World Academy of Science, Engineering and Technology 78 2011

Aerobic Treatment of Oily Wastewater: Effect

of Aeration and Sludge Concentration to
Pollutant Reduction and PHB Accumulation
Budhi Primasari, Shaliza Ibrahim, M Suffian M Annuar and Lim Xung Ian Remmie

local source of economic development, especially in those

Abstract—This study is aimed to investigate feasibility of the remote and rural areas.
aerobic biological process to treat oily wastewater from palm oil food Apart from being one of the Malaysia’s main sources of
industry. Effect of aeration and sludge concentrations are studied. income industry, this industry especially the vegetable oil
Raw sludge and raw wastewater was mixed and acclimatized for five
(edible oil) processing plant is a main contributor of oily
days in a stirred tank reactor. The aeration rate (no aeration, low;
1.5L/min and high rate; 2L/min) and sludge concentration (3675, effluent discharge. The wastewater is high in organic content,
7350, and 11025mg/L of VSS) were varied. Responses of process which resulting in a chemical oxygen demand and also high in
were pH, COD, oil and grease, VSS, and PHB content. It was found oil and grease. Approximately, 10 to 25 m3 of wastewater is
that the treatment can remove 85.1 to 97.1 % of COD and remove generated per metric ton of product from seed dressing and
12.9 to 54.8% of oil & grease. The PHB yield was found to be within edible fat and oil processing.
0.15% to 2.4% as PHB/VSS ratio and 0.01% to 0.12% as PHB/COD
Among all the pollutants in the effluent, oil and grease is
removed. The higher aeration results a high COD removal and oil &
grease removal, while experiment without aeration gives better PHB one of the most complicated pollutants to be removed due to
yield. Higher sludge concentrations (11025mg/L VSS) give higher the characteristics of these waste waters which contain a very
removal of oil & grease while moderate sludge concentration high volume of oil-in-water emulsions as their basic
(7350mg/L VSS) give better result in COD removal. Higher PHB contaminant. These emulsified oil droplets are sheltered from
yield is obtained in low sludge concentration (3675mg/L). spontaneous coalescence into larger flocs, thus making oil
separation by simple gravity a difficult and time consuming
Keywords—oily wastewater, COD, PHB, oil and grease
process.
Due to enormous scale of this industry, the palm oil related
I. INTRODUCTION
effluents have been increasing at an alarming rate, and the

M ALAYSIA is considered one of the largest palm oil

exporters in the world with a total 4.3 million hectare of
palm oil plantations and over 400 palm oil mills throughout
effects are detrimental to both the environment and human
race. In overcoming these issues, measures to counter
pollution from vegetable oil related effluents have been
the country. The palm oil sector provides an important element deployed by the Malaysia Government. In order to regulate the
in the rural economy of Malaysia by providing a significant discharge from the palm oil related industry as well as to
source of employment in large scale. In general, there are exercise other environmental control through the enactment of
upstream and downstream industries which are related to palm Environmental Quality Act (EQA), 1974. In addition, the
oil sector. In the upstream industries, there are palm oil Environmental Quality (Sewage and Industrial Effluents)
plantations and palm oil mills while the downstream industries Regulations, 1979 which states the Maximum Effluent
include the food industries and non-food industries such as Parameter Limits has been incorporated in regulating the
biodiesel industry, oleo chemicals, pharmaceutical and others. industrial effluent quality.
These downstream industries like mills, local services and Usually, the problems of this oily effluent pollution have
support to the plantations and mills as well as providing the been overcome with the use of anaerobic and facultative
digestion to treat the waste. This is due to the anaerobic
B. Primasari is with the Department of Civil and Environmental treatment of wastewater has been considered to have a number
Engineering, University of Malaya, Malaysia (phone: 60-3-79677647; fax:
60-3-796753185555; e-mail: bprimasari@um.edu.my) and also affiliated of advantages over the conventional aerobic process. It saves
with Water Laboratory, Department of Environmental Engineering, the energy needed for aeration, converts organic pollutants
University of Andalas, Padang, Indonesia. into methane gas, a readily useable fuel, needs a low nutrient
S. Ibrahim, is with the Department of Civil and Environmental
Engineering, University of Malaya, Malaysia (phone: 60-3-79674458; fax:
requirements and produces low biomass.
60-3-796753185555; e-mail: shaliza@um.edu.my). To treat oil and grease rich waste waters biologically the
M.S.M. Annuar, is with the Institute of Science Biology, University of most important criteria to be met are stable and active biomass
Malaya, Malaysia (phone: 60-3-79674458;e-mail: and proper sludge concentration. Hence, this paper is aimed at
suffian_annuar@um.edu.my).
L. X. I. Remmie, is with the Department of Civil and Environmental achieving the goal by using a simple anaerobic batch reactor to
Engineering, University of Malaya, Malaysia (phone: 60-173473489). treat the oily wastewater through assessing the effects of

