Edible oil degradation by using yeast coculture of ... - Springer Link

Appl Microbiol Biotechnol (2009) 82:351–357 DOI 10.1007/s00253-008-1834-2

ENVIRONMENTAL BIOTECHNOLOGY

Edible oil degradation by using yeast coculture of Rhodotorula pacifica ST3411 and Cryptococcus laurentii ST3412 Daisuke Sugimori

Received: 16 May 2008 / Revised: 15 December 2008 / Accepted: 17 December 2008 / Published online: 8 January 2009 # Springer-Verlag 2009

Abstract To develop a microbial treatment of edible oilcontaminated wastewater, microorganisms capable of rapidly degrading edible oil were screened. The screening study yielded a yeast coculture comprising Rhodotorula pacifica strain ST3411 and Cryptococcus laurentii strain ST3412. The coculture was able to degrade efficiently even at low contents of nitrogen ([NH4–N]=240 mg/L) and phosphorus sources ([PO4–P]=90 mg/L). The 24-h degradation rate of 3,000 ppm mixed oils (salad oil/lard/beef tallow, 1:1 w/w) at 20°C was 39.8%±9.9% (means ± standard deviations of eight replicates). The highest degradation rate was observed at 20°C and pH 8. In a scaled-up experiment, the salad oil was rapidly degraded by the coculture from 671±52.0 to 143±96.7 ppm in 24 h, and the degradation rate was 79.4%±13.8% (means ± standard deviations of three replicates). In addition, a repetitive degradation was observed with the cell growth by only pH adjustment without addition of the cells. Keywords Oil degradation . Coculture . Rhodotorula . Cryptococcus . Wastewater treatment

Introduction Edible oil is released into wastewater from food processing industries and restaurants. This oil has a very high biological oxygen demand and remarkably pollutes public

D. Sugimori (*) Department of Industrial System, Faculty of Symbiotic Systems Science, Fukushima University, 1 Kanayagawa, Fukushima 960-1296, Japan e-mail: [email protected]

water bodies such as rivers, lakes, and seas. In general, the oil present in wastewater is removed using a grease trap, but it does not get completely eliminated. Consequently, wastewater containing large amounts of oil often leaves the trap as an efflux and contaminates public water bodies. Further, since accumulation and putrefaction of the oil in the trap leads to malodor and sanitary problems, the accumulated oil and scum must constantly be physically removed. However, the oil again accumulates immediately, and this problem remains to be solved. Microorganisms capable of degrading edible oil would be useful to solve the above-mentioned problems. Thus far, there are many reports on the microbial degradation of edible oil (Okuda et al. 1991; Bednarski et al. 1994; Wakelin and Forster 1997; Suzuki et al. 2001; Matsumiya et al. 2007); however, these studies were carried out by using rich media, and the condition was somewhat different from that of the practical wastewater from restaurants and food processing industries. In fact, the author’s previous investigation showed that the content of inorganic nitrogen and phosphorus in wastewater from a restaurant of a university was low: Total nitrogen and phosphorus content was 60±30 and 11±4.5 mg/L, respectively. The author previously reported on Acinetobacter sp. strain SOD-1 that is capable of degrading salad oil on a basal medium and has practical uses (Sugimori et al. 2002). However, the development of efficacious microorganisms is not advanced yet. Strain SOD-1 and other microorganisms exhibited poor activity toward animal oil and a short lifetime when stored as cell formulations. Further studies are required to exploit microbial preparations for the treatment of oil-contaminated wastewater, especially those exhibiting strong activity toward animal oils as well as vegetable oils at low nitrogen and phosphate concentrations, and having a long lifetime in storage.

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In this study, the author isolated two kinds of yeasts capable of effectively degrading even animal oils as well as vegetable oils on a minimal medium containing low content of nitrogen and phosphate. In this report, the author describes the degradation features and the lifetime in storage.

