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Original ArticleTreatment of Palm Oil Mill Effluent by

Thermotolerant Polymer-Producing FungiPoonsuk Prasertsan, Haslinda Binmaeil

Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Thailand

ABSTRACTPalm oil mill effluent (POME) had acidic pH (pH 4.5) and high C/N ratio that was suitable for fungal treatment. Treatment of POME (15 − 51 g/L) by Humicola insolens D2, Thermomyces lanuginosus E4, and Rhizopus oryzae ST29 for 5 days indicated the optimum concentration of 22.5 g/L soluble COD. R. oryzae ST 29 was most efficient in removal of COD (60.0%), oil & grease (98.6%), and total solids (52.9%). The strain grew well (16.9 g-biomass/L) and also produced the highest biopolymer (26.9 mg/g biomass) with the simultaneous removal of solids from POME. The maximum treatment efficiency and enzyme production (814 U/mL CMCase and 1,550 U/mL xylanase) were achieved after 4 days cultivation. R. oryzae ST 29 was selected for optimization studies which revealed the supplementation of 0.025% fertilizer (46% urea) and the initial pH of 4.5. Under the optimum condition, the treatment increased to 80% COD removal with simultaneous increase of biopolymer by about 2 folds (52.2 mg/g biomass). Therefore, bioaugmentation of R. oryzae ST 29 in POME not only enhanced the treatment efficiency but also generated biomass, enzymes, and biopolymer (MW 17,700 Daltons) as bioproducts.

Keywords: palm oil mill effluent, thermotolerant fungi, biopolymer, Rhizopus oryzae, wastewater treatment

INTRODUCTION

Palm oil industry is one of the three major agro-industries in southern Thailand and consisted of oil palm plantation, palm oil mill and refinery [1]. In Thailand, palm oil mill generated 0.5 − 1.2 m3 effluent per tonne of fresh fruit bunch (FFB) or in average 0.87 m3/t FFB [1,2]. Intensive studies for clean technology in palm oil mills in Thailand reported the average figure of 0.56 m3 (POME) per ton of fresh fruit bunch (FFB) [3]. Palm oil mill effluent (POME) is the mixed effluent generated from two major sources; sterilizer and de-canter or separator during the extraction of palm oil. POME is an acidic brownish colloidal suspension consisting of high organic content (75 − 96 g-COD/L, 22 − 54 g-BOD/L), high total solids (51 − 105 g/L including 24 − 77 g/L suspended solids) with oil & grease (8 − 26 g/L), and high temperature (70 − 80°C) [4–6]. POME also contains many minerals and heavy metals such as N (3,140 mg/L), P (517 mg/L), K (3,900 mg/L), Mg (1,255 mg/L), Cu (7 mg/L), Zn (14 mg/L) and Fe (516 mg/L) [7]. Total solids in POME include sus-

pended solid, dissolved solid, and lignocellulosic wastes [8–10]. The oil & grease in wastewater had to be removed to prevent its attachment to water treatment units, avoid problems in the biological treatment and consistent with water-discharge requirements [11].

Industrial wastewater is generally treated by physical, chemical and biological methods. Biological treatment of POME to reduce the wastewater strength is one potential method that can be adopted to alleviate the pollution prob-lem faced by the palm oil industry. Many researchers have reported the use of various micro-organisms to treat food processing wastewater and POME [12] such as lipolytic bac-teria and fungi [13]. Anaerobic digestion and pond systems are efficient in reducing organic matter and solid floatation on the surface of ponding system especially in the first and second ponds is observed in all palm oil mills. The major disadvantage of ponding system was the requirement of large area due to the high hydraulic retention time (HRT). To shorten the HRT and decrease the area needed, the efficiency of many aerobic microorganisms were tested. Yarrowia lipo-

Corresponding author: Poonsuk Prasertsan, E-mail: poonsuk918@yahoo.comReceived: July 31, 2017, Accepted: November 23, 2017, Published online: June 10, 2018Copyright © 2018 Japan Society on Water Environment

Journal of Water and Environment Technology, Vol.16, No.3: 127–137, 2018doi: 10.2965/jwet.17-031

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lytica NCIM 3589, a marine hydrocarbon-degrading yeast, gave a COD reduction of about 95% with a retention time of two days [10]. Many fungi, such as Trichoderma viride, T. harzianum, and Myceliophtora thermophila were used for treatment of POME [10,12]. The fungi have the ability to convert dissolved and suspended organic matter into a mycelium that is high in protein content and can be readily recovered by simple filtration or screening [12]. Thus, using biological treatment of POME offers an alternative solution to reduce the total suspended solids and organic load content of the effluent [14].