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various sludge concentrations, pH and acclimatization periods by the cost of substrate (molasses, glucose and propionate) and
on the oily effluent quality. Also, due to the nature of the extraction of the polymer from the cells. Furthermore, the need
wastewater, the microbial activity throughout the treatment for sterilization and sterile fermentation systems is prevented.
will result the accumulation of biodegradable polyester, It is well known that activated sludge submitted to transient
Polyhydroxybutyrate (PHB) which can be used in the conditions, mainly caused by discontinuous feeding and
manufacturing of biodegradable plastics. In short, the variations in electron acceptor presence, can store large
proposed reactor will not only reduce the detrimental effects of amounts of PHA. This accumulation is the key factor in the
the effluents, but the end products of this reaction will benefit competition for substrate between microorganisms in the
the general public. activated sludge processes [7].
During the wastewater treatment, bacteria can produce PHA is synthesized and intracellularly accumulated in most
storage granules of PHB (poly-b-hydroxy butyric acid). PHB bacteria under unfavorable growth condition such as limitation
is microbial poly-esters, and also called as bio-plastics, as they of nitrogen, phosphorus, oxygen or magnesium in the presence
are biodegradable polymer. PHB is stored in the cell of excess supply of carbon source [4] and [8]. Strategies are
cytoplasm by a microorganism under stress conditions caused still being developed to simulate conditions for efficient
by limitation of nutrients. Industry use pure microbial cultures production of PHAs. [8] and [9]. Some bacteria such as A.
and pure substrate (mostly glucose) to produce PHB. A high eutrophus, A. Latus and mutant strain of Azotobacter
production cost process is caused by the substrate cost and vinelandii are known to accumulate PHA during growth in the
energy for sterilization and extraction of the polymer, absence of nutrient limitation.
therefore, so far production cost of polymer. Therefore, still Several factors need to be considered in the selection of a
ten times of the conventional plastics. Using mixed cultures microorganism for the industrial production of PHA such as
can reduce cost of production of PHB, because of cheap the ability of the cell to utilize an inexpensive carbon source,
substrate and simple (non-sterile) reactors are used. Using growth rate, polymer synthesis rate and the maximum extent of
mixed culture, selection of microorganism occurs based on its polymer accumulation of a particular cell based on the
capability, to store PHB. The metabolism of PHB storage is substrate. Some workers have derived equation that predicts
the availability of electron donor and acceptor or because the the PHA yield on carbon sources [10] which could be used for
non-continuous availability of substrate for the microorganism. the preliminary calculation of PHA yields. In order to reduce
Cooking oil wastewater (from palm oil factory) can be the overall cost, it is important to produce PHA with high
considered as an alternative, cheap substrate for PHB productivity and yield.
production. The COD moderate content (1500-5000 mg/L) Several methods such as fed-batch and continuous
and high oil and grease content (80-100 g/L) will be an cultivations have been carried out to improve productivity [4]
advantage as a substrate. and [8]. Only three prominent PHAs [PHB, poly (3-
Bioplastics are attracting much interest as alternatives to hydroxybutyrate-co-3-hydroxyvalerate) and poly (3-
traditional plastics. They are biodegradable and can be formed hydroxyhexanoate-co-3-hydroxyoctanoate)] had been
from renewable resources. Among the biodegradable plastics produced to a relatively high concentration with high
polyhydroxyalkanoates (PHAs), and in particular, the productivity. Recently, workers have been exploring
copolymer poly -(hydroxybutyrate-hydroxyvalerate) cultivation strategies involving inexpensive, renewable carbon
[P(HBHV)], are the most promising. P(HB-HV) has similar substrates in order to reduce production cost and obtain high
properties to polypropylene. It can be processed in the same productivity [4], [11] - [14]. Recovery of PHA should also be
way, and it could have the same wide range of application [1]. considered because it significantly affects the overall process
However, until now production of P(HB-HV) has proven to be economics. The last stage of PHB production involves
much more costly than traditional oil-derived plastics and this separating the polymer from the cells. To do this a solvent of
has hindered its use becoming more widespread [2]. aqueous extraction can be used. In the aqueous process, the
Poly -hydroxybutyric acid (PHB) is the most extensively cell walls are broken, and the polymer is then extracted and
studied PHA, produced in nature in the presence of excess purified. The aqueous process is less expensive, but the
carbon by bacteria as storage granules providing food, energy process reduces the polymer molecular weight.
and reducing power [3]. PHB has properties similar to The research project is primarily intended to treat oily
petroleum derived synthetic plastics like polypropylene (PP) wastewater, and simultaneously to produce bio-plastic in terms
and is completely biodegradable in the environment. of PHB (poly hydroxy butyric acid). The stirred tank reactor
Much effort has been spent in optimizing the poly-b- was used, and to enhance pollutant reduction and PHB
hydroxybutyrate (PHB) production using pure substrates and accumulation, aeration was applied. Effect of sludge
pure cultures [4] - [5]. However, the product (PHB) cost is still concentration, i.e. number of microbes worked for treatment,
around ten times higher than that of conventional plastics [6]. and effect of aeration, i.e. amount of aeration supplied were
The use of mixed cultures and cheap substrates (waste investigated.
materials) can reduce costs of PHA production by more than
50%. The major costs in the PHA production are determined

