Wal. Sci. Tech. Vol. 23. Kyoto. pp. 701-708. 1991.

0273-1223/91 $0·00 + ·50

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DENITRIFICATION WITH BIOGAS AS

EXTERNAL CARBON SOURCE
M. Werner and R. Kayser

Technical University of Braunschweig, P. O. Box 3329, D-3300

Braunschweig, FRG

ABSTRACT

Denitrification with biogas consisting of approximately 60% methane was tested

over one year in three types of lab-scale reactors. Denitrification of sani­
tary landfill leachate with biogas as a sole carbon source was found to be
possible. Denitrification rates from 60 mg NOs-NIl ' d with an activated
sludge reactor, 150 mg N03-N/l • d with a trickling filter and 550 mg N03-N/l
. d with a fluidized bed reactor were obtained. The oxygen concentration
played a significant role in process optimization. Not only high
concentrations of oxygen in the liquid medium but also low oxygen
concentrations in the aeration gas inhibited denitrification.

KEYWORDS

Denitrification; Methane; Biogas; Sanitary landfill leachate; Activated

sludge; Trickling filter; Fluidized bed reacto�

INTRODUCTION

In the past, first preference of wastewater treatment in Germany was the

removal of oxygen-consuming substances, such as BODs and ammonia-nitrogen. At
the present time great attention is paid to the removal of nitrogen from
wastewater to reduce nitrate problems in groundwater and to avoid eutrophi­
cation of surface water, especially of the North Sea.

Biological nitrogen removal is one of the most economic and effective means of
nitrogen reduction. Normally there is no problem to achieve extensive nitri­
fication and denitrification. However, some wastewaters with high nitrogen
concentrations do not contain enough organic carbon to get complete
denitrification (e.g. some industrial wastewaters, landfill leachates).

In these cases external sources of organic matter have to be added to optimize

denitrification and minimize nitrogen effluent levels. Methanol is mainly used
as the hydrogen donor for denitrification, because of certain advantages. But
for complete denitrification 3 kg Methanol per 1 kg N03-N (DM 1. 30 ($ 0.65)
per 1 kg N) is necessary, (Henze and Bundgaard, 198 2), so that it can be a
cost-intensive factor for wastewater treatment.

The future of denitrification as a means of removing nitrogen from wastewater

depends on the availability of an effective but inexpensive carbon source.

701

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 702 M. WERNER and R. KAYSER

In recent years some investigations about denitrification with methane gas

were published (Harremoes and Henze, 1 971 ; Davies, 1 973; Sollo et aI, 1 976).
The results have shown that denitrification with methane as a sole carbon
source is possible, but only very small denitrification rates were obtained.

The exact biochemical pathway is yet unknown. Mechsner and Hamer (1 98 5) have
reported that denitrification with methane will be carried out by a methano­
trophic/methylotrophic bacterial association. Methane-oxidizing (methanotro­
phic) bacteria are considered to be strict aerobic and most species are able
to grow only on methane. The aim of the methanotrophic bacteria is the
oxidation of methane via methanol to carbon dioxide and water according to
equation (1 ). Under unknown environmental conditions excess methanol is
produced, which can be used by methylotrophic bacteria for denitrification.

CH4 + 202 ----> C02 + 2H2 0 (1 )

1- > CH30H for denitrification

DESCRIPTION OF THE RESEARCH PROJECT

At the Technical University of Braunschweig, investigations on denitrification

with biogas were performed. Objective of this research project was the use of
landfill gas for denitrification of landfill leachate.

Biogas from landfills is produced during an anaerobic fermentation process

comparable to biogas production in sludge digestion. Landfill gas has a
content of approx. 60% methane and 40% carb on dioxide. The total gas produc­
tion rate of a landfill amounts to 3 20 m3 biogas per ton refuse over more t han
35 years (Hoeks, 1 98 3). Consequently landfill gas is an inexpensive, readily
available and long-term reliable carbon source.

Landfill leachate from "old" landfills (age >5 years) is characterized by

relative low levels of COD «3000 mg/l) and of BODs «300 mg/l). The most
significant pollutant of these leachates is nitrogen. The concentration of
Total Kjeldahl Nitrogen (TKN) varies from
700 - 1 500 mg/l. There is no problem
to achieve extensive nitrification, but denitrification is limited because of
the lack of a carbon source. In most cases denitrification efficiencies of
less than 1 0% are attained. To obtain greater rates, an external carbon source
like methane must be added.