Materials and methods Materials The salad oil used for the experiments was a commercial vegetable oil consisting of canola (rapeseed) and soybean oils and was obtained from The Nisshin Oil Mills (Tokyo, Japan). The lard used was commercial pig oil obtained from Snow Brand Milk Products (Tokyo, Japan). The beef tallow was purchased from Nacalai Tesque (Kyoto, Japan). All other chemicals were of the highest grade. Screening of oil-degrading microorganisms The minimal medium used for screening and the oil degradation experiments was comprised of 1.0 g (NH4)2SO4, 0.5 g K2HPO4, 0.1 g NaCl, 0.1 g MgSO4•7H2O, 0.1 g CaCl2•2H2O, 0.03 g FeCl3•6H2O, and 0.1 g yeast extract per liter of distilled water (pH 7.2–7.5 without adjustment). The microorganisms were screened by an enrichment technique as follows. In a test tube containing 5 mL of the minimal medium, 50 μL of mixed edible oils (salad oil/lard/beef tallow, 1:1 w/w) was added, and the mixture was sterilized by autoclaving. Samples collected from soil in Fukui, Japan were suspended in sterile saline. The supernatant was inoculated in the tube and then was incubated at 20°C with shaking (170 strokes/min). Successive cultures were transferred to the fresh medium at a 1:100 ratio. After three successive transfers, the cultures were stored at −80°C, and no microorganisms were isolated. Assay for oil degradation At each concentration of 0.5%, each preculture of strain ST3411 and strain ST3412 were added to the 100 mL minimal medium containing 0.3% the mixed oils in a 500-mL flask. The cultures were incubated at 20°C for 24 h on a rotary shaker (130 rpm). After cultivation, the cultures were autoclaved for 15 min at 121° C; the cells were disrupted by sonication for 5 min at 80 W, followed by acidification with 6 M HCl. The residual lipids in the culture were extracted twice using 40 mL of nhexane. The n-hexane layer was collected and neutralized with distilled water followed by dehydrating with anhydrous MgSO4. The obtained solution was filtered, and n-hexane was removed by evaporation. The residue was exactly weighed. The degradation rate was determined on the basis of the equation Degradation rate (%)=100×(A−B)/A, where A is the weight of the residue for a control experiment without inoculation of the cells and B is the weight of the residue for the cultures.

Appl Microbiol Biotechnol (2009) 82:351–357

Isolation and construction of a coculture system The microorganisms were isolated on minimal medium agar plate containing 0.5% salad oil. The oil degradation ability of the isolates and cocultures was investigated by the above-mentioned assay. The effects of pH, temperature, oil content, and initial inoculum dose of cells on the oil degradation by the coculture of strain ST3411 and strain ST3412 were further investigated. The number of colonyforming units (CFUs) was determined using nutrient agar plates supplemented with 25 μg/mL chloramphenicol. When at each concentration of 0.5%, each preculture of strain ST3411 and strain ST3412 was added to the 100 mL minimal medium; the initial cell density of strain ST3411 and strain ST3412 was 5.7×105 and 3.0×105 CFU/mL, respectively. The ratio of initial cell density in the other inoculum experiments was also the same as that (about 2:1) of 0.5% of each inoculation. Taxonomic characterization of the isolates Strain ST3411 and strain ST3412 were taxonomically identified by TechnoSuruga (Shizuoka, Japan). Scaled-up experiments in an open system To the flask containing the 100 mL minimal medium containing 0.3 g mixed oils, 0.5 mL of each preculture of strain ST3411 and strain ST3412 was transferred. The culture was incubated at 20°C for 48 h on the rotary shaker and then transferred to a 15 L bath (200 mm×350 mm×210 mm) with 8 L of the minimal medium containing 8 mL salad oil. The temperature was maintained at 20°C with a temperature controller. The dissolved oxygen (DO) concentration was maintained at approximately the saturated state by aeration at 5 L/min. Before sampling, the culture solution was thoroughly homogenized at 24,000 min−1 (Ika Ultra-Turrax T25, Ika Labortechnik, Staufen, Germany). The oil content was measured by the n-hexane extraction method. Chemical oxygen demand (CODMn) was measured using a spectrophotometer DR/2500 (Hach Company, Loveland, USA) and the Hach method (modified method 10067) using KMnO4. A fed-batch experiment was carried out by every 24-h addition of 8 mL salad oil without adding any other supplement and the cells. The pH was adjusted to 7.0 with 1 M NaOH at 24-h intervals. Preparation of microbial formulation and examination of storage stability Strain ST3411 and strain ST3412 were cocultivated for 48 h at 20°C and harvested by centrifugation. The obtained cells were washed twice with physiological saline. The cell paste was aseptically suspended in sterilized dispersion medium (80 wt.% skim milk, 4 wt.% sodium glutamate, 4 wt.% sodium ascorbate, and 0.85 wt.% NaCl) and lyophilized. The lyophilizate was powdered and transferred to a sterile conical tube followed by incubation


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at 40°C. The oil degradation ability of the microbial formulation was periodically measured by the above method. Then, 100 mg of the stored lyophilizate powder was transferred to the 100 mL minimal medium containing 3,000 ppm mixed oils as an open system.

