Thermophilic Anaerobic Digestion of Livestock Waste- The Effect of Ammonia

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Appl Microbiol Biotechnol (1993) 38:560-564

ppliedMicrobiology

Biotechnology© Springer-Verlag 1993

Thermophilic anaerobic digestion of l ivestock wastethe effect of ammonia

I. Angelidaki B. K. Ahring

The Anaerobic Microbiology/BiotechnologyResearch Group, Department of Biotechnology,Building 223, The Technical University ofDenmark, 2800 Lyngby,Denmark

Received: 3 April 1992/Accepted: 16 September 1992

Abstract. Ammonia concentrations of 4 g N/1 or more

inhibited thermophilic digestion of cattle manure. A sta-ble digestion of cattle manure could be maintained with

ammonia concentrations up to 6 g N/1 after 6 months o foperation. However, the methane yield was reduced and

the concentration o f volatile fatty acids increased from 1

to 3 g/1 as acetate, compared to controls with an am-monia concentration of 2.5 g N/1. The temporary strong

inhibition following an one-step increase in ammoniaconcentration was reduced by applying a gradual in-

crease. The specific methanogenic activity of ammonia-inhibited reactors (6 g N/l) with acetate or hydrogen as

substrate was reduced by 73 and 52 , respectively.Tests of ammonia toxicity on the acetate- and hydrogen-

utilizing populations showed a higher sensitivity of theaceticlastic compared to the hydrogenotrophic metha-

nogens; the specific growth rate for the aceticlastic me-

thanogens was halved at ammonia concentrations of

3.5 g N/l, compared to7 g N/1 for the hydrogenotrophic

methanogens.

Introduction

Inhibition during anaerobic digestion of livestock wasteis often caused by high ammonia concentration. In addi-

tion to ammonia (NH3 + NH~-) livestock waste containscompounds that readily release ammonia when de-graded, e.g. urea and proteins. Especially for swine and

poultry manure, the to tal ammonia concentration is oft-en higher than 4 g N/1 (Angelidaki and Ahring 1991).

Many investigations have dealt with the ammonia in-hibition level, but the results are conflicting and havebeen obtained under different conditions, such as thepH, temperature and inoculum used. McCarty (1964) re-ported ammonia inhibition to occur at concentrationsfrom 1.5 to 3.0 g N/1 at pH levels above 7.4, whereasinhibition occurred for all concentrations higher than

Correspondence to B. K. Ahring

3 g N/1 at all pH levels tested. Likewise Koster and Let-

tinga (1984) reported ammonia inhibition to occur at1.7 g N/1 at pH 7.5.

Much higher inhibitory levels have however been re-

ported by other authors. Van Velsen (1979) showed inbatch experiments, with inoculum adapted to high con-

centrations of ammonia, that methanogenesis occuredafter a lag phase at ammonia concentrations as high as

5 g N/1.

Only a few investigations have dealt with ammoniainhibition at thermophilic temperatures. Zeeman et al.

(1985) repor ted an initial inhibition at 1.7 g N/1 at 50 ° C.

Hashimoto (1986) found ammonia inhibition at about2.5 g N/1 for both mesophilic and thermophilic reactors

when the reactors were not previously acclimatized toammonia. However, the corresponding values was 4 gN/1 for thermophilic reactors previously acclimatized to

ammonia concentrations between 1.4 and 3.3 g N/1. Intheir experiments the effluent pH was approx. 7.2.

Free ammonia (NH3) has been suggested as the activecomponent causing ammonia inhibition. A level of

80 mg N/1 of f ree ammonia has been proposed as the

minimum inhibitory level (Koster and Lettinga 1984; DeBaere et al. 1984). McCar ty and McKinney (1961) andBraun et al. (1981) found 150 mg N/1 to be the inhibito-

ry free ammonia concentration. As the free ammoniafraction increases with temperature and pH, the am-

monia level tolerated at high pH and thermophilic tem-peratures would be expected to be low. Biogas reactorsoperating with livestock waste often have a high pH(about 8) and, especially at thermophilic temperatures,the free ammonia concentration will be up to ten times

higher than the free ammonia concentrations reportedas inhibitory.