In order to raise nitrogen removal,biogas was used as the external carbon

source instead of methanol or other carbon-containing fluids.

MATERIALS AND METHODS

E xperimental units

Three types of experimental units were used during the investigations. All
units were provided with pH-adjustment, semi-continuous measurement of
oxygen and methane in the gas-cycle and semi-continuous monitoring of
oxygen in the liquid culture medium. The leachate was fed with variable-speed
peristaltic pumps to the reactors. The units were operated at room temperature
between 18 "C and 21"C.

The first type was a completely-mixed, continuous-flow activated sludge

reactor with internal sludge recycle and external gas cycle to supply the
bacteria with methane and oxygen (Fig. 1). The closed activated sludge
reactors had an aeration basin of 16 L and a integrated settler volume of 6.9
L. It was aerated by fine bubble diffusors to insure adequate mixing.

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 Denitrification with biogas 703

Acid or
caustic �;;=:::!����jWI L"
EffLuentJ,..H\----f:H-jH
..

sett ling
compartment

Fig.!. Schematic illustration of the activated sludge reactor

The second type of reactor was a closed trickling filter with external water
and gas cycle (Fig. 2). The leachate and the gas were continuously pumped in
counter-current flow through the column. The reactor was filled with plastic
strips as filter media and had a reactor volume of 4,8 L.

Outlet gas

O.
addition

gas
cycle

Fig.2. Schematic illustration of the trickling filter

The third type was a fluidized bed reactor (Fig. 3). The unit consists of two
columns. Column ! was the biological fluidized bed reactor where biomass was
grown on sand with particle size in the range of 0.5 to 1.0 mm. The system was
operated in an upflow mode. L iquid passed through the column expanded the bed
to a height of 55 cm. Column 2 was the gas-dissolution reactor with a gas
cycle dissolving methane and oxygen in the liquid medium.

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 704 M. WERNER and R. KAYSER

.1 OutLet gas
effluent rll 1� =====;1
pH
controller

..

gas dissolution •. . .

reactor O2 addition

CH4 addition

influent

liquid medium
cycle

Fig. 3. Schematic illustration of the fluidized bed reactor

L eachate characteristics

Biologically treated leachate was taken from a full-scale sanitary landfill

leachate treatment plant in regular intervals and stored at 5'C until use. The
concentrations of the treated landfill leachate are given in Table 1. During
the investigation period ( more than 1 year) complete nitrification was almost
achieved; denitrification was not possible due to the lack of organic matter.

TABLE 1 Chemical Characteristics of leachate

(Average concentrations in mg!l)

Parameter Concentration

COD 700
DOC 240
BODs < 10
NH4-N < 1
N03-N 85 0
Cl 15 00
S04 380
Alkalinity (as CaC03) 5 10

Biogas characteristics

The landfill gas taken from the landfill site Braunschweig had an average
composition of 60% methane and 40% carbon dioxide. Moreover, the gas included
a high content of chlorinated hydrocarbons (Werner and Olderdissen, 1988). The
measured concentration varied in the range from 400 to 550 mg Cl/m3•

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 Denitrification with biogas 705

Experimental design

The reactors were started up with biomass from nitrifying activated sludge
plants treating sanitary landfill leachate. Orthophosphoric acid was added to
the influent. No further pretreatment was performed as nutrient supply.

Batch process with high methane concentrations in the gas cycle was used
initially to generate a methanotrophic/methylotrophic bacterial association.
After 2 month a continuous-flow operation was initiated. E ffluents of the
reactors were analyzed twice a week.

Batch tests were conducted to determine denitrification kinetics.

RE SULTS AND DISCUSSION

Results of batch tests

The results of a batch test are shown in Fig. 4. Activated sludge

from a lab-scale unit was fed with nitrified landfill leachate and aerated
with a gas composed of methane and oxygen.