For POME, its unique characteristics are high temperature with high solid content, high organic matter and oil. Research at pilot scale project entitled oil recovery from palm oil mill wastewater revealed that oil recovery could be improved by using decanter-polishing separator process (40% oil reduc-tion) which is more efficient than the separator-polishing separator process (14% reduction) [1] and this process has been used till present. Since no chemical is added during the oil extraction process, chemical treatment should be avoided as it could cause problems of chemical residues and the dis-posal. Due to its high solid content, it is not appropriate to use high treatment efficient up-flow-anaerobic sludge blanket (UASB) system for the treatment of POME. Total solids re-moval by sedimentation does not occur due to its emulsion property. Breaking the emulsion of POME by using enzymes such as pectinase to break down pectin was not successful as it may involve many other enzymes. The successful case oc-curred when applied xylanase enzymes for oil recovery and solids separation [15]. However, application of the enzymes to industrial level in palm oil mill is not practical as it is too expensive and need knowledgeable personnel. Therefore, this research work proposed a novel method in harvesting total solids from POME by aggregation to biopolymer and mycelium of polymer-producing fungi. Biopolymers are defined as polymers produced by living organisms, in this case fungi. This investigation involved the selection of the polymer-producing fungal strain, optimization studies on POME concentration, nitrogen source and concentration, as well as initial pH to obtain the highest treatment efficiency and biopolymer production from the selected fungal strain. The higher production of biopolymer was anticipated to increase the fungal aggregation to total solids in the POME and higher treatment efficiency.

MATERIALS AND METHODS

MicroorganismsHumicola insolens D2 and Thermomyces lanuginosus E4

were isolated from the wastewater sample of Para-rubber wood processing factory and rice husk sample of rice mill, respectively, and identified by observing on cell morphology (Binmaeil and Dalee, unpublished data). This work was con-ducted at Yala Rajabhat University, Thailand. They had the optimum termperature at 55°C. Rhizopus oryzae ST29 was isolated from samples taken from palm oil mill and identi-fied by using both cell morphology and molecular technique based on internal transcribed spacer (ITS) gene [16]. This fungal strain had the optimum temperature of 45°C, the same temperature as previously studied [16].

Effect of POME concentrations on growth and treat-ment efficiency of the three fungal strainsCharacteristics of POME

The POME was taken from Trang Palm Oil Co., Ltd. in Trang Province, Thailand and kept at −20°C until used. Characteristics of the POME were determined on color, pH, chemical oxygen demand (total COD and soluble COD), total solids (TS), suspended solids (SS), oil & grease [17], total nitrogen by total Kjeldahl nitrogen method and total phos-phorus by DIN 38402 A51 method [18].

Comparison on treatment efficiencyStarter culture was prepared by adding 10 mL of 0.1%

Tween 80 onto PDA slant of 5 days old culture of H. insolens, T. lanuginosus and R. oryzae ST 29. The concentration was adjusted to 2.4 × 106 spore/mL before using as the starter culture. The POME was diluted with deionized water in the ratio of 1:0, 1:1 and 1:2, to obtain the soluble COD concentra-tion of 51.1 g/L, 22.6 g/L and 15.0 g/L, respectively. They were sterilized (by autoclaving at 121°C for 15 min) before used. The starter culture of each fungi was inoculated (10% (v/v) of 2.4 × 106 spore/mL) into three concentrations of POME (supplemented with 0.06% NH4NO3) and incubated on a shaker (200 rpm) for 5 days at their optimum growth temperatures at 55, 55 and 45°C, respectively (Table S1). The samples were determined for biomass and soluble COD removal. The POME concentration giving the highest treat-ment efficiency was selected for further studies.

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Time course on treatment and enzymes production from the three fungal strains in the optimum concen-tration of POME

The starter culture of the three fungal strains were pre-pared as described above. They were inoculated (10% (v/v)) into POME (added with 0.06% NH4NO3) at the optimum concentration and incubated on a shaker (200 rpm) for 5 days at their optimum temperatures. Samples were taken every 24 h to determine for pH, biomass, COD and total solids, followed the methods as described in the Standard Methods for the Examination of Water and Wastewater [17]. For de-termination of enzymes activity, the culture broth samples were centrifuged (10,000 rpm for 10 min) and the clear supernatant (crude enzyme) was used for determination of enzyme activity. The lipase activity was determined as described by [19] while the activities of carboxymethylcel-lulase (CMCase), xylanase and pectinase were determined following the methods as described earlier [20–22] and the reducing sugars were measured by Somogyi-Nelson method [23].

