Biological Denitrification Using Saw Dust as the Energy ... - eWISA

BIOLOGICAL DENITRIFICATION USING SAW DUST AS
THE ENERGY SOURCE
H.A. Greben, M. Tjatji and S. Talma
Division for Water, Environment and Forestry Technology, CSIR, PO Box 395, Pretoria 0001.
E-mail: hgreben@csir.co.za
ABSTRACT
Nitrate contamination is one of the major problems in groundwater, which is increasingly becoming
a threat to groundwater supplies. Nitrogen in groundwater also results from human excreta, ground
garbage and industrial effluents, particularly from food processing plants. In order to remove nitrate
from a water source, such as ground water the biological denitrification technology can be applied
for which a carbon and energy source is needed. The aim of this study was to construct an easy to
operate reactor to treat the NO3 contaminated (ground) water to such qualities that it can be used
as drinking water for cattle. To achieve this objective it was investigated whether saw dust as an
alternative carbon source for the bacterial denitrification, could be used. For this purpose a low
maintenance reactor, which was inoculated with anaerobic digester sludge, was fed with nitrate
rich (100-200 mgN/L) water. A 100% nitrate removal was observed when the feed water contained
200 mg/ NO3-N at a Hydraulic Retention Time (HRT) of 1 day, operating a stable reactor system.
It was shown that similar nitrate removal rates were obtained when the reactor was operated at
room or mesophilic temperatures.
Keywords: biological denitrification, COD, groundwater, nitrate, sawdust
INTRODUCTION
Nitrate contamination is recognised as one of the major problems in groundwater utilisation
worldwide (Kaiser 2001). In Southern Africa elevated nitrate concentrations are increasingly
becoming a threat to groundwater supplies (Tredoux 1993, Tredoux et al., 2001). Nitrate in drinking
water for animal and human consumption is not recommended for health reasons. The World
Health Organization has set a limit of 10 mg/ℓ NO3 - (as N) for human consumption and 100 mg/ℓ
NO3 - (as N) for animals. For humans, the main nitrate risk is associated with the development of
methaemoglobinaemia (in children under one year of age), the development of gastric cancers,
birth defects and abortions (Terblanche 1991). Cattle and pigs can die of high nitrate (>300 mg N/ℓ)
consumption. Even moderate consumption can influence the health and milk production of milk
cows and the health of other affected animals, e.g. game.
Increased nitrate concentrations can be caused by the oxidation of ammonia (NH3) in the soil by
the nitrifying bacteria. Ammonia is often added to the crops as a nitrogen-containing chemical
fertiliser. In Taiwan, North America as well as in some European countries, pig and cattle manure is
often used as a fertiliser, which results in high nitrate levels in the run off and in the ground water of
the affected area’s, which causes high concentration of NO3 - in river and lake waters. The high
nitrate concentration generates eutrophication, often followed by fish-kills due to oxygen depletion.
Nitrogen in ground water can also be caused by human excreta (pit-latrines), sewage disposal,
cattle kraal seepage and industrial effluents, particularly from food processing. In the arid parts of
southern Africa, there is also a substantial nitrate contribution from natural sources (Heaton 1984)
that are concentrated because of the high aridity of the area. Presently, there is not much local
evidence of groundwater contamination from overuse of fertilizers (Tredoux 1993). There is,
however, enough evidence to presume that land use patterns contribute to high groundwater
nitrate levels (Heaton 1985)
Proceedings of the 2004 Water Institute of Southern Africa (WISA) Biennial Conference 2 –6 May 2004
ISBN: 1-920-01728-3 Cape Town, South Africa
Produced by: Document Transformation TechnologiesOrganised by Event Dynamics