Results Screening of oil-degrading microorganisms Thirty-three cultures were obtained from 91 soil samples. The culture no. 341 from soil sample of playing field of Fukui National College of Technology that exhibited the highest degradation rate was selected. Three microorganisms were isolated from this culture and designated as ST310, ST3411, and ST3412. Table 1 shows the result of reconstruction of the coculture system composed of strains ST3411 and ST3412 that effectively degraded the edible oil mixture. Both strains were multiplied via multipolar budding and did not form sexual reproductive organs. The nucleotide sequence of the 28S rDNA-D1/D2 region of strain ST3411 showed 99.8% identity with those of Rhodotorula mucilaginosa SY-246 (BLAST accession no. AB193175) and Rhodotorula sp. SY-96 (BLAST accession no. AB026006). In addition to this, strain ST3411 elaborated nitrate. Since R. mucilaginosa is not able to elaborate nitrate, ST3411 was assigned as Rhodotorula pacifica based on the results of biochemical analysis (Table 2; Kurtzman and Fell 1998). The nucleotide sequence of the 28S rDNA-D1/D2 region of strain ST3412 showed 100% and 99.8% identity with those of Cryptococcus laurentii CBS139 (BLAST accession no. AF075469) and C. laurentii KCTC7831 (BLAST accession no. AF257269), respectively. ST3412 was assigned as C. laurentii based on the results of biochemical analysis, especially sucrose (positive), maltose (positive), melibiose (positive), raffinose (positive), melezitose (positive), Darabinose (positive), rhamnose (positive), and D-glucosamine (negative; Table 2; Kurtzman and Fell 1998). Table 1 Edible oil degradation ability of isolates and their coculture Strains ST310 ST3411 ST3412 ST310 + ST3411 ST310 + ST3412 ST3411 + ST3412 ST310 + ST3411 + ST3412

24-h degradation rate/%a 17.8 32.2 18.5 19.9 20.3 43.0 40.9

a The degradation rate was determined in 24-h cultivation at 3,000 ppm mixed edible oil (salad oil/lard/beef tallow, 1:1 w/w) and 20°C

Table 2 Morphological and physiological characteristics of R. pacifica ST3411 and C. laurentii ST3412 Characteristic

Strain ST3411

Strain ST3412

Cell shape Colony color Growth at 25°C 28°C 30°C 37°C 40°C Starch production Fermentation of glucose Utilization of Glucose Galactose L-Sorbose D-Glucosamine D-Xylose D-Ribose L-Arabinose D-Arabinose L-Rhamnose Sucrose Maltose Treharose Cellobiose Salicin Melibiose Raffinose Melezitose Inulin Soluble starch Erythtiol Inositol D-Glucitol D-Gluconate 2-Keto-D-gluconate D-Glucuronate Succinate Methanol N-Acetyl-D-glucosamine Hexadecane Nitrate Nitrite Tolerance to 50% glucose 10% NaCl/5% glucose 0.01% cycloheximide 0.1% cycloheximide Requirement of Vitamin free Thiamin

Rods Pink∼orange

Spheroids White

+ + + − − − −

+ + + NT NT + −

+ S W − + NT + NT S + + + + + − + + − − − − NT S NT − S − − − L L

+ + − − + S + + + + + NT + + + + + − NT + S S + + + + − + NT − −

− − − −

− − − −

− +

− +

− negative, + positive, W weak positive, S slowly positive in several weeks, L positive after latent periods of 2 weeks, NT not tested

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Table 3 Lipid degradation ability of the coculture of strain ST3411 and strain ST3412 Lipids

24-h degradation rate/%a

Salad oil Lard Beef tallow Mixed oil Oil ballb BDF production wastec

36.3±2.1 26.7±1.0 24.8±6.9 39.8±9.9 29.1±7.6 20.2

Characterization of oil degradation by coculture Cultures of some microorganisms cause emulsification in the nhexane extraction of lipids, resulting in failure to extract lipids completely. In this study, no emulsification was observed in the n-hexane extraction. The coculture of strain ST3411 and strain ST3412 efficiently degraded various kinds of oils (Table 3). Efficient degradation was observed at temperatures between 20°C and 30°C around pH 8 at below 3,000 ppm oil content and with 0.25–1% of each inoculum (Fig. 1). As shown in Fig. 1c, the degradation rate decreased with the initial oil content, suggesting that the degradation process could be inhibited by substrate oil. The oil degradation activity was remarkably decreased at below 0.1% and above 2% of each inoculum (Fig. 1d), indicating that lower initial cell concentration would be insufficient for the effective degradation, and excessive inoculum dose presumably inhibits the cell growth and the oil degradation through deterioration of state of the culture. The oil was degraded rapidly after approximately 15 h; besides, both strains grew well for over 48 h (Fig. 2).