In the present study we examined the effects of addi-tion of dif ferent ammonia concentrations and the possi-bility of adaptation to ammonia during anaerobic ther-mophilic digestion of cattle manure in continuously fedlab-scale reactors. The specific methanogenic activity(SMA) of an uninhibited and an ammonia-inhibited

reactor are reported. Finally, we examined the effect ofvarious ammonia concentrations on thermophilic aceti-



561

c l a st i c a n d h y d r o g e n o t r o p h i c m e t h a n o g e n s i n b a t c h e x -

p e r i m e n t s .

Mater ia l s and meth ods

in th is exper iment were compared wi th those f rom contol reactors

wi th 2 .5 g N/1 ammonia . In both exper iments the ext ra ammonia

was added to the feed as NH4C1. Duplicate reactors were operated

for each concentra t ion tes ted. As the var ia t ion between dupl ica te

reactors was in genera l smal l (< 10 ) mean values are repor ted.

Cont inuous ly f ed reac tor exper iment s SMA tes t

Substrate. Cat t le manure , obta ined f rom a Danish biogas plant

was used as subst ra te and was provided in one batch for each of

two exper iments . The natura l amm onia conc entra t ion of the f irs t

batch was 1.5 g N/1. The bat ch used fo r the second expe riment

had a h igher ammonia content and was therefore di lu ted wi th tap

water resul t ing in a f inal ammo nia concentra t ion of 2 .5 g N/1.

Data of the two batches are g iven in Table 1 .

The exper iments were performed in s ix 4 .5-1 , automated lab-

scale reactors with a workin g volu me of 3 1 as shown in Fig. 1. The

reactors h ad a slowly mov ing stirring blade (60 rpm), which was

act ivated for 1 min everys 3 min. Gas p roduct ion was measured

automat ica l ly wi th a gas meter as previously descr ibed (Angel idaki

et al . 1992). The re actor temp eratu re was kept at 55 ° C, and the

retention time (RT) was 15 days.

Experimental design. Two series of reactor exper iments were per-

formed. In the f i rs t exper iment the ef fec t of adding ammonia to a

level of 4 and 6 g N/1 was compared to the perform ance of cont rol

reactors wi th an ammonia concentra t ion of 1 .5 g N/1. In the sec-

ond ex per iment the ef fec t of 6 g N/1 was compared to the ef fec t of

a gradual ly increas ing concentra t ion of ammonia . The N concen-

t ra t ion was increased a t in tervals of 30 days (corresponding to 2

RT) an d the levels were 2.5, 3, 4, and 5 g N/1. The results obta ined

Table 1. Data on cat t le manure used

Subst ra te data Exper i - Exper i -

men t I merit II ~

Tot al solids ( ) 6.4 5.9

Volatile solids ( ) 4.5 4.2Tota l n i t rogen (g N/ l ) n .d . b 3 .7

Am mon ia ni t rogen (g N/ l ) 1 .5 2 .5

pH 7.6 7.8

Volatile fatty acids 7.4 7.6

~ After d i lu t ion wi th tap water

u Not determined

The SMA of the control reactors and the reactors receiving 6 g N/1

amm onia were compared. The tes ts were performed in 58 ml se-

rum vials conta ining 21 ml BA med ium (Angelid aki et al . 1990)

adjus ted to pH 7.9 (corresponding to the pH of the reactors) wi th .

Na OH and a gas phase of N~/CO~ (approx• 90: 10) in order to

keep the pH at 7 .9-8 .0 dur ing the exper imenta l per iod. Aceta te

(30 mM) or 200 kPa of H~/COa (80:20) were applied as substrates

and the methane produced was compared to via ls wi thout sub-

s t ra te added. The via ls were inocula ted anaerobical ly wi th 25

(v/v) reactor content and were incubated in a shaking water bath

at 55 ° C . T he S MA was es t imated as the in i t ia l methane produc-

t ion ra te per gram biomass (vola ti le sol ids , VS). The mean act iv i ty

found in the control v ia ls (wi thout subst ra te addi t ion) was sub-

t rac ted f rom act iv i t ies found in the exper imenta l v ia ls .