Denitrification is a process in which the oxidised nitrogen substances, i.e. nitrates and nitrites are
reduced to nitrogen gas, such as N2O and N2, when a proton donor (energy source) is available. In
most biological denitrification systems, the nitrate polluted waste water (e.g.domestic sewage)
contains sufficient carbon (organic matter) to provide the energy source for the conversion of
nitrate to nitrogen gas by the denitrifying bacteria. To treat groundwater, in which the nitrate
contents may be as high as 100 mg NO3-N/ℓ with low dissolved carbon content, an additional
proton acceptor is required. Generally, methanol is used as well as a more complex product such
as molasses.
In order to promote nitrate removal in remote areas, it is not advisable to use complex mechanical,
maintenance requiring equipment (Clark and Tredoux in preparation). Robertson et al., (2000) as
well as Robertson and Cherry (1995) and Blowes et al., (2000) demonstrated passive in situ nitrate
removal methods that are mechanically simple and do not require significant additional
maintenance. They showed the use of nitrate reactive permeable sub surface barriers to passively
remove nitrate from septic system effluents. These barriers were installed as layers downstream of
conventional septic systems infiltration beds and as vertical walls intercepting horizontally flowing
septic system plumes. The barriers contained waste cellulose solids, such as sawdust and leaf
compost, which provided the carbon source for heterotrophic denitrification (Delwiche 1981, Nitrex
2002). It is envisaged that in South Africa a market exists for an easy to operate nitrate removal
system, which can effectively reduce the nitrate concentration of the ground water to levels
acceptable for the drinking water of cattle and other farm animals.
The aim of this study was to investigate the suitability of saw dust as the carbon and energy source
for the biological nitrate removal, using an easy to operate reactor.
MATERIALS AND METHODS
Feed Water
Artificial nitrate rich water was used as feed water for the continuous operated reactor, which
contained initially 100 mg/ℓ nitrate (NO3-N), thereafter 200 mg/ℓ. During the first period of the study
(till day 109) a weak NaHCO3 solution was added to the feed water in order to maintain the reactor
pH between 7 and 8. Once the denitrification process was in operation, the addition of the NaHCO3
solution was omitted.
Reactor(s)
During the first part of the experiment, one reactor was operated consisting of a Perspex reactor
(R1) of which the volume was 2ℓ (Fig. 1).
Figure 1. The anaerobic reactor for denitrification.
The feed water entered the reactor at the bottom, while the effluent was discharged at the top of
the reactor. The HRT was kept at 24 h during the first 109 days of the experiment. This reactor was
operated at 30 °C. After 110 days of the experiment, a second reactor (R2) was introduced. This
reactor was operated under the same conditions as the first reactor, however the second reactor
was operated at room temperature (Table 1). Reactor R1 was inoculated with micro organisms,
obtained from the anaerobic digester at the Daspoort Sewage Plant, Pretoria, South Africa.
Reactor R2 was inoculated with half of the biomass from Reactor R1.

Carbon and Energy Source
During all experiments, the carbon and energy source consisted of wood saw dust, obtained from
the CSIR campus garden service, as a product obtained from cutting down different trees.
Experimental Procedure
The following parameters were tested:
• feed water nitrate concentration
• feed water flow rate
• reactor temperature
• feed water pH/alkalinity
The biological denitrification experiment was divided in six different experimental periods, which
are shown in Table 1. During period “start” the biomass in R1 was adapted to the nitrate rich
environment during a start up period of 3 months. This period was followed by period 0, during
which period, R1 contained the biomass to which 25 grams of saw-dust was added regularly. Every
time the nitrate concentration in the reactor was removed to levels < 10 mg/ℓ, a fresh nitrate
solution of about 200 mg/ℓ was added to the reactor. When a constant reduction in the nitrate
concentration in the reactor could be observed, the reactor was operated continuously. Whenever
it was noticed that the nitrate removal in the reactor was less effective, fresh saw dust (25 g per
time) was added to the reactor (on days 6, 22, 30, 46, 64 and 84). During periods 1-3, the HRT
was maintained at 24h, while during period 4 the feed flow rate was doubled, which resulted in
HRT of 12h. On day 110 of the experiment, the biomass of reactor R1 was divided over two
reactors: R1, which was operated at 30 °C and R2, which was kept at an ambient temperature of
approximately 22 °C. Both reactors received 25 g of saw dust each on day 110 (Table 1).
Exp.
Period
Days
Table 1. The experimental periods during continuous operation.
HRT (h)
Feed Nitrate Conc.
(mg/ℓ)
Temperature Motivation
Start 1-85 Stationary 100
Room temperature Test Denitrification
concept
0 85-121 Batch 250
Initiate NO3
reduction
1
1-31
Continuous Operation
24 100 Test flow
2 32-88 24 200
Increase NO3
3 123-133 24 125
R1 30 °C
R2 room temp.
Temperature
effects
4 145-152 12 150
Test high flow
(lower HRT)
Days 89-109 no results reported. Days134-142 are omitted due to feed water inconsistency
RESULTS AND DISCUSSION
During the start period, the concept of denitrification using saw dust as the carbon and energy
source was investigated. After approximately 3 month of operation, the denitrifying bacteria had
adapted to nitrate reduction utilising the degradation products of the saw dust as the carbon and
energy source. During the batch period (period 0, days 85-121), total nitrate removal was obtained.
After feeding the reactor continuously, during period 1, nitrate was removed consistently from an
average concentration of 100 mg/ℓ to 60 mg/ℓ (Fig.2). During this period the average nitrate
removal rate was 83 mg N/d.

