J . Chem. Tech. Biotechnol. 1991, 52, 135-143

Inhibition of Growth and Acetate Uptake by Ammonia in Batch Anaerobic Digestion

Hector M. Poggi-Varaldo, Judy Tingley & Jan A. Oleszkiewicz*

Environmental Engineering Division, Department of Civil Engineering, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada

(Received 1 February 1990; revised manuscript received 16 August 1990; accepted 16 August 1990)

ABSTRACT

Ammonia inhibition 5f unacclimated anaerobic mesophilic mixed cultures was studied in batch mode. Bacterial growth rate and speciJic acetate-uptake rate were aflected by the concentration of un-ionised ammonia (UAN) in a three-stage pattern: initial inhibition, plateau andjinal inhibition stage. The un-competitive inhibition model provided the most reasonable data j i t . The stage pattern of inhibition suggested that more than one mechanism of UAN inhibition are acting on the mixed culture. The efects of UAN on the length of initial lag-time also followed a three-staged curve. High concentration of UAN resulted in acetate release. The biological basis and origin of the acetate release were not determined.

Key words: acetoclastic bacteria, ammonia, anaerobic digestion, inhibition kinetics.

NOTATION

A-MPB Acetoclastic methane producing bacteria HAc Acetic acid or acetate, mg dmP3 as acetic acid H-MPB Hydrogenotrophic methane producing bacteria Ki Inhibition coefficient in uncompetitive model eqn (3) (mg dm-3 UAN) MPB Methane producing bacteria SOC Soluble organic carbon (mg dmP3 carbon) TAN Total ammonia nitrogen (mg dm-3 N)

* To whom correspondence should be addressed.

135

J . Chem. Tech. Biotechnol. 0268-2575/91/$03.50 0 1991 SCI. Printed in Great Britain

H . M . Poyyi-Varaldo, J . Tinyley, J . A . Oleszkiewicz 136

UAN UAN,

V O A Y

Pi ,max ; P m a x

Un-ionised ammonia nitrogen (mg dm-' N) Cut-off concentration, the UAN level beyond which there is no more growth (mg dmT3 N ) Volatile organic acids Empirical exponent related to the shape and concavity of the curve in eqn (2) (dimensionless) Maximum specific growth rate in the inhibitor-spiked syringe, and in the control syringe, respectively (day- ')

1 INTRODUCTION

Considerable effort has been devoted to kinetic analysis of acetate catabolism under inhibitory conditions. In the case of ammonia inhibition, the un-ionised ammonia species (UAN) seems to play the main role,'v2 but the effects on the kinetics of acetate consumption and growth of acetoclastic methane producing bacteria (A-MPB) are far from conclusive. It was suggested that the inhibition of hydrogenotrophic methane-producing bacteria (H-MPB) by ammonia was the principal cause of cessation of methanogenesis., Other research indicated that the A-MPB are the most sensitive and thus most easily inhibited by ammonia in mixed culture^.^-^ Naguib6g7 showed that acetate catabolism with addition of ammonia-releasing co-substrate followed a stimulatory competitive-like pattern in substrate-amended sediments. Bhattacharya & Parkin' demonstrated an uncompetitive-like pattern of ammonia inhibition of continuous completely mixed bench-scale digesters.

In this work results of a batch mesophilic study of inhibition by ammonia of mixed culture of acetoclastic methanogens will be reported in terms of the effects of UAN on lag-time and kinetic parameters of both the maximum specific growth rate of A-MPB production and the specific acetate uptake rate (AUR).

2 EXPERIMENTAL

Experiments were conducted in 100 cm3 PerfectumTM glass syringes, according to Sobkowicz and Klemm (private communication, Badger Engrs, Inc., Cambridge, MA, USA) as modified elsewhere.' Syringes contained 60cm3 of a mixture prepared as follows: 12 cm3 of seed anaerobic sludge from high rate digesters at the Winnipeg North End wastewater treatment plant, mixed with 48 cm3 of a stock solution, comprising (g drn-,) KH,PO, (0.4), K,HPO, (0.4), NaHCO, (1.9), CuCO, (0.2) and MgCl, .6H,O (1.725) together with sodium acetate (2.733). A trace elements solution,' and varying amounts of a NH,Cl were added and pH-adjusted deionised water was used to make up a final volume of 60 cm3. The syringes were incubated in a water bath at 35°C.

