite was produced (at maximum 40 mg N/I), but it decreased to below 1 mg N// as the denitrification process stabilized (from day 51 to 120, Fig. 2a). The ammonium concentration in the effluent was around 8 mg N/I from day 51 to 77, but it increased to 13 mg N/I when yeast extract was added (from day 80 to 120, Fig. 2a). Molecular nitrogen was the only nitrogen compound detected in the gas phase from day 51 to 120. The nitrogen mass balance showed that the nitrogen measured in the influent could be accounted for in the reactor effluent (Table 4). The oxidation-reduction potential (ORP) was around - 164 mV from day 51 to 77, but increased to - 128 mV when yeast extract was added.

The denitrification yield was 0.25 g N/g COD without yeast extract and 0.28 g N/g COD with yeast extract (Table 2). The maximum denitrification rate of the enrichment was found to be 76 mg N/g TSS. h, whereas the apparent denitrification rate was calculated to be 48 mg N/g TSS . h (Table 3).

The microscopic examinations showed the enrichment to be strongly dominated by gram-negative, rod-shaped, motile bacteria. Similar rod-shaped bacteria were found

TABLE 1. Average values of COD, TSS, ORP and different nitrogen species in the intkent and the effluent from the long-term cultivations of denitrifying bacteria on different carbon sources in chemostats

Carbon source and COD in COD out experimental period (mg/l)

Nysgl;in NO?-gyOout N02--N out NH4+-N in NH4+-N out TSS out ORP (mg/l) m m (mg/0 (mg/0 (mg/l) (mg/l) (mV)

Acetic acid (day 5 l-77)a

Acetic acid (day 80-120)b

Crude syrup (day 5 l-77)

Crude syrup (day 80-120)b

Hydrolyzed starch (day 5 l-77)

Hydrolyzed starch (day 80-120)b

Methanol (day 5 l-77)

Methanol (day 80-108)b

Methanol (day 113-120)E

1376 (s=20)

1561 (s=52)

1413 (s=43) 1607

(s=67) 1486 (s=38) 1708

(s=92) 1423

(s=42) 1620

(s= 16) 1620

(s= 16)

(s=5i) (s =Z)

210 (s=53)

196 (s= 17)

249 (s=27)

245 (s=33)

815 (s=l81)

534 (s=91)

(s ,:A)

464 127 (s=34)d (s=16)

(s=4&) (s %t)

143 (s=20)

(s %)

461 (s = 30)d

(s Y3)d

452 (~=23)~

(2%) 306

(s=57) 158

(s=30)

(s =2:4)

0.03 (s=O.Ol)

0.05 (s=O.O3)

(szf2.3) 0.65

(s=2.7)

(S %4)

0.06 (s=O.O4)

0.06 (s=O.Ol)

0.05 (s=O.O2)

(s %%)

(s _5445)d (SZ.1,

(s =Y.2)

(s =?oy (s =7.0) 7.7

(s =5:.0)d

(s=3.3)

(s zi.6) 2.7

(s=3.6)

(S,5:.l)d (s =361.0)

(s=~:.S)

(s =z.O)

272 -164 (s=61) (s=15)

352 ~ 128 (s==60) (s= 16)

307 -86 (s=80) (s=24)

517 -55 (s=81) (s=7)

277 - 128 (s-=63) (s=45)

441 -97 (s=49) (s=7)

(S L$l3) (s-$

328 - 155 (s==73) (s=8)

a Without yeast extract. b With yeast extract. c With yeast extract and HRT increased from 24 h to 36 h. d Average values for the total experimental period (day 51-120). Abbreviations: COD =chemical oxygen demand; HRT = hydraulic retention time; ORP = oxidation reduction potential; s = standard deviation;

TSS = total suspended solids.

280 LEE AND WELANDER J. FERMENT. BIOENG.,

to dominate on both cultivation media under aerobic and anoxic conditions. The dominating true denitrifying strain (70% of the isolates obtained on the agar plates) was tentatively identified as Pseudomonas alcaligenes subgroup A (Table 5). The other less common true denitrifying strain was identified as Acinetobacter johnsonii by the Biolog Identification System, but the similarity index (0.47) was only moderately acceptable. Finally, a nitrite-producing, fermentative bacterium, Aeromonas hydrophila, was also isolated from the reactor.