a

Means ± standard deviations of eight replicates for mixed oil and three replicates for others b Lipids accumulating in oil/water separator of sewage c n-Hexane extracts were 1,184 ppm in 3,000 ppm waste of BDF production

R. pacifica strain ST3411 and C. laurentii strain ST3412 were deposited as FERM AP-21121 and FERM AP-21122, respectively, in International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology in Japan.

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Fig. 1 Effects of culture conditions on the mixed oil degradation in flask cultivation. a Effect of temperature (bar indicates standard deviations of eight replicates); b pH profile; relationship between oil content (c) or initial inoculum dose of cells (d) and degradation rate. Except for c, the oil content was 3,000 ppm mixed oil

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Time (h) Fig. 2 Time course of cell growth and the 3,000 ppm mixed oil degradation in flask cultivation of the coculture at 20°C. Closed circle, oil degradation (bar indicates standard deviations of three replicates); cell growth (open square, strain ST3411; open triangle, strain ST3412)

Scaled-up experiments, preparation of microbial formulation, and storage stability The salad oil was rapidly degraded by the coculture in scaled-up batch experiments, and the degradation rate was 79.4%±13.8% (means ± standard deviations of three replicates; Table 4). The CODMn value decreased from 252 to 154 mg/L, indicating the effects of cell growth and oil degradation. The pH value changed from 7.62 to 4.21, suggesting that organic acids may have been produced because fatty acids were not detected by thin-layer chromatography. In the fed-batch treatment, the degradation was repeatedly observed with the good cell growth (Fig. 3). The oil degradation activity was maintained for over 50 days at 40°C (Fig. 4), indicating that the microbial formulation of strain ST3411 and strain ST3412 is likely stable after long-term storage.

It is noteworthy that the coculture of strain ST3411 and strain ST3412 efficiently degraded animal oils such as lard and beef tallow as well as vegetable oil at even 20°C, neutral pH, and low content of nitrogen and phosphorous. The author’s previous investigation showed that the total nitrogen and phosphorus content in wastewater from a university restaurant was 60±30 and 11±4.5 mg/L (means ± standard deviations of ten replicates), respectively. On the other hand, when the contents of nitrogen and phosphorus sources in the culture medium were below 240 mg-[NH4–N]/ L and 90 mg-[PO4–P]/L, no microorganism was obtained in the screening study. Further work is needed to solve the problem of nitrogen and phosphorus content. However, the properties of the coculture should be preferable to treatment of lipid-contaminated wastewater in grease trap because their condition is close to that of the practical wastewater. Under the same conditions, the 24-h degradation rate of the lipiddegrading bacterium Acinetobacter sp. strain SOD-1 and Acinetobacter sp. strain CL-3, which we previously developed, was 5.7% and 5.2%, respectively. These strains required substantial amounts of ammonium salt and phosphate for lipid degradation and showed poor degradation of animal oils (Sugimori et al. 2002). Okuda et al. reported that Bacillus sp. strain 351 degraded above 80% of 5,000 ppm various kinds of oils at 30°C in 24 h; however, the medium used in the experiments contained high concentration of ammonium salt and phosphate (Okuda et al. 1991). Matsumiya et al. reported that Burkholderia sp. strain DW2-1 degraded 85% of 10,000 ppm salad oil at 30°C in 24 h and 77.4% of 10,000 ppm beef tallow during a 48h cultivation, but the synthetic wastewater medium containing peptone and beef extract was used for the investigation (Matsumiya et al. 2007). Sugimoto et al. reported that Acinetobacter sp. strain SK0402A degraded 83.5% of 2,000 ppm salad oil at 22°C in 24 h on the synthetic wastewater medium (Sugimoto et al. 1994). These reports did not show the degradation of animal oils and effect of oil content on the degradation rate. The lipid degradation ability of these bacteria is high, though the condition of experiments, viz. temperature and medium composition, would be to some extent different from that of the practical wastewater. Thus,

Table 4 Oil degradation by the coculture system in scaled-up experiment Time (h)

0 24

Cell growth (CFU/mL) ST3411

ST3412

3.8×107 2.0×107

2.4×106 1.01×109

pH

DO (mg/L)

