Biological Denitrification of Potable Water Using Coconut Shells in a ...

BIOLOGICAL DENITRIFICATION OF POTABLE
WATER USING COCONUT SHELLS IN A
FLUIDISED BED
Abstract
Nitrate concentrations in surface water and especially in ground water are a rapidly growing
environmental problem. Concerns of possible health consequences have led to the adoption of a
stringent nitrate standard (50 mg nitrate (NO - 3 ) per litre) in water for human consumption (WHO,
1984). Biological denitrification processes offer a potential alternative to the relatively costly and
in some cases ineffective physicochemical treatment processes. Biological denitrification utilises
bacteria to denitrify the aqueous nitrates in the absence of oxygen. This research will examine the
use of coconut shells as the carbon source for denitrifying bacteria, as well as providing a
medium on which the bacteria can grow.
The focus of this research is on the physical and environmental factors that influence the
effectiveness of biological denitrification. This research is attempting to characterise these
factors, and in doing so, evaluate and optimise the performance of the fluidised bed reactor. It is
envisaged that design guidelines for determining the most effective temperature, hydraulic
residence time, and nitrate loading to operate the fluidised bed bioreactor, will be developed.
The scientist who is content merely to carry out his work, without stopping to ask himself, `To what
effect, for good or ill among men, is my work being applied?’ is no more than a ditch digger with a
diploma in his pocket. Frank Dalby Davison, `Literature’, Soviet Culture: Selection of Talks at the
Cultural Conference, 1942

Introduction
Nitrate (NO - 3 ) is a naturally occurring form of nitrogen found in the soil. Nitrogen is essential to
all life, and most crop plants require large quantities to sustain high yields. The formation of
nitrates is an integral part of the nitrogen cycle in our environment. A moderate amount of nitrate
is a harmless constituent of food and water. Plants use the nitrates from the soil to satisfy nutrient
requirements, and may accumulate nitrate in their leaves and stems. The nitrate is released in a
soluble form when the plant degrades.
Due to its high solubility, nitrate can also leach into groundwater. Nitrate pollution of drinking
water constitutes an important and rapidly growing environmental problem. As a result of human
activities, the nitrate contamination of groundwater is becoming more prevalent. In agricultural
regions the extensive and uncontrolled use of fertilisers often leads to nitrate leaching into the
groundwater. Similarly the discharge of ammonia-rich effluents into the soil is another
significant source of nitrate pollution, typically from poorly designed or maintained municipal
sewage treatment networks.
High concentrations of nitrate in water can cause methemoglobinemia or blue baby syndrome, a
condition especially found in infants under six months. The stomach acid of an infant is not as
strong as older children and adults, which causes an increase in bacteria that can readily convert
nitrate to nitrite (NO - 2 ). The nitrite is absorbed into the blood, and haemoglobin (the oxygencarrying
component of blood) is converted to methemoglobin which is unable to carry oxygen
(White et al. 1973). This results in reduced oxygen supply to vital tissues, such as the brain.
Methemoglobin in infant blood cannot be changed back to haemoglobin, a process that normally
occurs in adults. Severe methemoglobinemia may result in brain damage and death by oxygen
starvation.
Pregnant women, adults with reduced stomach acidity, and individuals deficient in the enzyme
that changes methemoglobin back to normal hemoglobin are all susceptible to nitrate-induced
methemoglobinemia. The obvious symptom of methemoglobinemia is a bluish colour of the
skin, particularly around the eyes and mouth. Other symptoms include headache, dizziness,
weakness and difficulty in breathing.
Bottle-fed infants are predisposed to sudden infant (cot death) death syndrome, a condition of
oxygen starvation, from which about 3 per 1000 infants from most European societies die
(Money 1978). Many explanations for the occurrence of the syndrome have been offered, but
none appear satisfactory. Because of the similarity in symptoms it is possible that high
methaemoglobin levels may predispose infants to sudden death syndrome (WHO 1978).
2