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II.MATERIALS AND METHODS D. Analytical methods

Accumulation of PHB is measured using methods explained
A. Oily wastewater and Sludge
by Law Slepecky in Slepecky [15]. Chemical parameters, i.e.,
Sample of sludge and wastewater was collected from COD, VSS, oil and grease, were analyzed in accordance with
Golden Jomalina Food Industry Sdn. Bhd. Golden Jomalina methods in the standard methods (APHA, 1999).
Sdn. Bhd. produces refined palm oil and palm kernel oil
products which include shortening, margarine, frying palm oil, III. RESULTS AND DISCUSSION
milk fat replacement, vegetable ghee and cooking oil. The
industry also produces oil products for application in dairy A. COD removal
products, coloring and as food ingredients. These products are The COD removal percentage is shown in Fig 1. COD
exported to Japan, Hong Kong, Europe and United State of removal was found in range of 85.1 to 97.1%. The graph
America. The sludge is collected from an aerobic tank, and shows that high aeration rate give highest removal percentage
oily wastewater is obtained directly from an outlet of the for chemical oxygen demand (COD). It can be seen that
factory. Characteristics of wastewater and sludge are shown in removal of COD increased with increasing aeration. The air
Table 1 below. supplied helps in degradation of organic matters in wastewater
by the oxidation process.
TABLE I
CHARACTERISTIC OF WASTEWATER AND SLUDGE
Parameter Sludge Wastewater
TSS (mg/L) 17762.5 322.5
VSS (mg/L) 14700 215
COD (mg/L) 46246.5 3115
pH 6.48 6.30
Temperature (
) 23.8 22.5
Oil & Grease (mg/L) - 92.3
PHB (mg/L) 40.12 -