Ef fec t s o f ammo nia on methanogen ic popula t ions

The ef fect of d i f ferent concentra t ions of ammonia on acet ic las t ic

and hydrogenotrophic methanogenic popula t ions was tes ted in

batch exper iments , us ing 5 (v/v) d iges ted manure as inoculum

in BA medium (con tent of amm onia in BA medium was 0 .26 g

N/ l ) . The pH in these exper iments was adjus ted to 7 .2-7 .3 , con-

s idered to be the opt imum pH for these bacter ia l groups . As sub-

s t ra te, 3 0mM aceta te or 200 kPa H2/COz (80:20) was appl ied.

For the aceta te ser ies, the range of co ncentra t ions tes ted was 0 .26-

13 g N/1 and 0 .26-20 g N/1 for the H2/CO z ser ies . The speci fic

growth ra tes of the acet ic las t ic or hydrogenotrophic popula t ions

were es t imated by a semi- logar i thmic plot of methane product ionversus t ime. Each exper iment was run in t r ip l ica te and a l l exper i -

ments were repeated.

Analy t i ca l methods

VS, to ta l sol ids and pH were determined us ing s tandard methods

(American Publ ic Heal th Associa t ion 1985) . CH4, CO2 and vola-

t i le fa t ty ac ids (VFA) were measured by gas chromatography as

3.

~t~

_-?_i_ . _ . _ . _ i .

i3

2 6 7

Fig. 1. The reactor set-up: 1 subst ra te

flask on a magnetic stirrer; 2, pump; 3reactor ; 4 water bath; 5 control ler ; 6gas-separating bottle; 7, gas meter



562

0 .4

>~ 0.3~ . ~

~ o12>.

r

~ 0.1

0

~

~@ ~~ , ~ : : ~ . ; - ~ ' ~ 4

~ ~ ' ~ ~ ' ~ ' ~ ' ~ ~ - ~ t

~ v ~ v % v ]~ ............................................................................................................................................................... ~ ~ ~

/ ~ I I I •

<

>

10

b ~ i i i

0 10 20 30 40 50

Time days)

Fig. 2a, b. Continuous reactor experiment I. a Methane yield (5days average), b Volatile fatty acids (VF A) concentration (calcu-lated as acetate). The r rows indicate when the addition of am-monia was started: ~, control (1.5 g N/l); V , 4 g N/I, ~ ', 6 g N/1.VS, volatile solids

0.4A

>i

~ 0.3

__.m 0 2~

(9e

. ~ 0 . ]

0

~0

8

E~ 6

LL

> 4

I I I I I / /

, ................

I I I ~ ~ / ~

I I I I ~ /~/ ~

...........................................................................................................................................................................

r \................................................. 7 ~ ; ~ ...................~- -- -~ -. -. .. .. -~ v~ ...........................

4 ~ -~ ~. ~ _ ~ - . ~ ,.- ~ ~ ................

~ T , - - - . , - ~ - ~q I '~1 I I / / / /~

0 20 40 60 80 100 180 200

T i m e d a y s )

Fig. 3 a, b. Continuous r eacto r experiment II. a M ethane yield (5days average), b VFA concentration (calculated as acetate). A m-mon ia was introduced at da y 7 at a concentration of 6 g N/I o rstepwise from 3 to 5 g N/1. Th e r rows indicate the stepwise in-crease in ammo nia concentration to 3, 4 and finally 5 g N/1 in thereactors receiving increasing concentrations of amm onia: t , con-trol (2.5 g N/l); V , increasing a mmonia concentrations; ~' , 6 gN/1

previously described (Angelidaki et al. 1990). Total amm onia con-

tent was determined by the Kjeldahl method.The free ammo nia concentration was calculated fro m theequilibrium relationship:

[T-NH3][NHa]

where [NH3] and [T-NHa] are the free and the total ammoniaconcentrations, respectively, and ka the dissociation constant, withthe value of 38.3.10 -1° at 55 ° C; during calculations, the appr o-priate pH values were used.