NO3 Conc.(mg/L)
250
200
150
100
50
0
0 20 40
Time (d)
60 80 100
NO3 In NO3 Out
Figure 2. The biological denitrification during a period of 86 days.
When the feed nitrate concentration was increased to 200 mg/ℓ, the biomass in the nitrate removal
reactor (R1) took 11 days to acclimatise to the increased nitrate concentration (day 31-42). From
day 43 onwards, improved nitrate removal could be observed (Fig.2). After day 50, total nitrate
removal was obtained. The average nitrate removal rate during period 2 was 267 mg N/d, which is
a 3-fold increase on that of period 1. This improved removal rate can be ascribed to a better
performance of the denitrifying micro organisms after adaptation, as well as to the higher nitrate
concentration in the feed water. When a higher concentration nitrate is available for the denitrifying
organisms, these bacteria will out-compete the methanogenic bacteria for the available COD,
which is used by both groups as the carbon and energy source.
The overall denitrification percentage rate is given in Figure 3.
Nitrate % removal
120
100
80
60
40
20
0
0 20 40 60 80 100
Time (d)
nitrate % removal
Figure 3. The nitrate % removal during experimental periods 1 – 2
The Denitrification Results as Obtained During Periods 3 and 4
Effect of Temperature
During periods 3 and 4 two reactors were operated. The addition of NaHCO3 was stopped in both
reactors, as the reactor pH’s were stable due to the formation of alkalinity during the denitrification
process. During period 3, reactor R1 was operated at 30 °C and reactor R2 at room temperature.
The results from both experiments are given in Figure 4.
The graphs in Fig. 4 indicate that although the NO3 concentration in the feed water differed by
about 50 mg/ℓ, the treated water in both cases had a nitrate concentration less than 20 mg/ℓ. These
results indicate that the reactor temperature and pH had no direct influence on the de-nitrification
process. This is an important finding as it means when this technology will be applied in the field,
the reaction will continue when neutral, nitrate rich ground water is fed to the reactor during an
ambient temperature of 20-22°C.

NO3 conc. (Mg/L)
Denitrification in R1 and R2 at 30 and 22 C
300
250
200
150
100
50
0
122 124 126 128
Time (d)
130 132 134
Feed R1 Treated water R1 Feed R2 Treated water R2
Figure 4. The denitrification in R1 and R2 at 30 and 22 °C, respectively.
Effect of HRT
The results of period 4 (HRT of 12h) is given in Figure 5.
It can be observed from the graphs in Fig. 5 that a shorter HRT (12H) was not beneficial for the
biological NO3 removal. Three days after starting the reactors at a HRT of 12h, the denitrification
process ceased in both reactors. The COD in the reactors was < 10 mg/ℓ, indicating that as soon
as the woodchips were fermented to a usable energy source, the fermentation products (COD)
were washed out at the faster feed rate. In addition, the microorganisms seemed to washout,
because when a biomass sample was taken from both reactors and looked at under the phase
contrast microscope, only a few denitrifying bacteria could be observed (Photos 1 and 2).
NO3 conc. (Mg/L)
Denitrification in R1 and R2 when HRT is 12h
150
100
50
0
144 145 146 147 148 149 150 151 152 153
Time (d)
Feed R1 Treated water R1 Feed R2 Treated water R2
Figure 5. The denitrification in R1 and R2 when the HRT is 12h.
Photo 1. Microorganisms in R1
(1000 x magnification).
Photo 2. Microorganisms in R2
(1000 x magnification).

Reactor Cod
The COD utilisation in combination with the nitrate reduction (from day 49-102) is given in Figure 6,
from which the COD utilisation and the nitrate concentration in the feed and in the treated water
can be observed. It can be seen that the COD utilisation coincided with the nitrate reduction, which
indicates that the available COD is used for the denitrification process. From day 90 onwards, the
COD is < 50 mg/ℓ, which caused the denitrification process to come to a halt since there was not
enough carbon and energy source for the microorganisms to reduce the available nitrate. This
finding confirms that the denitrification process is dependent on a certain minimum concentration of
COD present in the reactor. The COD pattern in the reactors was not monitored consistently during
the different experimental periods and no further evidence of the relationship between the
denitrification and COD concentration in the reactor can be shown.
Figure 6. The COD concentration in reactor R1, during the continuous operation.
Fresh saw dust was added to the reactor as soon as it was observed that the nitrate concentration
in the reactor increased and thus that the denitrification process became less effective. Each
addition of sawdust comprised 25 g and it was added to the reactor on days 6, 22, 30, 46, 64 and
84. On Day 110, reactor R1 was split into R1 and R2 and each reactor received 25 g of saw dust.
The relationship between adding the saw dust and the denitrification efficiency during the different
periods is given in Table 2.
Table 2. The relationship between the nitrate removal and saw dust added.
Period Average
mgNO3/d
removed
Total gNO3
converted
during period
g saw dust
added
% Efficiency
g N/g saw dust
1 (1-31) 83 mg NO3/d 1.25 50 2.5
2 (32-88) 267 3.7 75 4.9
Split reactors R1 R2 R1 R2 R1 R2 R1 R2
3 (123-133) 274 382 1.0 1.3 50 50 2 2.6
4 (145-152) 78 60 0.2 0.2 25 25

Table 3. The COD concentration degrading 25 g/ℓ sawdust with time
Day COD mg/ℓ
0 466
5 84
9 225
13 10
17 10
22 191
24 63
29 104
41

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