Daily gas production was measured by plunger displacement. Gas composition was determined on a Gow-Mac 550 gas chromatograph (GC) equipped with a thermal conductivity detector at 1 l O T , and a stainless steel column

Inhibition of growth and acetate uptake by ammonia 137

1.80 m x 25 mm outside diameter packed with Poropak Q 80/lOO mesh at 55°C. The injector temperature was maintained at 95°C. Helium was the carrier gas. Volatile organic acids (VOA) were determined on a Gow-Mac 750 GC furnished with a flame ionisation detector at 200°C, and a borosilicate glass column 1.80 m x 2 mm internal diameter packed with Carbopak 80/120/B-DA/4% Carbowax 20M at 175°C. The injector was kept at 200°C. The carrier gas was nitrogen. Soluble organic carbon (SOC) was analysed using a Dohrmann DC-80 Carbon Analyser fitted with an infrared detector. Ammonia was determined by distillation in a Tecator Model 1002 distillation unit followed by nesslerisation, in accordance with the Standard Methods." Total and volatile suspended solids, pH and other analyses were performed according to Standard Methods." Complete analyses were performed initially on the stock remaining in the volumetric flasks and at the end of the run. 0.2 cm3 samples for liquor analysis were collected twice a week.

Un-ionised ammonia nitrogen concentration (UAN) in the syringes was estimated from the total ammonia concentration (TAN) and the average pH of each syringe, using eqn (1),l and assuming the ammonia ionisation constant pK, at 35" equal to 8-35.

3 RESULTS AND DISCUSSION

Figure 1 shows a typical histograph of SOC, acetic acid (HAc) and biogas produced in a syringe with 602mgdm-3 UAN. Lag-times defined as the times to reach 5 cm3 of biogas or a 5% utilisation of HAc in the cultures were of similar duration. An apparent release of acetate was observed during the lag-time for all syringes with UAN above 213mgdmP3 UAN. Figure 2 shows the effect of UAN on lag-time and the data suggest essentially a staged pattern. This differs from other batch ammonia inhibition studies." The discrepancy could be due to the fact that a step-wise increase of ammonia level in the same batch reactor was used in those studies," thus accomplishing acclimation. Three stages may be defined in this study: an increasing stage, followed by a plateau phase, and a third stage of rapid increase of the lag-time with increasing concentration of the un-ionised ammonia (Fig. 2).

Maximum specific growth rate values (estimated from biogas cumulative production data") were plotted against UAN concentration (Fig. 3). Their patterns indicated a steep first inhibition phase, followed by a plateau, and a final inhibition phase. Two models were fitted to the experimental results: Luong's model,I3 and the uncompetitive model,I4 shown in eqns (2) and (3), respectively.

138 H . M . Poygi-Varaldo, J . Tingley, J . A . Oleszkiewicz

A HAC Biogas A

h 1200

h

-40 2 U

Time (days)

Fig. 1. Typical evolution of biogas production, soluble organic carbon and acetic acid concentrations with time, during the batch incubation of a syringe containing 602 mg dm-3 un-ionised ammonia

nitrogen.

Unionised ammonia (mg N dm-3)

Fig. 2. Effect of un-ionised ammonia concentration on the duration of the lag phase, defined as the time during which the first 5 cm3 of biogas are produced.

Inhibition OJ growth and acetate uptake by ammonia

- Luong ----- Uncompetit ive

I I I L I

G 100 200 300 400 500 600 700 Unionised ammonia (mg N dm-3)

139

1

Fig. 3. Maximum specific growth rate of acetoclastic methanogenic bacteria fitted to: a staged- Luong inhibition model: 1st stage, 0.20*[1- (UAN/209)]0'958; 2nd stage, 0.086; 3rd stage, 0.20*[1-(UAN/721)]1'621; (eqns (6), (8) and (9) of Table 1 respectively); and a staged un-competitive-like inhibition model: 1st stage, 0,24/[1+ (UAN/61.6)]; 2nd stage, 0.086; 3rd stage,

0.24/[ - 11.5 + (UAN/35.6)]; (eqns (7), (8) and (9) of Table 1 respectively).

where

pi,max, pmax = maximum specific growth rate in the inhibitor-spiked syringe and in the control syringe, respectively (day- l ) ,

is no more growth (total inhibition conditions) (mg dm-3), y =empirical exponent related to the shape and concavity of the curve

pi,max versus UAN,

UAN, = ammonia cut-off concentration, the UAN level beyond which there

K , =inhibition coefficient (mg dm- UAN).