Effect of nitrogen source and concentration on growth, treatment efficiency and biopolymer production from the selected fungal strain

The effect of three nitrogen sources on growth, treatment efficiency and biopolymer production from the selected strain was studied. NH4NO3 (Fluka Co., Ltd., Buchs, Switzerland), urea (Rankem Co., Ltd., Gujarat, India) and fertilizer (46–0-0 or 46% urea, commercial grade purchased from local fertilizer shop) at the concentration of 0.1% N were added with the amount of 2.8, 2.14 and 2.17 g/L (w/v), respectively, into the POME. The starter culture (2.4 × 106 spore/mL) was inoculated (10% (v/v)) and incubated on a shaker (200 rpm) for 4 days at the optimum temperature. Blank was prepared using the same method but no addition of nitrogen source into POME. The nitrogen source giving the best results was selected to find its optimum concentration. The selected nitrogen source was added into the POME at the concentra-tions of 0, 0.01, 0.025, 0.050, 0.075, 0.1 and 1% (w/v). The experiment was conducted as described above. Samples were taken every 24 h to determine for biomass, soluble COD and biopolymer. The nitrogen concentration giving the best results was chosen for further studies. The biopolymer was determined by recovery from the biomass or dried cell weight after centrifugation and lyophilized. The crude bio-polymer was dissolved in 1% sodium dodecyl sulfate (SDS) (in the ratio of 1:12.5 (w/v)), at room temperature for over-night with stirrer, centrifuged and washed the precipitate

with deionized water and centrifuged to remove insoluble materials for three times. The precipitate was lyophilized, then dissolved in 1 M NaOH (in the ratio of 1:30 (w/v)) at room temperature for 2 − 3 h with stirrer, then centrifuged and washed with 1 M NaOH for three times. The biopolymer was precipitated from the supernatant by addition of cold ethanol in the ratio of 1:4 (supernatant:cold ethanol) at 4°C overnight, centrifuged and dried (103°C, 24 h) [24]. The biopolymer obtained was determined for molecular weight (MW) using gel permeation chromatography (GPC, Waters 600E, Milford, USA) and particle size using zeta potential analyzer following the procedures as described by Leal et al. [24]. GPC was determined using ultrahydrogel linear 1 col-umn (Waters, Milford, USA) with Refractive Index Detector (Waters 2410, Milford, USA).

Effect of initial pH on growth, treatment efficiency and biopolymer production from the selected fungal strain

The effect of initial pH was studied by adjusting the pH of POME to pH 3.0, 3.5, 4.0, 4.5, 5.5, 6.0 and 6.5 using NaOH or HCl before autoclaved. The original pH of the POME (pH 4.5) was used as a control. The starter culture was prepared as above and inoculated (10% (v/v)) into POME supplement-ed with the optimum concentration of the selected nitrogen source at different initial pHs. The cultivation was carried out on a shaker (200 rpm) for 4 days at the optimum tem-perature. Samples were taken every 24 h to determine for biomass, soluble COD and biopolymer as described above.

RESULTS AND DISCUSSION

Effect of POME concentration on treatment efficien-cy of the three fungal strains

Characteristics of the POME in this study were similar to those from the others reported earlier (Table 1). It had acidic pH (4.5) and contained high organic matter (144 g/L COD), high total solids and suspended solids (71.5 g/L and 34.2 g/L, respectively) with lower amount of oil & grease (10 g/L), total nitrogen (1.2 g/L) and phosphorus (0.5 g/L). The difference in POME characteristics in various studies was due to the difference in the quality of raw material, method of oil extraction, the efficiency of the process and the sampling time, degree of oil extraction and volume of water used during the milling process [25]. Since there was no chemical added during the oil extraction process at high temperature, POME was considered to be a very good source for growth of microorganism [26].