Denitrification with crude syrup After 65 d of operation, the COD concentration in the reactor stabilized at around 210mg/l (Fig. lb, Table l), and no sucrose, glucose or fructose could be detected in the effluent. The nitrate concentration in the reactor was around 164mg N/I. The addition of yeast extract from day 77 did not show any improved effect on the denitrification process. The nitrite concentration was initially high (around days 14 to 39, at maximum 106mg N/I), but from day 50 it was below 1 mg N/I, except for a transient increase around days 69 to 75 (at maximum 40mg N/I, Fig. 2b). After the addition of yeast extract a significant increase of nitrite (to 13 mg N/f) was observed on day 90, but after this it remained below 1 mg N/I for the rest of the experimental period. The ammonium concentration in

50

45 a

110 Acetic acid i A.a.with y.e. loo

-90

-80

- 70 5 ??

-6O- z

- 50 i -402

- 30

- 20

- 10

0 0 IO 20 30 40 50 60 70 80 90 100110120

Time (d)

50

45 c Hydrolyzed starch! H.s. with y.e.

IO 20 30 40 50 60 70 80 90 100110120 Time (d)

the effluent was 12mg N/I from day 51 to 77, but it decreased to 8 mg N/I when yeast extract was added (Fig. 2b). During the experiment, molecular nitrogen was the only nitrogen compound found in the gas phase from day 51 to 120. The nitrogen mass balance showed that the nitrogen measured in the influent could be accounted for in the reactor effluent (Table 4). The ORP was around -86mV from day 51-77, but increased to -55 mV when yeast extract was added.

The denitrification yield was 0.26g N/g COD without yeast extract and 0.22g N/g COD with yeast extract (Table 2). The maximum denitrification rate of the enrichment was found to be 48 mg N/g TSS. h, whereas the apparent denitrification rate was calculated to be 26 mg N/g TSS . h (Table 3).

The microscopic examinations showed the enrichment to be strongly dominated by motile and rod-shaped, gram-negative bacteria. No significant difference in the composition of the microflora was obtained between the different cultivation media and incubation conditions. Two true denitrifying isolates, both possibly belonging to Pseudomonas, were obtained (Table 5). In addition, two types of nitrite-producing, fermentative bacteria (Enterobacter asburiae and Klebsiella ozaenae) were isolated.

Crude syrup / C.S. with y.e. II 0

0 10 20 30 40 50 60 70 80 90 100110120 Time (d)

80

70 -5 E 60-

50 5 40 E

z 30

40 =1 m 35 .S 30 z 5 25 .3 520

5 15

10 i

0 10 20 30 40 50 60 70 80 90 100110120 Time (d)

FIG. 2. Concentrations of ammonium and nitrite in the effluent from the denitrifying enrichments on different carbon sources versus time. (a) Acetic acid; (b) crude syrup; (c) hydrolyzed starch; (d) methanol. Symbols: *, ammonium; 0, nitrite. The concentrations of ammonium and nitrite in the influent were 50 mg N/I and 0 mg N/I, respectively. Together with nitrate, the total nitrogen content was 500 mg N/I. From day 77 the total nitrogen content in the influent was increased from 500 to 517 mg N/I by adding growth factors in form of yeast extract (y.e.). The HRT in all reactors was 24 h, except for in the methanol enrichment in which the HRT was increased on day 108 to 36 h (Fig. 2d). A summary of all results is given in Table 1.

VOL. 82. 1996 DENITRIFICATION WITH DIFFERENT CARBON SOURCES 281

TABLE 2. Average growth and denitrification yields and COD mass balances for the long-term cultivations of denitrifying bacteria on different carbon sources in chemostats

Carbon source HRT Period Growth yield (Y) (days x-ty)

Denitrification yield (YDN) (h) (g TSS/g COD removed) (g NOJ--N removed/g COD removed)

Acetic acid 24 51- 71 Acetic acidb 24 80-120 Crude syrup* 24 51- 77 Crude syrupb 24 SO-120 Hydrolyzed starcha 24 51- 1-l Hydrolyzed starchb 24 80-120 Methanolb 24 80-108 Methanolb 36 113-120

0.20 (s=O.O5) 0.24 (s=O.O4) 0.26 (s=O.O6) 0.35 (s =0.05) 0.22 (s=O.O5) 0.30 (s=O.O3) 0.24 (s=O.O6) 0.26 (s=O.O4)

0.25 (s=O.Ol) 0.28 (s=O.O2) 0.26 (s =0.05) 0.22 (s=O.O3) 0.19 (s=O.O2) 0.20 (s=O.O2) 0.27 (s=O.Ol) 0.28 (s=O.O02)

COD mass balancef (g COD oxidized + biomass/

g COD removed)

1.02 1.14 1.11 1.14 0.87 0.99 1.11 1.14

Abbreviations: COD= chemical oxidation demand; HRT = hydraulic retention time; s = standard deviation; TSS = total suspended solids. a Without yeast extract. b With yeast extract (corresponding to 195 mg/l COD). c Calculated as: [Y . 1.4]+ [YDN. 2.861. Assumptions (31): 1 g TSS = 1.4 g COD; 1 g N09--N oxidizes to 2.86 g 02.