Healthy, well-nourished adults can consume fairly large amounts of nitrate with few known
health effects. In fact, most of the nitrate we consume in our diet is from raw or cooked
vegetables. The nitrate is readily absorbed and excreted in the urine. However prolonged intake
of high nitrate levels has been linked to gastric problems due to the formation of nitrosamines.
These N-nitrosamine compounds have been shown to cause cancer in test animals (Self and
Waskon, 1992).
Concerns over possible health consequences has led to the adoption of a stringent nitrate standard
(50 mg nitrate (NO - 3 ) per litre) 1 in water for human consumption (WHO, 1984). In the United
States, the Environmental Protection Authority (USA EPA) has adopted an allowable nitrate
concentration of 10 mg/L of NO - 3 -N (EPA, 1986). In New Zealand the Ministry of Health has a
Maximum Acceptable Value for nitrate in drinking water of 50 mg/L nitrate (Ministry of Health).
In New Zealand, Rosen (1997) assessed 72 national sites from the National Groundwater
Monitoring Network (NGWP), and found that 6% of these sites had concentrations greater than
11.3 mg/L of NO - 3 -N. Similarly Painter et al. (1997) found that the following regions typically
had nitrate concentrations greater than 10 mg/L of NO - 3 -N; the Amuri Plains, Ashburton, Ashley
catchment, Hamilton Basin, Heretaunga Plains, Lincoln, Pukekohe, Takapau Plains, and the
Waimea Plains.
The removal of nitrates is an expensive process and for this reason, nitrate standards are often not
enforced, even in countries where the standard has been officially adopted. Inexpensive and easy
to apply nitrate technologies are therefore urgently required. Biological processes offer a
potential alternative to the relatively costly and in some cases ineffective physicochemical
treatment processes that currently exist.
1 Equivalent to 14mg/L NO 3 - -N
3

Background
Water is an essential element in the maintenance of all forms of life. To sustain life the available
water must have specific characteristics. Water quality is defined in terms of those
characteristics.
Since World War II and the dawn of the 'chemical age', water quality has been heavily impacted
world-wide by industrial and agricultural chemicals. Eutrophication of surface waters from
human and agricultural wastes, and nitrification of groundwater from agricultural practices has
affected large parts of the world.
Literature reviews (Bouwer and Crowe, 1988, Gayle et al., 1989, Kapoor and Viraraghavan,
1997) have identified that removal of nitrates from drinking water is an important and developing
area of research. They identified a need to further optimise current treatment technologies as well
as to develop new and emerging technologies.
Biological denitrification processes offer a potential alternative to the relatively costly and in
some cases ineffective physicochemical treatment processes. Nitrate is a stable and highly
soluble ion, with low potential for coprecipation or absorption. These properties make it difficult
to remove using conventional water treatment technologies such as lime softening and filtration.
More sophisticated technologies such as – chemical denitrification, ion exchange, reverse
osmosis, electrodialysis, catalytic denitrification – can be used to remove nitrates from drinking
water. However these methods have several disadvantages ranging from excessive operational
costs, problem associated with the waste disposal of by-products, and operational limitations.
One innovative and emerging technology is biological denitrification, Bouwer and Crowe (1988)
identified that detailed research is required to evaluate nitrate removal rates, with respect to
substrate concentrations (a rate limiting nutrient to growth), pH, and temperature. The influence
of toxic organic compounds on reactor performance is also still unknown. In addition, they
identified concerns with the deterioration of the microbiological characteristics of biologically
denitrified water.
This research differs from current research in this field because the coconut shell has a dual role,
both as the substratum (the support surface on which the microbial cells grow), as well as the
substrate, since the coconut shell’s providing the organic carbon source for the bacteria. Existing
research has considered supplementing the biological process with an external carbon source
such as methanol, ethanol, or acetic acid. Thus all the current biological denitrification design
relationships are unable to be applied to this research.
4