7.62 4.21

8.73 8.08

The scaled-up experiment was performed using 1,000 ppm salad oil at 20°C n-Hexane extracts (means ± standard deviations of three replicates)

a

n-Hexa (mg/L)

671±52.0 143±96.7

COD (mg/L)

252 154

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a

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n-Hex (ppm)

800

600

400

200

0 0

50

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150

Time (h) 11

b 10

strain TI-E degraded 65.6% and 57.9% of 250 ppm olive oil and beef tallow, respectively, at 15°C in 24 h on a minimal medium (Taki and Inoue 2000). The coculture of ST3411 and ST3412 degraded 74.5% of 361 ppm mixed oil at 20°C in 24 h (Fig. 1c), suggesting that the lipid degradation ability of the coculture is higher than that of Pseudomonas sp. strain TI-E. When both strains of ST3411 and ST3412 were cocultivated for 48 h during the preculture, the maximal degradation was observed in the main culture. Meanwhile, no difference was observed in cell growth between the coculture and individual pure cultures. This result indicates that the development of symbiosis most probably results in the maximal degradation. As shown in Fig. 1, the coculture is applicable to temperature between 20°C and 30°C and to pH between 7 and 8. In particular, the degradation activity is highly susceptible to pH of the medium. The coculture was able to degrade effectively at below 800 ppm oil contents, which is close to the oil content of the practical lipid-contaminated wastewater. The seeding experiment suggested that effective degradation will be achieved when the initial cell density is between 3 and 11×105 CFU/mL of ST3411 and between 1.5 and 6×105 CFU/mL of ST3412. In the scaled-up experiments, the coculture degraded the salad oil rapidly and repetitively without supplementation of the cells and nutrition, and it can therefore potentially be 35

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Time (h) Fig. 3 Salad oil degradation during fed-batch treatment in scaled-up experiments by using the coculture. a Time course of oil degradation (arrow indicates addition of 8 mL salad oil); b cell growth (closed circle, strain ST3411; closed square, strain ST3412)

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their bacteria might not be able to exert the degradation ability at the condition of the practical wastewater. To the author’s knowledge, this is the only report that has published about microbial lipid degradation under conditions close to practical wastewater: Taki and Inoue reported that Pseudomonas sp.

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Storage time (d) Fig. 4 Time course of the oil degradation ability of the microbial formulation comprising strain ST3411 and strain ST3412. The lyophilizate powder of the coculture was incubated in the sterile conical tube at 40°C


applied to the lipid-contaminated wastewater treatment. A preservation of an enzyme reagent is examined by assay of the enzyme activity after 60-day storage at 37–40°C. It is thought that this storage corresponds to 1-year storage at room temperature. The coculture formulation exhibited high storage stability, strongly indicating that the handling of the formulation is easier than that of conventional formulations or microorganisms. Acknowledgment A part of this research was supported by the “Research for Promoting Technological Seeds” from Innovation Plaza Miyagi, Japan Science and Technology Agency.

References Bednarski W, Adamczak M, Kowalewska-Piontas J, Zadernowski R (1994) Biotechnological methods for the up-grading and modification of animal waste fats. Acta Biotechnol 14:387–393

357 Kurtzman CP, Fell JW (1998) The Yeasts, a taxonomic study. Amsterdam, Netherlands: Elsevier Matsumiya Y, Wakita D, Kimura A, Sanpa S, Kubo M (2007) Isolation and characterization of a lipid-degrading bacterium and its application to lipid-containing wastewater treatment. J Biosci Bioeng 103:325–330 Okuda S, Ito K, Ozawa H, Izaki K (1991) Treatment of lipidcontaining wastewater using bacteria which assimilate lipids. J Ferment Bioeng 71:424–429 Sugimori D, Nakamura M, Mihara Y (2002) Microbial degradation of lipid by Acinetobacter sp. strain SOD-1. Biosci, Biotechnol, Biochem 66:1579–1582 Sugimoto Y, Maruoka T, Seo Y, Fujita M (1994) Microbe capable of degrading edible fatty oil and usage thereof. Japan Patent 06153922 Suzuki T, Nakayama T, Kurihara T, Nishino T, Esaki N (2001) Coldactive lipolytic activity of psychrotrophic Acinetobacter sp. strain No. 6. J Biosci Bioeng 92:144–148 Taki H, Inoue R (2000) Microorganism having oil and fat-decomposing activity and treatment of waste water. Japan Patent 2000270845 Wakelin NM, Forster CF (1997) An investigation into microbial removal of fats, oils and greases. Biores Technol 59:37–43