Denitrification with hydrolyzed starch The nitrate and COD concentrations in the effluent stabilized after about 40 d of operation (Fig. lc, Table 1) and no starch or glucose could be detected in the effluent. The nitrate concentration in the effluent was around 209 mg N/I and the COD concentration around 249 mg/l. The addition of yeast extract to the medium on day 77 improved the denitrification process somewhat (the nitrate concentration decreased to 160mg N/I), but not the COD removal. The nitrite concentration was high during the start-up period (around days 14 to 31, at maximum 76 mg N/I), and a second transient increase (at maximum 26mg N/I) was observed between days 75-78. But, after the addition of yeast extract from day 77, the nitrite concentration remained below 1 mg N/I (Fig. 2~). The ammonium concentration in the effluent was 1Omg N/l from day 51 to 77, but it decreased to 3 mg N/I when yeast extract was added (Fig. 2~). No other nitrogen compound besides molecular nitrogen could be detected in the gas phase from day 51 to 120. The nitrogen mass balance showed that the nitrogen measured in the influent could be accounted for in the reactor effluent (Table 4). The ORP was about - 128mV during the period of 51-77 d, but increased to -97 mV from day 80 to 120.

The denitrification yield was 0.19g N/g COD without yeast extract and 0.2Og N/g COD with yeast extract (Table 2). The maximum denitrification rate of the enrichment was found to be around 42 mg N/g TSS. h, whereas the apparent denitrification rate was calculated to be 27 mg N/g TSS . h (Table 3).

The microscopic examinations showed the enrichment to be strongly dominated by motile and non-motile rod- shaped, gram-negative bacteria. No significant difference in the composition of the microflora was obtained between the different cultivation media and incubation conditions. Two true denitrifying bacteria could be isolated (Table 5). One of them was identified as Agrobacterium radiobacter. The other denitrifying isolate could not be identified by the Biolog Identification System, but belongs possibly to Pseudomonas. Three nitrite-producing, fermentative bacteria were isolated and tentatively identified as E. asburiae, K. ozaenae and A. hydrophila.

Denitrikxtion with methanol The enrichment of denitrifying bacteria growing with methanol as the carbon and energy source proceeded slowly and still after 2.5 months of operation, nitrate (306mg N/I) as well as COD (815 mg/l) were found at high concentrations in the effluent (Fig. Id, Table 1). The denitrification process was somewhat improved by addition of yeast extract to the medium from day 77 (the average concentrations of nitrate decreased to 158 mg N/I, and the COD to 534mg/l). However, not until the HRT was increased to 36 h on day 108, did the denitrification process improve considerably. The nitrate and COD concentrations decreased to 26 mg N/l and 75 mg/l, respectively, and no methanol was detectable in the effluent. The nitrite concentration was below 1 mg N/l throughout the experiment (Fig. 2d). The average ammonium concentration in the effluent was, as compared with the other denitrifying systems, relatively high (38 mg N/I) throughout the experimental period (from day 51 to 120, Fig. 2d). No

TABLE 3. Maximum denitrification rates, apparent denitrification rates and maximum growth rates of the denitrifying enrichments on different carbon sources

Carbon source Maximum denitrification rate (r&a (mg NO1--N/g.TSS-h) Apparent denitrification rateb

(mg N03--N/g.TSS. h) Maximum growth rate of the systemc

(h-l)

Acetic acid 76 (s=7) 49 0.091 Crude syrup 48 (s=S) 26 0.083 Hydrolyzed starch 42 (s=2) 21 0.062 Methanol 30-45d 29 >0.042, CO.028 he

B Determined in batch tests on the biomass in the chemostats at the end of the experimental period, i.e. after day 120. Calculations are based on two batch tests.

b Apparent denitrification rates calculated from the chemostat experiments during the days 80-120 for acetic acid, crude syrup and hydrolyzed starch, and days 113-120 for methanol.

c Calculations based on the denitrification rate obtained from batch experiments and on the growth yield obtained from the chemostat experiments. Growth rate (h-l)= [r ~~.2.86~Y]/[1-Y.1.4].1OOO.Assumptions(31): 1gTSS=1.4g02,and1gN03--Noxidizes2.86gO,.

d Denitrification rate calculated based on the maximum growth rate of the system. e Growth rate estimated from the chemostat experiments, with nitrate and COD in excess.