B. Sludge
Fig. 1 Effect of aeration and sludge concentration on COD
The raw oily wastewater and SBR sludge from a palm oil removal
refinery factory in Kuala Langat, Selangor was collected as the
subjects for this study. These samples were collected using 25 Higher aeration provides more oxygen to oxidize and
liters containers and kept in the cold room (in the degrades the COD in the wastewater. The organic substances
Environmental and Public Health Laboratory, Department of are readily degraded to simple organics by microorganisms in
Civil and Environmental Engineering) at temperature range the treatment. Sludge concentration of 7350mg/L VSS is
from three ºC to four ºC for preservation. suitable for oily wastewater treatment as the sludge contains
C.Experimental adequate microorganisms to remove organic substances in
wastewater. It is comparable with previous study which stated
Acclimatization is a process for microorganisms in the
sludge to adapt to the new environment in wastewater and 3000-6000mg/L MLSS can achieve higher treatment
maintaining performance in different environmental condition. efficiency in activated sludge. The other operation parameters
The organisms adjust their morphological, behavior, physical can be varied to improve the removal efficiency, and the best
and chemical form so able to respond to the changes occurred. operational condition may apply in this study.
Acclimatization is carried out using a mixture of activated B. Oil and Grease removal
sludge, and wastewater collected in a 3:1 ratio in volume for
Fig. 2 shows oil and grease removal at different aeration
five days. Every acclimatization process was carried out in a
three liter stirred tank with a 6-blades up-pumping 45o-pitched rate. Oil and grease removal was found in range of 12.9 to
blade impeller at 200 rpm. 54.8%. It is obvious that from no aeration to higher aeration
The treatment process of oily wastewater was carried out in rate, the removal percentage increased. For run at sludge
variation of aeration rate (zero aeration, low aeration concentration of 7350mg/L VSS, the final effluent still contain
1.5L/min, high aeration 2.0L/min), and sludge volume (25%, high oil and grease concentration, as the removal percentage is
50%, and 75% equivalent to 4360mg/l, 8720mg/l, and low. Aerobic treatment of oil and grease is shown to be in low
13080mg/l VSS respectively). All the treatment run are carried efficiency when oily wastewater is not diluted because oil is
out in a three liters cylinder tank and stirred with a 6-blades long chain organics and hard to be degraded. In conclusion,
up-pumping 45o-pitched blade impeller. To provide aeration, oxygen provided can help degrade oil and grease better.
an air pump was utilized to provide different rate of air supply.

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Fig.3 Effect of aeration and sludge concentration PHB yield as

Fig.2 Effect of aeration and sludge concentration on oil and mgPHB/mg VSS
grease removal
Fig 4 shows the PHB yield in terms of COD removal varied
Oil and grease degrade better when oxygen is supplied by aeration rate. It is shown in the graph that treatment without
because it will react with organics and smaller of organic aeration produces a higher PHB yield in terms of COD
substances will be formed. Microorganisms can consume these removal. PHB yield per COD removed was found in range of
simple organics for their growth and form as biomass. Higher 0.01 to 0.12%. When there is no aeration in treatment,
sludge concentration indicates a high concentration of microorganisms may gain energy from intracellular
microorganisms, which reduce long chain organics better. polyphosphate and enhance the PHB production rate.
However, the removal percentage is still low for oil and Polyphosphate degradation lead to high PHB production rate
grease. Certain microorganisms can remove oil content well, as polyphosphate can provide sustainable energy. In addition,
but it may not exist in the sludge. Lower initial COD energy also gained from oxidation by a small amount of air
concentration in wastewater denotes oil and grease in entered when mixing in treatment without aeration.
wastewater has been reduced. The amount of oil is less, and a
better removal can be achieved as organics to the
microorganism ratio is decreased. A high sludge concentration
produces a higher removal of oil and grease.
C. PHB yield
Fig 3 shown below is the graph of PHB yield at variation of
aeration rate. It is indicated in the results that experiment runs
without aeration supplied to get better PHB yield compared to
low aeration and high aeration rate. PHB yield as PHB
accumulation per mass of sludge removal was found in range
of 0.15 to 2.4%. In most studies stated that with oxygen
supplied, PHB production in activated sludge is higher than
those without oxygen supplied. Small amount of organic
substrate is oxidized and provide energy to microorganisms.
However, the energy tends to utilize for cell growth rather than Fig.4 Effect of aeration and sludge concentration PHB yield as
PHB accumulation. In addition, air supplied should be mgPHB/mgCOD removed
controlled as the percentage of air in activated sludge will
affect the formation of PHB within the bacteria cell. Under the Lower sludge concentration (3675mg/L VSS) can yield
aerobic conditions, excess of organic carbon is required to more PHB per substrate provided is due to availability of
improve the accumulation of PHB. Excess of organic carbon is excess substrate. With a low or a high aeration, sludge
available in the wastewater, but it probably is utilized for other concentration of 7350mg/L VSS produce a higher PHB yield.
purposes result in low yield of PHB. Organic substrates are oxidized and provide energy for
microorganisms to produce PHB. Energy supply is adequate
for PHB accumulation compared to excess energy in lower
sludge concentration (3675mg/L VSS), which tended to
accumulate as other forms of energy.
For PHB yield in terms of PHB/VSS and PHB/COD
removal, treatment without aeration can give a better result in