Results

Ad d i t i o n o f a m m o n i a t o a t o t a l c o n c e n t r a t i o n o f 4 o r

6 g N/1 to the feed o f rea c to rs fe d wi th ca t t l e man ure

con ta in ing 1 .5 g N/ I (a t day 7 ) resu l ted in a decrease in

the methane y ie ld a f te r 3 weeks (F ig . 2a) . The methane

y ie ld decreased f rom appro x . 0 .2 to 0.05 1 CH 4/g VS fo r

b o t h r e a c t o r s , c o r r e s p o n d i n g t o 2 5 % o f t h a t o f th e c o n -

t ro l r e a c t o r s r e c e i v i n g n o e x t r a a m m o n i a . T h e d e c re a s e

wa s f a s t e r f o r t h e r e a c t o r s w i t h 6 g N / 1 a m m o n i a (F ig .

2 a ) . T h e VF A c o n c e n t r a t i o n fo r b o t h r e a c t o r s r e c e i v i n g

a d d i t i o n a l a m m o n i a i n c re a s e d a s t h e m e t h a n e y i e l d d e -

creased (Fig . 2b).

In t h e s e c o n d e x p e r i m e n t t h e b a s i c l e v e l o f a m m o n i a

wa s 2 . 5 g N / 1 . T h e m e t h a n e y i e l d a n d t h e VF A c o n c e n -t ra t ion were the same as fo r the con t ro l reac to rs in the

p re v i o u s e x p e r i m e n t , s h o wi n g t h a t 2 .5 g N /1 a m m o n i a

h a d n o a p p a re n t e f f e c t o n t h e b i o g a s p ro c e s s c o m p a re dto man ure wi th 1 .5 g N/1 amm on ia (F ig . 3a , 3b ) .

As fo und in the f i r s t exper imen t , add i t ion o f 6 g N/1

resu l ted in a decreased methane y ie ld , to approx . 0 .05 1

C H4 / g VS . T h e VF A c o n c e n t r a t i o n i n c re a s e d f ro m

abo u t 1 g /1 to above 4 g /1 as ace ta te . H owe ver , a f te r ap -

p rox . 2 RT (30 days ) the methane y ie ld increased aga in

to app rox . 0 .1 1 CH g/g VS and the V FA leve l decreased

to 3 g /1 as ace ta te (F ig . 3a , b ) . I n the re ac to rs rece iv ing

s u b s t r a t e w i t h a g r a d u a l l y i n c re a s i n g c o n c e n t r a t i o n o f

am mo nia , 3 g N/1 d id no t resu l t in any changes in me-

t h a n e y i e l d a n d VF A c o n c e n t r a t i o n c o m p a re d t o t h e

con t ro l s . A dd i t ion o f 4 g N/ l , howe ver , resu l ted in an

i n c re a s e i n t h e VF A c o n c e n t r a t i o n , f o l l o we d b y a d e -c r e a s e i n m e t h a n e y i e l d . Af t e r 2 R T t h e m e t h a n e y i e l d

par t i a l ly recovered a l though the VFA leve l s t i l l in -

creased .

Wh en 5 g N/1 was in t roduced , se r ious p rocess fa i lu re

o c c u r r e d a n d t h e m e t h a n e y i e ld d ro p p e d t o 0 . 1 1 C H g / g

VS, i . e . the same level as the reac to rs rece iv ing 6 g N/1

from day 7 . At the end o f the exper imen t (200 days ) ,

r e a c t o r s w i t h b o t h s t r a t e g i e s o f a m m o n i a i n c re a s e ( i n -

s tan t and g radual ) s tab i l i zed a t a methane y ie ld o f 0 .15 1

CH 4/g VS an d a VFA conce n t ra t io n o f 3 g /1 (F ig. 3a ,

b).In bo th exper imen ts the bas ic pH leve l in the reac to rs

wa s a p p ro x . 7 .9 . Ac c u m u l a t i o n o f VF A i n t h e i nh i b i te dreac to rs resu l ted in a lower ing o f pH to approx . 7 .5 .