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The effect of POME concentrations was investigated to find the maximum treatment efficiency of the three fungal strains after 5 days cultivation. The optimum POME con-centration was 22.6 g/L COD for all three fungal strains (Table 2), giving the highest COD removal of 20.0, 40.0, and 60.0%, respectively. The optimum POME concentration was obtained by diluting the POME in the ratio 1:1 or 50% dilu-tion. POME contained not only organic matter but also some inhibitors such as phenol (270 − 350 mg/L) [26]. Phenol and its derivatives are not only difficult to degrade or assimilate by microorganisms but also toxic and possess phytotoxic property to the germination of duram wheat seed [27] and maize (Zea mays L.) seeds [28]. The treatment efficiency of R. oryzae ST29 was about 3 fold and 1.5 fold higher than those of H. insolens and T. lanuginosus, respectively. These results were directly correlated to the growth of the three fungal strains with the highest biomass of 4.06, 7.96 and 16.9 g/L, respectively. The treatment efficiency of R. oryzae ST29 was higher than that of Rhizopus sp. ST4 (54.9% COD removal) [29,30] and activated sludge process in treating diluted POME (5000 mg/LCOD) at 24 h HRT (27% COD removal) [31].

Time course on treatment and enzymes production from the three fungal strains at the optimum con-centration of POME

H. insolen, T. lanuginosus, and R. oryzae ST29 were cul-tivated at their optimum temperatures of 55, 55 and 45°C, respectively, in POME (added with 0.06% NH2NO3) at the optimum concentration on a shaker (200 rpm) for 5 days. R. oryzae ST29 was confirmed to be the most efficient and gave higher COD removal (72.5% at 4 days) (Fig. 1) than the previous report (66%) [29] as well as by H. insolen (28.6%) and T. lanuginosus (30.6%). Growth of R. oryzae ST29 (18.3 g/L) was 2.3 and 4.4 times higher than those of H. in-solen (7.9 g/L) and T. lanuginosus (4.13 g/L). The pH values during cultivation of R. oryzae ST29 slightly increased (from 4.5 to 5.2 − 5.5) while those of H. insolen and T. lanuginosus were rather stable (in the range of 4.4 − 4.6). The total solids were reduced by 66.4%, 20.5% and 11.4%, respectively. It was observed that R. oryzae ST29 produced biopolymer, therefore, it was anticipated that the highest total solids removal was due to the formation of biopolymer that could simultaneously harvest the total solids in POME as well as the mycelium. This resulted in spontaneous sedimentation

Table 1 Characteristics of palm oil mill effluent.Parameter This study 1 2 3 4Color Brown Brown Brown BrownpH 4.5 4.6 4.2 − 4.8 4.2 − 4.7 4.2 − 4.5Total COD (g/L) Soluble COD (g/L)

143.9 50.8

52.9 -

90.0 − 179 -

95.5 − 112 -

75.2 − 96.3 -

Total solids (g/L) 71.5 36.4 51.2 − 105 68.9 − 75.3 35.0 − 42.0Suspended solids (g/L) 34.2 11.6 24.3 − 76.8 44.7 − 47.1 8.5 − 12.0Oil & grease (g/L) 10.0 4.7 5.7 − 57.5 8.8 − 10.1 8.3 − 10.6Total nitrogen (g/L) 1.2 0.5 0.8 − 10.0 1.3 − 1.5 0.8 − 0.9Total phosphorus (g/L) 0.5 - - - 0.09 − 0.12Remarks; 1: Prasertsan et al. [4]; 2: Pechsuth et al. [24]3: Choorit and Wisarnwan [37] 4: O-Thong et al. [6]-: Not Determined

Table 2 Effect of POME concentration soluble COD on growth and treatment of the three fungal strains after 5 days incu-bation on a shaker (200 rpm) at their optimum temperatures.

Dilution ratio

Initial COD (g/L)

H. insolens T. lanuginosus R. oryzae ST29Biomass

(g/L)COD removal

(%)Biomass

(g/L)COD removal

(%)Biomass

(g/L)COD removal

(%)1:0 51.1 3.95 5.88 4.06 20.0 6.44 35.31:1 22.6 4.06 20.0 7.96 40.0 16.9 60.01:2 15.0 3.02 0.004 7.35 30.1 7.51 40.0

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and clearer culture filtrate compared to those of the other two fungal strains.