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both terms. Microorganisms gain energy from degradation of weight distribution control in a fed-batch culture of Alcaligenes
eutrophus," Journal of Fermentation and Bioengineering, vol. 76, pp.
polyphosphate to produce PHB. The rate of PHB production is 465-469, 1993.
higher in anaerobic condition, although higher PHB content [6] A.S.M., Chua, H. Takabatake, H. Satoh, and T. Mino. “Production of
can be accumulated in aerobic condition. High aeration can polyhydroxyalkanoates (PHA) by activated sludge treating municipal
result in PHB degradation and low yield of PHB. Excess wastewater: effect of pH, sludge retention time (SRT), and acetate
concentration in influent,” Water Res. 37(15). Pp. 3602-3611.2003.
energy provided from oxidization of organics is used for other [7] N. Azbar, (2004). A Review of Waste Management Options in Olive Oil
purposes such as microorganism’s growth. Low sludge Production. Crit Rev Env Sci Tech. vol. 34, no. 3, pp 209–247
concentration accompanied by high concentration of [8] G. Du and J. Yu, "Metabolic analysis on fatty acid utilization by
Pseudomonas oleovorans: mcl-poly(3-hydroxyalkanoates) synthesis
wastewater as the ratio is 1:3 in volume. Thus, carbon versus -oxidation," Process Biochemistry vol. 38, pp. 325-332, 2002.
substrate concentration is higher in smaller population of [9] G. Du, J. Chen, J. Yu, and S. Lun, "Continuous production of poly-3-
microorganisms. The substrate is enough for bacterial growth hydroxybutyrate by Ralstonia eutropha in a two-stage culture system,"
Journal of Biotechnology, vol. 88, pp. 59-65, 2001.
and lead to better yield of PHB. There is higher COD [10] J. Wang and J. Yu, "Kinetic analysis on inhibited growth and poly(3-
concentration in wastewater without dilution, so the PHB yield hydroxybutyrate) formation of Alcaligenes eutrophus on acetate under
is higher as the organics are adequate for PHB production. The nutrient-rich conditions," Process Biochemistry, vol. 36, pp. 201-207,
2000.
organics are utilized to produce PHB with energy provided.
[11] D. Byrom, "Production of poly-[beta]-hydroxybutyrate: poly-[beta]-
hydroxyvalerate copolymers," FEMS Microbiology Letters, vol. 103, pp.
IV. CONCLUSION 247-250, 1992.
[12] T. Tsuge, K. Tanaka, and A. Ishizaki, "Development of a novel method
The experiment shows that a high removal percentage of for feeding a mixture of L-Lactic Acid and Acetic Acid in Fed Batch
COD and O&G can be obtained when a high aeration rate is Culture of Ralstonia eutropha," Journal of Bioscience and
applied in the treatment process. The COD and O&G removal Bioengineering, vol. 91, pp. 545-550, 2001.
[13] Y. Poirier, "Production of new polymeric compounds in plants " Current
increased when higher aeration rate is used. Accumulation of Opinion in Biotechnology, vol. 10, pp. 181-185, 1999.
PHB is decreasing when higher aeration is applied. The PHB [14] P. Kahar, T. Tsugea, K. Taguchi, and Y. Doi, "High yield production of
yield in process without aeration is higher than those with polyhydroxyalkanoates from soybean oil by Ralstonia eutropha and its
recombinant strain," Polymer Degradation and Stability, vol. 83, pp. 79-
aeration. Low aeration can gain more PHB accumulation 86, 2004.
although the yield is low. The aerobic treatment is efficient [15] J. H. Law and R. A. Slepecky, "Assay of Poly-b-hydroxybutyric acid,"