TABLE 4. Nitrogen mass balances around the denitrifying enrichments on different carbon sources in the chemostats during 7-10 d at the end of the experiment

Carbon source and experimental period

Bio-N

(mtm/d)

NO,--N

(mzd)

232 (s=18)

N03--N gut

(mid4 21.2

(s=2.74) 73.3

(s=5.0) 68.3

(s=5.7)

NOz--N out

(mgkl) 0.02

(s=O.O2)

N2-N out

(w/d) 211

(s=28) 153

(s=25) 156

(s=27) 133

(s=56)

NI$+-N

(mlfl/d)

(sZ.2)

(S=:,

NHd+-N out

(mg/d)

(SZL,

4.85 (s=l.O)

0.26 (s=O.l5)

Bio-N c r out in out

(mg/d) (mg/d) (mg/d) Acetic acid

(day 110-120) Crude syrup

(day 110-120) Hydrolyzed starch

(day 110-120) MeOH

(day 113-120)

(s 9bT4,a 20.3 268 261

(s =4.2)b 31.6 265 263

(s=3.0)b 27.7 259 252

(s =2.4)b 16.2 173 168

(s=1.7)b

231 (s= 15)

0.03 (s =O.OOS)

0.02 (s=O.O03) (SZS)

150 (s=7.7) (s =Y83

0.02 (s=O.o05) $0.3,

a Organic nitrogen in the yeast extract. b Calculated as 12% of TSS (total suspended solids). Abbreviation: s = standard deviation.

other nitrogen compound besides molecular nitrogen could be detected in the gas phase from day 51 to 120. The nitrogen mass balance showed that the nitrogen measured in the influent could be accounted for in the reactor effluent (Table 4). The ORP was - 155 mV from day 51 to 108, but increased to -120mV from day 113 to 120.

The denitrification yield for the last experimental period (days 113 to 120) was calculated to be 0.28 g NOsN/g COD (Table 2). The maximum denitrification rate was estimated to be around 30-45 mg N/g TSS. h, whereas the apparent denitrification rate was calculated to be 29 mg N/g TSS .h (Table 3).

During the first period when the denitrification process was poor, the microflora was composed mainly of rod- shaped, gram-negative bacteria. A low amount of appendaged bacteria, possibly Hyphomicrobium spp., could be observed and after the increase in HRT, these

bacteria became dominant. Four different bacteria were isolated, of which three were true denitrifying bacteria (Table 5). However, none of these could be positively identified by the Biolog Identification System. The dominating appendaged bacterium was only able to use one of the 95 carbon sources used in the Biolog Iden- tification system (formic acid, after 96 h incubation). Based on its morphology and physiology (21) it is proposed to be a Hyphomicrobium sp. This isolate was found to dominate only on the anaerobically incubated methanol-medium agar plates, whereas the other three isolates were obtained from the aerobically incubated methanol-medium and TGE agar plates. The second denitrifying strain is possibly a Pseudomonas. The third denitrifying strain possibly belongs to the coryneforms, due to its special morphology (gram-positive, pleomor- phic, clubbed forms, metachromatic granules, 21). Even though this coryneform bacterium grew well on TGE

TABLE 5. Tentative identifications of isolates from the denitrifying enrichments on different carbon sources in the chemostat experiments

Carbon source True denitrifiers (end product Nz)

Similarity Oxidation of index with the substances in

Biolog ISa the B;k$g ISb fi

Nitrite producers (Nor- to NOz-)

Similarity Oxidation of index with the substances in

Biolog ISa the B;~J& ISb n

Acetic acid Acinetobacter johnsonii genospecies 7cs d

Pseudomonas alcaligenes subgroup AC. s

Crude syrup Pseudomonas alcaligenes subgroup BC

Psatdomonas corrugatac Hydrolyzed starch Agrobacterium radiobacter

subgroup Bd+ e Pseudomonas corrugatac

0.47

0.70

21

33

0.27 33 Enterobacter asburiaecs f 0.57 69

0.40 63 0.65 68

0.39 52

Methanol Hyphomicrobium SP.~, i - 1 Pseudomonas delqfieldiic 0.21 38 Coryneform bacteriumss j - 0