It should be noted that the fitting using Haldane kinetics does not allow us to differentiate between non-competitive or un-competitive behaviour, since the dependence of the apparent saturation coefficient on ammonia concentration can not be assessed. The uncompetitive case and denomination was selected based on earlier work.

If the models are forced to fit the data in the full range of UAN, eqns (4) and ( 5 ) are obtained (Table 1). Equations (6)-(10) in Table 1 describe a staged fitting of the growth inhibition curves, breaking the curve into three stages: 0-1 13 mg dm-3 UAN, a rapid inhibition of growth; 114-540 mg dm-3, plateau of constant slow growth without the negative effects of un-ionised ammonia; and 541-700mgdm-3 UAN, the last stage of rapid inhibition of growth. The

140 H . M . Poygi-Varaldo, J . Tinyley, J . A . Oleszkiewicz

TABLE 1 Comparison of Growth Rate Models

Model U A N Formula Equation correlation runge coeficient

Luong

U ncompetitive

Luong

Uncompetitive

Constant plateau

Luong

Uncompetitive

0-700

0-700

0-1 13

0-113

113-541

541-700

541-700

0.18 UAN

1 +- 216

~ ( 5 )

o+( UAN ,,,I O Y 5 * ] (6)

0.24 , UAN (7) I+-

61.6

0.086 (8)

o.*o[ 1 -(g)"""] (9)

0.24 UAN

- 11.5 +- 35.6

0.8 1

0.84

0.9 1

0.97

-

0.98

0.98

improvement in the fit is expressed in significantly improved correlation coefficients (Table 1) and illustrated in Fig. 3. Both models displayed a similar level of agreement with the experimental data. The only advantage of Luong's model over the un-competitive model could be the prediction of a more plausible cut-off concentration of UAN in the third stage with cessation of biological activity (approximately 710 mg dm-3-not validated in this work), while the second model predicts a less realistic continuous reduction of activity with no break at all.

Both staged models present few shortcomings. Luong's model predicts a fictitious cut-off UAN concentration in the first stage (209 mg dm-3), while the un-competitive model shows a negative coefficient in the denominator of the third stage formula due to the geometric shift of the asymptote of the hyperbola with respect to the y axis. The K i values of the un-competitive staged model obtained in this work (Table 1, eqns (5) and (10)) are of the same order of magnitude as the value that was reported," 1600 mg dm-3 as TAN or 18 mg dmP3 as UAN (Bhattacharya, S. K . , Private communication, 1989).

In another study" a two-stage linear fitting was used to correlate the batch methane production rate of ammonia-inhibited cultures. No discussion on biological or mechanistic implications was presented. From the biological point of view, the need for a staged modelling of the data strongly suggests that at least

Inhibition of growth and acetate uptake by ammonia

0

141

,O Unionised ammonia (mg N dm-3)

Fig. 4. Effect of un-ionised ammonia concentration on acetic acid release during the lag phase. Acetic acid release was defined as:

(Peak concentration in the lag phase - Initial concentration)*100 (Initial concentration)

in terms of acetic acid concentrations.

two different mechanisms of inhibition act on A-MPB (and/or that distinct subgroups of A-MPB in the mixed culture have different sensitivities to ammonia), related to different levels of the toxicant UAN. A cation exchange-distortion caused by ammonia as a probable cause for inhibition of MPB was reported.I6 It would be likely that this mechanism is acting in the first stage. The inhibition in the third stage could be related to osmotic effects of the ammonium salt (plain ionic concentration increase effect) or to a mechanism of inhibition by ammonium ion at the level of the bacteria membrane site where methane synthesis occurs16 but this second interpretation would require further research (Patel, G. B., Private communication, 1989). The staged modelling with unstructured models points out the deficiencies of such type of representations and stresses the need for the development of structured models for application in anaerobic process ~imulat ion. '~

At UAN concentrations larger than 213 mg dm-3 the concentration of acetate in this study increased beyond the initial acetate contents of the cultures and peaked near the end of the lag period, in what will be called here acetate release. Whether the acetate was of intracellular origin, produced by A-MPB18 or produced by acidogenic bacteria degrading complex material already present in the seed portion of the culture and subsequently released to the liquor, cannot be distinguished and needs more investigation. The acetate release seemed to increase proportionally to the UAN concentration (Fig. 4). Only the release of the syringe containing 498 mg dm-3 UAN departed from the general trend.