and feasible in oily wastewater treatment but not suitable for Journal of Bacteriology, vol. 82, pp. 33-36, 1961.
accumulation of PHB. [16] G. Braunegg, G. Lefebvre, and K. F. Genser, "Polyhydroxyalkanoates,
biopolyesters from renewable resources-Physiological and engineering
Sludge concentration indicates the MLVSS of the activated aspects," Journal of Biotechnology, vol. 65, pp. 127–161, 1998.
sludge. It is found that sludge concentration of 7350mg/L VSS [17] E. Y. Lee and C. Y. Choi, "Biosynthesis and Biotechnological
produces the highest COD removal while best result for Production of Degradable Polyhydroxyalkanoic Acid," Biotechnol.
Bioprocess Eng, vol. 2, pp. 1-10, 1997.
removal of O&G obtained when the sludge concentration was [18] B. Qu and J. Liu, "Determination of optimum operating conditions for
11025mg/L VSS. The yield of PHB can be seen to be highest production of polyhydroxybutyrate by activated sludge submitted to
when sludge concentration is 3675mg/L VSS. Low sludge dyna[1]mic feeding regime," Chinese Science Bulletin, vol. 54, pp. 142-
149, 2009.
concentration (3675mg/L VSS) can achieve a high removal of [19] G.-C. Du, J. Chen, H.-J. Gao, Y.-G. Chen, and S.-Y. Lun, "Effects of
COD and O&G as well as PHB yield. However, accumulation environmental conditions on cell growth and poly-b-hydroxybutyrate
of PHB is higher when sludge concentration is high. Further accumulation in Alcaligenes eutrophus," World Journal of Microbiology
& Biotechnology, vol. 16, pp. 9-13, 2000.
study should be carried out to investigate the treatment
[20] L. S. Serafim, P. C. Lemos, M. G. Albuquerque, and M. A. Reis,
efficiency and PHB accumulation when a lower sludge "Strategies for PHA production by mixed cultures and renewable waste
concentration is applied, materials," Applied microbiology and biotechnology, vol. 81, pp. 615-
28, Dec 2008.
[21] K. Johnson, R. Kleerebezem, and M. C. van Loosdrecht, "Influence of
REFERENCES the C/N ratio on the performance of polyhydroxybutyrate (PHB)
[1] S. Lee and J. Yu. Production of biodegradable thermoplastics from producing sequencing batch reactors at short SRTs," Water research,
municipal sludge by a two-stage bioprocess. Resour Conserv Recy., vol. 44, pp. 2141-52, Apr 2010.
vol. 19, pp. 151-164, 1997 [22] S. Shahhosseini, "Simulation and optimisation of PHB production in
[2] H. Salehizadeh and M. C. M. Van Loosdrecht, "Production of fed-batch culture of Ralstonia eutropha," Process Biochemistry, vol. 39,
polyhydroxyalkanoates by mixed culture: recent trends and pp. 963-969, 2004.
biotechnological importance," Biotechnology Advances, vol. 22, pp.
261-279, 2004.
[3] M. C. M. Van Loosdrecht, J. J. Beun, and J. J. Heijnen, "Poly-[beta]-
hydroxyalkanoate metabolism in activated sludge," in Advances in
Water and Wastewater Treatment Technology, M. Tomonori, H.
Keisuke, T. Satoshi, and S. Hiroyasu, Eds., ed Amsterdam: Elsevier
Science B.V., 2001, pp. 239-248.
[4] S. Y. Lee, K. S. Yim, H. N. Chang, and Y. K. Chang, "Construction of
plasmids, estimation of plasmid stability, and use of stable plasmids for
the production of poly(3-hydroxybutyric acid) by recombinant
Escherichia coli," Journal of Biotechnology, vol. 32, pp. 203-211, 1994.
[5] H. Shimizu, S. Tamura, S. Shioya, and K.-i. Suga, "Kinetic study of
poly-d(-)-3-hydroxybutyric acid (PHB) production and its molecular

176