Since the composition of POME was mainly water, oil and cellulosic wastes [8,9], the three fungal strains had to produce enzymes such as CMCase, xylanase, pectinase and lipase, to degrade the substances for their growth during cultivation in POME (Fig. 2). R. oryzae ST29 produced the highest CMCase activity (815 U/mL) and specific CMCase activity (92.3 U/mg protein) at 4 days cultivation. This was 2.95 times lower than that of the purified enzymes from the other strain of R. oryzae (273 U/mg protein) [32]. H. insolen and T. lanuginosus showed lower CMCase activity (430 and 531 U/mL, respectively) and specific CMCase activity (50.8 and 62.7 U/mg protein, respectively). The xylanase activity of R. oryzae ST29, showed the highest activities (1570 U/mL) followed by those of H. insolen and T. Lanuginosus (1250 U/mL and 821 U/mL, respectively) at 4 days cultivation. Unlike other enzymes, pectinase activity was highest from T. lanuginosus (930 U/mL) followed by R. oryzae ST29 and

H. insolen (890 U/mL and 681 U/mL, respectively). Lipase activity, however, was not detected from any fungal strain. This may be due to the acidic pH of POME (pH 4.5) as R. oryzae was reported to give the maximum lipase activity at pH 8 [33].

Effect of nitrogen source and concentration on growth, treatment efficiency and biopolymer pro-duction from R. oryzae ST29

The effect of three nitrogen sources (at 0.1% N) on growth, treatment and biopolymer production from R. oryzae ST29 cultivated in POME was studied (Fig. 3A). Ammonium nitrate, urea and fertilizer (46% urea) gave no significantly difference on mycelial growth (19.0, 18.5, and 18.1 g/L, respectively) but the biopolymer was highest (32 mg polymer/g biomass, or 5.78 g/L) in the presence of fertilizer (46% urea). The cells grown in ammonium nitrate and urea produced the biopolymers at 16.3 mg /g biomass (3.10 g/L), and 15.8 mg /g biomass (2.93 g/L), respectively. R. oryzae

Fig. 1 Time course on treatment of POME by three fungal strains cultivated at their optimum temperatures for 5 days.

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Fig. 2 Time course on enzymes production during treatment of POME by the three fungal strains cultivated at their optimum temperatures for 5 days.

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ST29 could reduce organic matter with the highest COD removal (60%) in the presence of fertilizer (46% urea) in POME, followed by urea and ammonium nitrate (55% and 50% COD removal), respectively. The organic and inorganic nitrogen sources showed significant impact on growth and

biopolymer production by microorganism. Organic N-sources provide the required vitamins, micronutrients and intermediate compounds for the molds and might act as stimulators and precursors essentially for optimum growth and exo-biopolymer production while inorganic nitrogen

Fig. 3 Effect of nitrogen sources (0.1% nitrogen) (A) and fertilizer (46% urea) concentration (B) on growth and treatment of POME (at the optimum concentration) by Rhizopus oryzae ST29 after 4 days incubation at 45°C.

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sources gave rise to relatively lower mycelial growth and exo-biopolymer production [34,35]. In this study, R. oryzae ST29 was found to give the maximum mycelial growth and biopolymer production in the presence of fertilizer (46% urea) as this fungus could use organic nitrogen sources better than inorganic nitrogen sources. These results were similar to previous studies in which the maximum mycelial growth and exo-biopolymer production by Cordyceps militaris was achieved when corn steep powder (1%) was employed as nitrogen source and the optimal concentration was 10 g/L. The mycelial growth and exo-biopolymer production were highest in the presence of organic nitrogens (corn steep liquor, corn steep powder, casein peptone M, meat peptone, tryptone, polypeptone, soy peptone and yeast extract), com-pared to the use of inorganic nitrogen (ammonium nitrate, ammonium chloride, ammonium phosphate and sodium nitrate) [32]. The mycelial growth and exo-biopolymer pro-duction by Auricularia polytricha were highest in medium added with yeast extract and found to be lower amount than in the medium added with inorganic nitrogen [36].

The influence of fertilizer (46% urea) at various con-centrations; 0, 0.01, 0.025, 0.050, 0.075, 0.1 and 1% (w/v) supplemented in POME, on mycelial growth, treatment and biopolymer production by R. oryzae ST29 were investigated (Fig. 3B). The optimum concentration was at 0.025% (w/v) giving the mycelial growth of 19.3 g/L, 65% COD removal and biopolymer production of 52.2 g/L. Growth at the other concentrations were not significantly different (18.27 − 19.18 g/L) while the biopolymer production were different in a wide range (10.9 − 40.7 mg /g biomass). The treatment efficiency (COD removal) was similar in the POME supple-mented with 0.025% and 0.050% (w/v) fertilizer (46% urea) (65% and 60% COD removal, respectively), followed by 0.075, 0.1, 1.0, 0.01 and 0% (55, 40, 35, 30 and 25% COD removal, respectively). Organic nitrogen was assimilated for the synthesis to the required protein, amino acid, micronu-trients and intermediate compounds for the microorganism and might act as stimulators and precursors essentially for optimum growth and biopolymer production. The low and high nitrogen leads to a significant effect on intermediate compound in the growth medium, which may negatively in-fluence fungal activities [34,37]. The optimum nitrogen con-centrations was 0.025% (w/v) fertilizer (46% urea), giving the highest mycelial growth (19.3 g/L biomass), maximum biopolymer production (52.2 mg-polymer/g-biomass) and could reduce organic matter with the highest COD removal (65%) by R. oryzae ST29.