142 H . M . Poggi-Varaldo, J . Tingley, J . A . Oleszkiewicz

4 SUMMARY AND CONCLUSIONS

Effects of increasing concentrations of ammonia on methanogenesis in unacclimated batch reactors were studied. The growth rate of acetoclastic methanogens calculated from gas production and specific acetate-uptake rate were inhibited by UAN in a three-stage pattern: rapid inhibition, plateau and further rapid inhibition. The inhibition pattern was best modelled using a three-stage model (eqns (7), (8) and (10)) consisting of two un-competitive-like equations and a plateau. The recently introduced Luong’s model did not fit the data better than the un-competitive model. The staged pattern of inhibition by UAN suggested that more than one mechanism of UAN inhibition acted on the mixed culture. Effects of UAN on lag-time also followed a three-staged curve. High concentration of UAN resulted in an initial release of acetate, confirmed by initial increase of dissolved carbon. The biological basis and origin of this release were not determined.

REFERENCES

1. Koster, I . , Characteristics of the pH-influenced adaptation of methanogenic sludge to ammonium toxicity. J . Chem. Technol. Biotechnol., 36 (1986) 445-55.

2. Wiegant, W. M. & Zeeman, G., The mechanism of ammonia inhibition in the thermophilic digestion of livestock wastes. Agric. Wastes, 16 (1986) 243-53.

3. Bhattacharya, S. K., P h D thesis, Department of Civil Engineering, Drexel University, 1986.

4. Heinrichs, D., Poggi-Varaldo, H. & Oleszkiewicz, J., Effects of ammonia on the anaerobic digestion of simple organic substrates. J . Enuiron. Engng, ASCE, 116 (1990)

5. Robbins, J . E., Gerhardt, S . A. & Kappel, T. J., Effects of total ammonia on anaerobic digestion and an example of digestor performance from cattle manure protein mixtures. Biol. Wastes, 27 (1989) 1-14.

6. Naguib, M., Methanogenesis in sediments of eutrophic lakes: 2 , Comparative investigations on the methane-precursor preference. Verh. Internat. Verein. Limnol., 22

7. Naguib, M., Kinetics of acetate and methanol conversion into methane in eutrophic sediments and its application in anaerobic systems. Water Sci Technol., 20(11/12) (1988)

8 . Bhattacharya, S. K. & Parkin, G. F., Modeling toxicity kinetics in complete-mix anaerobic systems. Paper presented at the CSCE-ASCE National Conf. Environ. Engng, Vancouver, BC, 1988.

9. Hilton, B. L. & Oleszkiewicz, J . A,, Sulfide-induced inhibition of anaerobic digestion. J . Enuiron. Engng ASCE, 114 (6) (1988) 1377-91.

10. APHA, Standard Methods for the Examination of Water and Wastewater, 16th edn. American Public Health Association, Washington, DC, 1985.

11. Koster, I . W. & Lettinga, G., The influence of ammonium-nitrogen on the specific activity of pelletized methanogenic sludge. Agric. Wastes, 9 (1984) 205-16.

12. Powell, G. E., Interpreting gas kinetics of batch cultures. Biotechnol. Letters, 5 (1983)

13. Mulchandani, A. & Luong, J . H. T., Microbial kinetics revisited. Enzyme Microb.

14. Parkin, G. F. & Speece, R. E., Modelling toxicity in methane fermentation systems.

698-71 1.

(1984) 951-5.

61-7.

437-40.

Technol., 11 (1989) 66-73.

J . Enuiron. Engng ASCE, 108 (6) (1980) 515-31.

Inhibition of growth and acetate uptake by ammonia 143

15. Bhattacharya, S. K. & Parkin, G. F., The effect of ammonia on methane fermentation

16. Sprott, D. & Patel, G. B., Ammonia toxicity in pure cultures of methanogenic bacteria.

17. Ataai, M. & Shuler, M. L. In Handbook on Anaerobic Fermentations, ed. L. E. Erickson

18. Westermann, P., Ahring, B. K. & Mah, R., Acetate production by methanogenic

processes. J. Water Pollut. Control Feder., 61 ( 1 ) (1989) 55-9.

System. Appl. Microbiol., 7 (1986) 358-63.

& D. Y. Fung. Marcel Dekker, New York, 1988, pp. 243-68.

bacteria. Appl . Environ. Microbiol., 55 (9) (1989) 2257-61.