2.5
liquid phase
Q.) 2
0
1.5
E
E

0.5
N02
0
0 5 10 15 20 25 30

120

100
gas phase
80
Q.)
0
60
E
E 40

20 CO2
0 --+-

0 5 10 15 20 25 30

time [h]

Fig. 4. Denitrification batch test using methane

JllST23-4/6-K

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 706 M. WERNER and R. KAYSER

As can be seen in the upper diagram a denitrification rate of 2.64 mmole N03/g
MLSS • d was achieved. For a limited time N02 concentration increased, but
was further reduced to nitrogen at the end of the test. The oxygen concen­
tration in the liquid culture medium was always zero.

The change in the gas concentration levels is typical for all batch tests. In
the denitrification phase the concentration of methane and oxygen decreased,
the concentration of carbon dioxide increased. In this period great amounts of
methane and oxygen were converted aerobically by methanotrophic bacteria to
carbon dioxide. At the same time carbon energy substrate (methanol) produced
by methanotrophic bacteria was used for denitrification by the methylotrophic
bacteria.

After complete reduction of nitrate the methane oxidation rate decreased

significantly. Only small amounts of methane and oxygen were further removed
and the carbon dioxide production was very low.

Results of lab-scale units

The reactors were operated over several months in 198 7 and 198 8 . Different
operational conditions were established, especially the influence of oxygen
was investigated. Table 2 shows maximum denitrificaton rates obtained.

Table 2 Maximum denitrification rates

Type of reactor Denitrification rate

Activated sludge reactor 60 mg N03-N/I d

Trickling filter 150 mg N03-N/I .

d

Fluidized bed reactor 550 mg N03-N/I d

The results from the activated sludge reactor were obtained with average
methane concentration of 45% and average oxygen concentration of 9% in the
aeration gas. The oxygen concentration in liquid medium during this period was
always below 1 mg/l.

Maximum denitrification rates at the trickling filter were attained with

oxygen concentrations of 18 % and methane concentrations of 50% in the gas
cycle. The oxygen concentration in liquid medium was in the range of 1 to 3
mg/l. The differences to the activated sludge reactor can be explained by the
greater thickness of the biofilm. In the t rickling filter a higher gradient in
oxygen concentration in the water-phase has to be established to supply all
bacteria with oxygen.

The highest denitrification rate of all units was obtained with the fluidized
bed reactor. As in the activated sludge reactor the oxygen concentration in
the liquid medium was always below 1mg/l. In the gas cycle the oxygen
concentration was approx. 12%, while the methane concentration ranged from 30%
to 40%. The results show the advantage of a fluidized bed with its high
treatment capacity because of high biomass concentration, great biomass
surface areas and good mixing of biomass and substrate.

According to equation (2) and (3), theoretically 0.8 3 Mole methane per mole
nitrate is neeclerJ for complete degradation from methane to methanol.

CH4 + 02 + 2W ----> (2 )

5CH30H + 6N03 ----> 3N2 + 5C02 + 7HzO + 60H- (3 )

However, during phases with high denitrification rates, up to 12 mole CH4 per
mole reduced N03 were consumed. This would indicate that only 7% of methane

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 Denitrification with biogas 707

was excluded as methanol and was available for denitrification. The main part
was used for bacteria build-up and decomposed aerobically to C02 and H2 0
according to equation (1).

Trace components in landfill gas did not inhibit the denitrification process.
Despite the high concentrations of halogenated hydrocarbons in the landfill gas
the same denitrification rates were achieved as if methane was used.

The only problem of using landfill gas is caused by the second main compound,
carbon dioxide (40%). The partial pressures of methane and oxygen in the gas
cycle are reduced by C02 resulting in lower denitrification rates. Therefore,
carbon dioxide should be chemically neutralized or stripped out.

Influence of oxygen on denitrification

Denitrification is an anoxic process and is suppressed in the presence of

oxygen. Henze Christensen and Harremoes (1977) reported that oxygen concen­
trations should be below 0.5 mg/l to achieve denitrification in suspended
cultures.

In contrast to these results, investigations from Hamer and Mechsner, (1984)

showed no inhibition in the presence of significant dissolved oxygen concen­
trations (up to 6. 4 mg/l).

However in a mixed culture of methanotrophic and methylotrophic bacteria the

oxygen concentration plays a significant role. Methanotrophic bacteria are
obligate aerobic and need oxygen for methane-oxidation but denitrification by
methylotrophic bacteria might be better in the absence of oxygen.