Aeromonas hydrophilae, f 0.62 49

Klebsiella ozaenae=s f Aeromonas hydrophila DNA

group 12ca f Enterobacter asburiae, f Kiebsiella ozaenaee, f Comamonas acidovoransc

0.46 64 0.39 29

0.46 65 0.54 66 0.58 33

a Similarity index with the strain library in the Biolog Identification System. Maximum similarity index is 0.96. Index values above 0.5 for plates read within 24 h are considered as acceptable.

b Amount (%) of oxidation of the 95 different carbon sources used in the Biolog Identification system. c Possibly acceptable identification only on the generic level (similarity index0.5). f Able to ferment glucose. s Clearly dominating, i.e. over 70% of the isolates on the plates. h Identification based on morphological and phenotypical parameters. i Dominating in the reactor at 36 h HRT and on the anaerobically incubated MeOH-medium plates. The other bacteria isolated from the MeOH

chemostat were obtained on the aerobically incubated agar media plates (TGE and MeGH). j This isolate was not observed by microscope in the chemostat. Abbreviation: IS=identification system.


plates, it could not utilize any of the 95 carbon sources solely with the mineral nutrient concentrations, which are used in the Biolog Identification System. Finally, a nitrite-producing bacterium, Comamonas acidovorans was also isolated, which was, in contrast to all other nitrite-producing bacteria in this study, unable to utilize glucose.

DISCUSSION

The results of the investigation clearly show that a complete (i.e. end product NZ, Table 4) respiratory denitrification process in chemostats under steady state conditions could be obtained with all of the four carbon sources (acetic acid, crude syrup, hydrolyzed starch, methanol) used in this study. For all four carbon sources it was found that growth factors in the form of yeast extract did not improve the denitrification process significantly, although it may possibly have contributed to less nitrite accumulation in the chemostats fed with the carbohydrate carbon sources. Yet, the characteristics of the carbon source had a significant influence on the denitrification process in terms of important parameters such as the denitrification rate, denitrification yield, sludge yield (Tables 2 and 3) and the microbial composition (Table 5).

The four carbon sources were all found to select different types of true (i.e. end product Nz) respiratory denitrifying bacteria and various amounts of nitrite- producing bacteria (Table 5). However, most of the bacterial identifications could only be tentatively performed, as the similarity indexes of the isolates with the strain library of the Biolog Identification System were generally low. This indicates that some of the isolates obtained in this study may constitute new species of denitrifying bacteria.

With acetate a high amount of a true denitrifying bacterium (Pseudomonas alcaligenes) was obtained. Another, less common true denitrifying bacterium was identified as A. johnsonii, but further investigations showed that the isolate obtained in this study was not identical with the type strain. Neither the type strain (22), nor most Acinetobacter strains isolated from the activated sludge in the nutrient removing systems (23) are true denitrifying bacteria.

Methanol also selected a high amount of a true denitrifying bacterium (Hyphomicrobium sp.). Although other true denitrifying bacteria (a Pseudomonas sp. and a coryneform bacterium) were also isolated from the methanol chemostat, these bacteria could not compete with Hyphomicrobium at the HRT of 24 h and with additional growth factors in form of yeast extract. Unknown strains of Pseudomonas, which are able to denitrify with methanol as a carbon source, have however been reported in literature (24). The main denitrifying coryneform bacterium previously reported in literature is Corynebacterium sp., whose true taxonomi- cal status is not yet established and furthermore is not a true denitrifying bacterium (1, 24).

Crude syrup and hydrolyzed starch stimulated different types of denitrifying bacteria (see summary in Table 5). Yet, the pattern of the microbial composition was similar, since both carbon sources, compared with acetic acid and methanol, selected for a more heterogeneous and metabolically versatile microflora consisting of true denitrifying bacteria and nitrite producing, fermentative

bacteria. This could already be observed in the chemostat experiments, as the average values of nitrite in the effluents were higher for these two carbohydrates than for acetic acid and methanol (Table 1). Even though no significant amounts of nitrite were accumulat- ed during the experimental steady state conditions, this type of microbial composition may be a potential risk for nitrite accumulation under less optimal conditions (25).