Biological Denitrification
Biological denitrification utilises bacteria to denitrify aqueous nitrates in the absence of oxygen.
The bacteria are heterotrophic (more correctly chemoheterotrophic), that require an energy
source, either in the form of organic carbon, carbon dioxide or sulphur. These denitrifying
bacteria are a form of anaerobic respirators, that use nitrate and/or nitrite as electron acceptors for
the oxidation of organic compounds when oxygen is absent, producing nitrogen gases (N 2 , NO,
and N 2 O). The microbial reduction of nitrate to gaseous nitrogen products is termed dissimilatory
denitrification or nitrate respiration. Generally denitrification is considered to be an anoxic
process, occurring in the presence of nitrate and the absence of molecular oxygen. The process
proceeds through a series of four steps, from nitrate to nitrogen gases.
Nitrate NO
−
3
→ NO
−
2
→ NO → N
2O
→ N
2
Nitrogen Gas
Surprisingly, most of the organisms known to denitrify are not strictly anaerobes, but rather are a
form of facultative organisms, which under anoxic conditions use nitrate as a final electron
acceptor for anaerobic respiration (Green et al., 1994). Gayle et al. (1989) discovered that most
investigators consider oxygen as an inhibitor of denitrification, some species have been reported
to denitrify in systems with oxygen tensions as high as 0.2 bar 2 .
Since denitrifying bacteria are heterotrophs, they utilise organic carbon to produce new biomass.
These organisms break down complex organic molecules in a series of steps. They gain most of
their energy from respiration by passing electrons along a series of electron acceptors (electron
transfer chain) to oxygen or in this case nitrate, which is reduced to nitrogen gases and water. In
this way chemical energy is converted to adenosine triphosphate (ATP) 3 . The yield of ATP per
unit of idealised organic substrate (glucose) is less when nitrate is the terminal acceptor, than if
oxygen is available, however anaerobic respiration still remains a very efficient energy yielding
process.
As mentioned, the organic carbon is chemically oxidised, while the nitrate ion is reduced to
ultimately gaseous nitrogen and water. Therefore the form of the organic carbon, can affect the
rate of denitrification (Æsøy et al. 1998).
Most of the experience with biological denitrification in drinking water has been in Europe.
These denitrification units are primarily anoxic biological granular media filters and fluidised-
2 1bar is equivalent to 0.9869 atm, where 1 atm is the pressure of 1m of water
3 ATP, is the intra-cellular chemical unit used to exchange energy between cellular functions
5

ed reactors. As in most potable water sources, the water is low in organic carbon and requires
the addition of an organic carbon source for denitrification. Methanol has frequently been
applied, however ethanol and acetic acid have also proved to be effective due to their
comparative low cost and reduced toxicity (Bosman and Hendricks, 1981).
While the operating costs of a biological denitrification reactor is substantial cheaper then other
physicochemical alternatives, the majority of the operating cost is associated with the
consumption of methanol. Therefore a bioreactor to denitrify contaminated drinking water, that
uses a cheaper, non-toxic carbon source offers significant advantages over the current options.
Carbon Sources
Researchers (Volokita et al., 1996a, 1996b) in Israel have investigated the denitrification of
drinking water using newspapers and cotton as the energy source. Their experiments involved
packing laboratory-scale columns with shredded newspapers or unprocessed short fibre cotton.
The media in the columns provided the sole chemical and physical substrate for the microbes.
These series of experiments examined the use of the simple cellulose component of these
mediums as the source of organic substrate needed by the heterotrophic bacteria. Since cellulose
is a basic component of all plant materials, it is an abundant and renewable resource.
Cellulose is a linear glucose polymer with hydrogen bonding between hydroxyl groups of
neighbouring parallel chains. Lignin 4 and hemicellulose occur in close association with the
cellulose fibres. The arrangement of these three organic molecules increases the ability of the
cellulose to resist enzymatic attack, thus only specific bacterial groups having the required
enzymes have the capacity to degrade cellulose.
This research makes use of a readily available, but an unutilised resource found in the most
‘developing’ or ‘third world’ countries in the Asia-Pacific region. Coconut shells are an organic
woody product, with similar properties to wood. In wood, cellulose constitutes about 50 percent
of the wood by weight. Lignin makes up about 23 to 33 percent of softwoods but only about 16
to 25 percent of hardwoods. Lignin is an insoluble, intractable material that is difficult to remove
from the wood as it binds the wood fibers together.
Hemicelluloses are associated with cellulose and make up about 15 to 30 percent of the wood by
weight. Ash-forming minerals are found in wood in very small quantities – perhaps 0.1 to 3
4 Lignin is the material that stiffens the cell-walls of woody tissue in plants
6