563

SM test

The activity of the aceticlastic and the hydr ogenotrophic

methanogenic populations was significantly lower in the

reactors receiving 6 g N/1 ammonia. The decrease in the

activity was higher (73070) for the aceticlastic popula tion

than for the h ydro genot roph ic methanogens (52070) (Ta-

ble 2). Microscopic examination of diluted samplesshowed numerous clusters of Methanosarc ina indicating

that this was the predominant genus of the aceticlastic

methanogens.

mmonia tox ic i ty experiment

The maximu m gr owth rates were 0.62 day -1 and

0.11 h - ~ for the aceticlastic and h ydro genot roph ic me-

thanogens, respectively. The inhibitory effect of am-

monia was in general stronger for the aceticlastic than

for the hydrogenotrophic methanogens with initial inhi-

bition occurring at an ammonia concentration of ap-prox. 2 g N/1 fo r the aceticlastic and 3.5 g N/1 for the

hydrogenotro phic methanogens (Fig. 4). Growth rates

were reduced to 50°70 of the uninh ibit ed value at 3.5 and

able 2 . S pec i f i c m e t hanogen i c a c t i v i t y ( S A M ) o f r e ac t o r s unde r

d i f f e r en t s t a t e s o f am m on i a i nh i b i t i on

R eac t o r s ~ S ubs t r a t e S M A R educ -

( g m o l / g V S / h ) t i o n ( ) b

Con t rol Aceta t e 23.3 + 3 .1

A m m o n i a A c e t a te 6 . 0 + 0 . 6 7 3

Con t rol H2/CO 2 24.0 + 1 .7A m m o ni a H 2 / C O 2 11 .5 -+ 2 . 0 52

VS, vo la t i l e so l ids

a T he con t r o l r e ac t o r r e ce ived 2 . 5 g N / 1 am m on i a i n t r oduced w i t h

t he ca t t l e w ast e w h i le t he am m on i a r eac t o r r e ce i ved am m o n i a t o a

to t a l l evel of 6 .0 g N/1

b R educ t i on o f t he S M A o f t he am m on i a r eac t o r com par ed t o t he

un i nh i b i t ed r eac t o r

1 0 0

~80

~ 6

O

m 40

>,-

~ 2 0-~t~

OO

~ i i i i i i

•

I

2 4 6 8 10 12 14

Ammonia g -N/ I )I f f I l I I

1 4 0 2 8 0 4 2 0 5 6 0 7 0 0 8 4 0 9 8 0

Free ammonia m g-N~)

Fig. 4 . Reduct ion in the speci f i c growth r a t e as a funct ion of am-m on i a : O , a ce t a t e ; O , hyd r ogen a s s ubs t r a t e

7 g N/1 (250 and 500 mg N/1 free NH3) for the acetate-

utilizing and hydrogenotrophic methanogenic bacteria,

respectively.

i scuss ion

Inhibition of the biogas process was observed when theammo nia concent ration was increased to 4 g N/1 or

more in the continuously fed biogas reactors. The me-

than e yield decreased to 25070, with bo th 4 and 6 g N/1

added, compared to the controls with 1.5 g N/1 ammon-

ia. When ammonia was introduced gradually, the proc-

ess was unaffected up to 3 g N/1 and only slightly af-

fected at 4 g N/l, with signs of recov ery after 1 RT. A t a

concentration of 5 g N/l, process performance was seri-

ously affected and reached the same reduced level as the

reactors f ed with 6 g N/1 from the start of the experi-

ment. After prolonged exposure to ammonia the reac-

tors with ammonia concentration of 5 or 6 g N/1 stabil-

ized at a level of 0.15 1 CH4/g VS and 3 g/1 VFA (Fig.3a, b).