Further comparison with other general isolations of bacteria from nutrient removing wastewater treatment systems shows that some of the genera found were similar to the findings in this study, such as Acinetobacter, Aeromonas, Enterobacteriaceae, Hyphomicrobium and Pseudomonas (e.g. 1, 6, 26). However, the validity of some of the bacterial identifications may have to be criti- cally reviewed. The classical phenotypical identification methods can often not identify environmental isolates, as only a minor part of the bacteria in the environment have been isolated and thoroughly characterized (27). In addition, the results of the bacterial isolations have to be questioned, as the present isolation methods do not give a representative picture of the true microbial community structure in activated sludge systems (28). Finally, it has recently been stressed that the full criteria for the determination of true respiratory denitrifying ability of bacteria has not been used in many studies (29).

In order to evaluate the validity of the measured yield values, a COD balance based on the oxygen equivalents was calculated by transforming the sludge and the denitrification yield into COD components. As can be seen in Table 2, the amount of formed products seems to balance the removed substrate fairly well. The obtained denitrification and sludge yields lie within the range reported in literature (7). Acetic acid and methanol both had similar and generally higher denitrification yields and lower sludge yields than crude syrup and hydrolyzed starch.

The measured denitrification rates indicate the maximum respiration rate, whereas the apparent rates also reflect process conditions such as the HRT or secondary processes. Denitrification rates which are typically reported in literature range from 1 to 20 mg N03--N/g VSS. h (3). The denitrification rates obtained in this study are comparatively high as they range from 26-76 mg N03-- N/g TSS.h (Table 3). This may be due to differences in the amount of viable biomass between dispersed, ex- ponentially growing bacteria in chemostats and recirculat- ed, heterogeneous activated sludge floes in full scale treatment plants. The apparent rates are generally lower than the measured ones (Table 3). For acetic acid this can be explained by the fact that the system is operated under carbon substrate limitation. The effluent from the systems operated on the carbohydrates (crude syrup and hydrolyzed starch) had a COD residue of more than 200 mg/l, and were consequently not operated under carbon substrate limitation. Instead, the differences in the rates can probably be attributed to hydrolysis processes of longer carbohydrate chains, which have to take place before they can be used in the denitrification.

The microbial system using methanol was operated under carbon substrate limitation during the last experimental period. The experiment showed that the growth rate was between 0.028 h-l and 0.042 h-l (Table 3). This suggests that the maximum denitrification rate should be around 30-45 mg/N03--N/g TSS . h. In order to verify


the low growth rate, a pure culture study on the dominating methanol-denitrifying bacterium (Hypho- microbium spp.) was performed. At 1YC the specific maximum denitrification rate was indeed found to be 32mg N/g TSS. h, and the growth rate 0.032 h-l (19).

6.

7.

Based on the denitrification rates and the sludge yields, the growth rates for the bacteria on the other three systems growing on the other three carbon sources (acetic acid, crude syrup and hydrolyzed starch) were roughly estimated to lie within a similar range of around 0.0624091 h-r (Table 3). It is interesting to notice that although the estimated growth rates for the systems operated on acetate and the two carbohydrates are fairly similar, the maximum denitrification rates differ significantly. The carbohydrates have a higher sludge yield, and consequently the denitrification yield will be lower. With the same type of reasoning it is logical that despite a low growth rate, the maximum denitrification rate for a system operated on methanol is, in comparison with the other carbon sources, relatively high.

8.

9.

10.

11.

12. To summarize this discussion, the carbohydrate

products had a higher sludge yield, a lower denitrification yield and stimulated not only denitrifying bacteria but also fermentative and nitrite producing bacteria. The hydrolysis of part of these carbon sources also seems to be rather slow. This means, that acetate and methanol appear to be more advantageous as carbon sources for denitritication in wastewater treatment plants. The methanol-denitrifying microbial system yielded a special- ized microflora with a substantially lower growth rate than the acetate-denitrifying system. A higher denitrification rate and also a faster response can consequently be expected with acetate than with methanol. Recent full scale studies have shown, though, that once an activated sludge system has been adapted to methanol, a good denitrification process can be obtained (30). The choice of acetic acid or methanol as an external carbon source for denitrification therefore has to be decided with respect to the possibilities and the needs of each wastewater treatment plant.

13.

14.

15.

16.

17.

ACKNOWLEDGMENT

This study was supported by the Swedish National Board for Industrial and Technical Development (NUTEK). Dr. Rajni Kaul at the Department of Biotechnology is acknowledged for linguistic advice.

18.

19.

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The Effect of Different Carbon Sources on Respiratory Denitrification Nautska et al