percent by weight. Extractives are not part of the wood structure, but they contribute colour and
odour to the wood (Infopedia, 1997).
Reactor Configurations
Traditionally biological denitrification processes utilise fixed bed biological filters. However
fixed bed reactors tend to suffer from clogging – where the growth of bacteria is sufficiently
large to block the pore spaces between the packed medium. This results in increased hydraulic
shear stresses in the adjacent pore pathways. If the shear stresses are sufficiently large the
bacteria attached to the support media is sheared off, thereby decreasing the efficiency of the
reactor. As indicated in Table 1, the current practice is to rigorously backwash the filter medium
in fixed bed reactors, to remove a proportion of the immobilised biomass.
Fluidised bed reactors offer superior performance compared to complete-mix and fixed-bed
biofilm reactors because the biofilm is evenly distributed throughout the reactor; while the liquid
regime of a fluidised bed offers all the advantages of a plug-flow reactor.
Since the media is in a turbulent fluidised state, clogging does not occur. Instead the hydraulic
conditions are kept symmetrical in the reactor – where the support particles order themselves
vertically up the reactor (i.e. the larger more dense particles settle near the bottom and the lighter
less dense particles rise to the top). Because fluidised beds do not suffer from clogging, it is to be
expected that they would be capable of greater denitrification rates.
Comparison of Different Biomass Support Reactors
Fixed/Expanded Beds
Fluidised Beds
Biomass control Back-washing Continuous particle removal and cleaning
Biomass recovery Washings collected in humus tank At high concentration external to reactor
Bed biomass attached
to substratum
Variable
Steady (variable if there is periodic
particle removal)
Reactor performance
(e.g.: carbon removal)
Variable
Steady (variable if there is periodic
particle removal)
Design
Requires prediction of the variation of
the bed biomass hold-up with time
Requires prediction of the variation of
the bed-particle biomass hold-up with
time
Table 1 Comparison of the different support media reactors. (Source: Cooper P.F. and B. Atkinson, 1981)
In fact, Bouwer and Crowe (1988) observed that fluidised bed reactors appear to offer the highest
nitrate-loading rate per unit volume, while overcoming the potential for clogging often
7

experienced in static bed reactors. Fluidised bed reactors however require greater recirculation of
the effluents to achieve sufficient nitrate removal, thus they require more process control.
Traditional biomass reactors, like fixed or expanded beds, tend to operate with variable biomass
concentrations, since periodic back washing of the biomass into humus collection tanks
decreases the mass of biomass. As Table 1 indicates, fluidised beds operate with a constant
concentration of biomass in the reactors.
8