In Fig. 5 and Fig. 6 the influence of the gas-cycle oxygen concentration on

den itrification rate and oxygen consumption is illustrated. The tests were
carried out with suspended cultures. Methane was supplied in excess.
,......,
"'C
II.) 1.2
� U)
0 U)
0:: �

C �
01
"-
a 0.8
:.:; I"')
0
0 0.6
Z
U
......
'c II.) 0.4

'c
�

0
0.2
II.) E
Cl
E
L......J 0
0 2 3 4 5 6 7 8 II 10

Oxygen Concentration [%02]

Fig. 5. Influence of oxygen concentration on
denitrif ication rate
,..... 70
c l'
a
U) 60
.;:; U)
a. � 50
E �
� 01
"-
III 40
C
oN
a 30
U
Q) 20
c
II.) 0
01
>- E 10
x E
0 L......J 0
0 10 20 30 40 50 60

Oxygen Concentrotion [%02]

Fig. 6. Influence of oxygen concentration on
oxygen consumption

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 701{ M. WERNER and R. KAYSER

The results described in Fig.5 show increasing denitrification rates with

rising oxygen concentrations in the aeration gas. At the same time the oxygen
concentration in mixed liquid was always below 1 mg/l. Oxygen concentrations
in the aeration gas below 2% inhibited the process and only endogenous
denitrification might occur.

The oxygen concentration in the liquid medium defined the upper limit of
oxygen concentration in the gas phase. Oxygen concentrations greater than
1 mg/l in suspended cultures inhibited denitrification and poor denitrifica­
tion rates were obtained.

As can be seen in Fig.6, increasing oxygen concentrations enhanced oxygen

consumption and thereby promoted methane oxidation according to equation 1.

The results showed that the production of excess methanol by the methanotro­
phic bacteria is dependent on the methane oxidation rate. With increasing
methane oxidation greater amounts of excess methanol are produced and used for
denitrification. Hence, optimum supply of methane and oxygen will be the main
point for process performance.

CONCLUSION

Biogas is an alternative carbon source instead of methanol for denitrification of

wastewater effluents. Biogas produced in great amounts in wastewater treatmen t
plants during sludge digestion or in landfills during anaerobic fermentation
has a content of approx. 60% methane and can be used for denitrification as a
sole carbon source. Hence, biogas is an effective, long standing reliable and
inexpensive carbon source to achieve complete denitrification.

ACKNOWLEDGEMENT

Financial support of the DFG (German Research Association) is acknowledged.

REFERENCES

Davies, T. R., (1973). Isolation of bacteria capable of utilizing methane as a

hydrogen donor in the process of denitrification. Water Research
Vol. I, 575 - 579.
Hamer, G., Mechsner, KI., (1984). Specialized bacterial associations for deni­
trification in integrated biotreatment process. Third European Congress
on Biotechnology, Munchen, 10-14.9. 198 4, 111-61 - 111- 68 .
Harremoes, P. , Henze Christensen, M., (1971). Denitrifikation med methan. Vand
l, Februar 1971. 7 - 11.
Henze Christensen, M., Harremoes, P. , (1977). Biological denitrification of
sewage: a literature review, Prog. Wat. Tech. , .!!, (4/5) 509- 555.
Henze, M., Bundgaard, E. ,(198 2). Bemessung von kombinierten Nitrifikations­
und Denitrifikationsanlagen. gwf-wasser/abwasser, �. 240 - 246.
Hoeks, J. , (198 3). Significance of biogas production in waste tips. Waste
Management and Research, l. 323 - 335.
Mechsner, Kl., Hamer, G. , (198 5) . Denitrification by methanotrophic/methylo­
trophic bacterial associations in aquatic environments . Denitrification in
the Nitrogen Cycle, Plenum Pub!. Corp. New York and London,. 198 5. 257-271.
Sollo, F. W., Muller, H.F., Larson, T. E ., (1976). Denitrification of wastewater
effluents with methane. Journal WPCF, Vol.48 , No. 7. 18 40-1842.
Werner, M., Olderdissen, G. ,(198 8 ). Removal of chlorinated hydrocarbons from
landfill gas. Proceedings 5th International Solid Waste Conference, Volume
�, 11. -16. 9. 198 8 , Copenhagen. 151 - 157.

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