The experiments clearly demonstrate that it will be

possible to obtain a stable digestion of manure with am-

monia concentrations exceeding 4 g N/1 after an initial

adaption period. However, the methane yield will be

lower (approx. 25 lower than for uninhibited reactors)

and the VFA level will be higher than in reactors with a

lower ammonia load.

Growth of methanogenic bacteria was inhibited by

ammonia levels above 2 g N/1 (Fig. 4), while a concen-

tration of 4 g N/1 was needed to affect the perf ormance

of the continuously fed biogas reactors. In a contin-

uously fed reactor inhibition is only detected when thereduction of the growth rates of the active biomass ap-

proaches the dilution rate used. In contrast a reduced

growth rate will directly affect the outcome of a batch

experiment.

The concentrations of free ammonia calculated for

the reac tor experiments are high. At 2.5 g N/I, corre-

sponding to the controls of the second experiment, the

calculated free ammonia concentration (pH=7.9) was

550 mg N/1. At 4 g N/1 ammonia , where the first signs

of inhibition occurred, the calculated free ammonia con-

centrat ion was appr ox. 900 mg N/1 (pH = 7.9). Howev-

er, the actual pH of the reactors dropped to approx. 7.7,

due to the accumulation of VFA, resulting in a free am-monia concentration of approx. 650 mg N/1. This con-

centration of free ammonia resulted in a reduction in the

growth rates of the aceticlastic methanogens to 20 , in

the experiments with ammonia toxicity (Fig. 4). Growth

rates at this level are still sufficient to retain the active

biomass within the reactor at the RT used in the reactor

experiments. However, ammonia concentrations higher

than 4 g N/1 resulted in growth rates of the aceticlastic

methanogens close to the RT, resulting in a decreased

methane yield and an increased VFA concentration inthe reactors.

Process instability due to ammonia resulted in VFA

accumulation, which again led to a lowering of the pHand thereby decreased the concentration o f free ammon-



564

ia in the reactor. This decrease in free ammonia could

explain the observed ability of the process to stabilize

even with high ammonia concentration and with a lower

but stable methane yield.

The SMA of the acetate-utilizing methanogens of the

ammonia-inhibited reactor decreased more than the hy-

drogenotroph ic populations. Both the SMA test and the

ammonia toxicity experiment showed that it is the aceti-clastic methanogens that are primarily affected by am-

monia. This result is in accordance with other reports

for mesophilic methan ogens Robbins et al. 1989; Bhat-

tacharya and Parkin 1989; Sprott and Patel 1986). How-

ever, Zeeman et al. 1985) and Wiegant and Z eeman

1986) suggested that the hydrogenotrophic methanog-

ens are more sensitive towards ammonia than the aceti-

clastic methanogens under thermophilic conditions,

which is not supported by our experiments.

Inhibition of the aceticlastic populations showed a

sigmoidal pattern. The same pattern of inhibition of the

aceticlastic populations was observed by Poggi-Varaldo

et al. 1991), who found that the bacterial growth rateand the specific acetate-uptake rate were affected by the

free ammonia concentration in a three-stage pattern: in-

itial inhibition, plateau and final inhibition. This inhibi-

tion pattern could indicate that two inhibition mecha-

nisms are involved, acting at different concentration lev-

els. The hyd roge notr ophi c populat ions exhibited, how-

ever, a more linear pattern of inhibition.

In large biogas plants a lowering of the gas yield has

serious economic consequences. Therefore, the ammon-

ia concentration should be checked and if possible kept

at 4 g N/1 or lower for maximum biogas production. If

high ammonia concentrations cannot be avoided, the re-

sults indicate that a longer RT would be beneficial.

Acknowledgements We thank Hector Garcia for his excellent as-sistance. This work was supported by grants from The Danish En-ergy Council, no. 1383/89-1 and from the Nordic Ministerial

Council.

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Thermophilic Anaerobic Digestion of Livestock Waste- The Effect of Ammonia