Scope and Statement of Objectives
Both Bouwer and Crowe (1988) and Kapoor and Viraraghavan (1997), have reviewed current
nitrate removal processes and concluded that biological denitrification offers a viable alternative
to current technologies. They identified however several research needs for biological
denitrification; these include the evaluation and optimisation of the performance of fluidised bed
and other reactors, and the determination of the kinds of natural and synthetic organic
compounds that can be biotransformed.
The scope of this research is distinctly different from existing research in this field. The coconut
shell has a dual purpose, not only is it a substrate (a rate-limiting nutrient for growth), it is also
the substratum (the support surface on which the microbial cells grow). Opposing gradients of
nitrate and organic carbon nutrients will occur within the bacterial layer. Predicting the rate of
bacterial growth and similarly the rate of denitrification are therefore complicated by the
availability of both the nitrate and organic carbon substrates.
Bouwer and Crowe (1988) identified fluidised bed reactors as offering the best loading rate per
unit volume. An up-flow fluidised bed reactor will be constructed, because it overcomes the
clogging problem experienced by static-beds. The reactor will utilise the denitrification
properties of anaerobic respiring bacteria consuming the pelletised shell of coconuts in an anoxic
environment.
The specific objectives of this research include:
1. The construction and operation of an anoxic fluidised bed reactor that would treat influent
concentrations in the range of 20-200mg/l Nitrate (NO - 3 ).
2. Characterising the performance of the fluidised bed in terms of the loading rates (nitrate and
carbon consumption), denitrification rates, and hydraulic residence times.
3. Determining the effluent quality in terms of the chemical oxygen demand, nitrate
concentration, suspended solids concentration, PO 3- 4 concentration, and dissolved oxygen.
4. Classifying the degradation of the coconut shell.
5. Assessing the resilience of the reactor to shock loading, either by varying the hydraulic
retention time, temperature, or the concentrations of the influent nutrients.
6. Investigating the influence of utilising the support particles as a carbon substrate source - the
advantage/disadvantage of this development and the effect of diffusion limitations to
produce local variations in the microbial species within the biomass.
7. Modelling the denitrification process, in particular the influence of the rate of bacterial
growth with the assimilation of nitrate and organic carbon nutrients.
9

Description of the Reactor
A continuous fluidised bed reactor will keep the coconut shell fragments in suspension. As
mentioned, the advantage of fluidised beds over packed reactors is that the electron acceptor
(nitrate enriched fluid) and the support medium (and substrate) are evenly distributed in the
reactor, thus allowing the biological denitrification process to proceed with greater efficiency.
The reactor is moulded from Pyrex® borosilicate glass and modular in construction. Borosilicate
glass was selected due to the ease of construction, as well as its high resistance to attack from
water, acids, salt solutions, and organic solvents. Each of the modular sections is flanged to allow
the modules to be easily connected and interchanged. A photo of the reactor configuration is
shown in Figure 1. Latex gaskets seal across the adjacent glass flanges – with a stainless steel
clamp providing the positive force to hold the flanges together.
A synthetic influent feeds the reactor at a rate of 0.033 to 23.5 L/day depending on the required
hydraulic residence time. A stock solution of concentrated nitrate and phosphorous that can be
added periodically and diluted in a known volume of deionized tap water, and stored in an 80L
influent storage container. A Masterflex® pump then pumps the influent via an inline pipe mixer
into the reactor. The inline pipe mixer will ensure that the recycle line and influent line are
sufficiently mixed so that a homogeneous solution flows to the reactor.
The homogeneous solution then enters the reactor through a diffusion chamber. A series of baffle
diffusers in the diffusion chamber provide uniform velocity in the tube of the reactor. The
Pyrex® glass reactor tubes being constructed from two flanged glass segments, allows various
combinations of reactor lengths to be erected.
The upflowing fluid holds the coconut shell pellets in a fluidised state within the reactor tube.
The reactor has been designed so that all the biological denitrification occurs within the tube of
the reactor. If however any of the media (coconut shell pellets) escapes the reactor tube, the
settling chamber will retain the media and return it back to the reactor tube. The increase in
cross-sectional area of the bowl-shaped settling reactor allows the velocity of the upflow fluid to
reduce sufficiently, so that the coconut shell pellets fall out of a fluidised state and settle back
into the reactor tube.
10

Figure 1 Photograph of the fluidised bed reactor and pump configuration.
Gauze filters on the inlet of the effluent and recycle lines will retain the majority of the biomass
and coconut shell fragments within the reactor preventing the biological process from occurring
outside the reactor. The recycle flow will be maintained by passing the line through a recycle
pump, and then back into the reactor. The effluent will run into a collected container for later
analyses.
11

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