It was found that R. oryzae ST29 could produce biopoly-

mer with the yield of 0.27 g/g biomass. The molecular weight of the biopolymer was 17,700 Daltons and particle size of 1,230 nm. Therefore, R. oryzae ST29 could reduce total sol-ids better than the other two strains. Biopolymer formation by this strain not only enhanced the solids (fiber, mycelium) and oil removal but also increased the treatment efficiency (% soluble COD removal) as solids, oil and biopolymer are organic matter as well. Therefore, the treatment efficiency by R. oryzae ST29 was 72.5% (soluble COD) with the reduction of 53.0% total solids and 98.7% oil & grease. Oil & grease in POME was in emulsion form and it dissolved at 45°C [29]. The oil and grease removal from POME were more likely to be the result from aggregation of the biopolymer together with the mycelium of the fungal strain. Besides, oil & grease could be removed from POME by enzymatic method us-ing the commercial xylanase (Meicellase) and enzymes extracted from Aspergillus niger ATCC6275 incubated at 40°C. This resulted in more than 99% oil & grease removal and COD removals of 76% and 69.4%, respectively [38]. In addition, nutrients in POME could be removed more than 50% with nitrogen and phosphorus removal of 61.5% and 50.0%, respectively. These results were similar to previous studies which pretreated POME by R. oryzae ST29 and could remove 52.6% total solids, 99.6% oil & grease, 39.2% nitrogen removal and 75% phosphorus [30].

Effect of initial pH on growth and biopolymer pro-duction from R. oryzae ST29

In order to investigate the effect of initial pH on mycelial growth and biopolymer production, R. oryzae ST29 was cul-tivated in the POME supplemented with 0.025% (w/v) fertil-izer (46% urea) with different initial pH (3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0 and 6.5) in shaked flask cultures. R. oryzae ST29 could grow at all initial pH tested (Fig. 4) with the optimum value at pH 4.5. It was reported that many species of fungi have more acidic optimum pH during submerged cultures [39,40]. This optimum pH value (pH 4.5) was very similar to that of Aspergillus carbonarius(4.0 − 4.5) [33,41,42]. In addition, the optimal pH for the mycelial growth and exo-biopolymer production was pH 5.0 for Paecilomyces ja-ponica and pH 6.0 for C.militaris [34,43]. The strain showed good mycelial growth with the highest biomass of 20.18 g/L and the maximum biopolymer production of 54.09 mg/g bio-mass (9.77 g/L) or about 2.2 folds higher than the previous experiment.

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CONCLUSIONS

Humicola insolens, Thermomyces lanuginosus and Rhizo-pus oryzae ST29 had the optimum growth temperature of 55, 55 and 45°C respectively. The optimum POME concentra-tion for growth of R. oryzae ST29 was 26.9 g/L soluble COD. This gave the highest biomass of 18.3 g/L and biopolymer of 26.9 mg/g biomass, with high oil and grease removal (98.7%), COD removal (60.0%), total solids reduction (66.4%) after 4 days cultivation. R. oryzae ST29 also produced the highest activities of CMCase (815 U/mL) and xylanase (1550 U/mL) while T. lanuginosus gave the highest pectinase activity (930 U/mL). Lipase activity was not detected. The optimum nitrogen source and concentration were found to be 0.025% fertilizer (46% urea) and optimum initial pH was 4.5. Under the optimum condition, R. oryzae ST29 exhibited treatment efficiency of 80% COD removal and 54.1 mg polymer/g biomass (9.77 g/L).

ACKNOWLEDGEMENTS

Thanks to Thailand Research Fund (Grant No.6080010) and Graduate School of Prince of Songkla University, Thai-land, for the financial support.

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