An Application of Wastewater Treatment in a Cold Environment and Stable Lipase Production of Antarctic Basidiomycetous Yeast Mrakia blollopis

Milk fat curdle in sewage is one of the refractory materials for active sludge treatment under low temperature conditions. For the purpose of solving this problem by using a bio-remediation agent, we screened Antarctic yeasts and isolated SK-4 strain from algal mat of sediments of Naga-ike, a lake in Skarvsnes, East Antarctica. The yeast strain showed high nucleotide sequence homologies (>99.6%) to Mrakia blollopis CBS8921T in ITS and D1/D2 sequences and had two unique characteristics when applied on an active sludge; i.e., it showed a potential to use various carbon sources and to grow under vitamin-free conditions. Indeed, it showed a biochemical oxygen demand (BOD) removal rate that was 1.25-fold higher than that of the control. We considered that the improved BOD removal rate by applying SK-4 strain was based on its lipase activity and characteristics. Finally, we purified the lipase from SK-4 and found that the enzyme was quite stable under wide ranges of temperatures and pH, even in the presence of various metal ions and organic solvents. SK-4, therefore, is a promising bio-remediation agent for cleaning up unwanted milk fat curdles from dairy milk wastewater under low temperature conditions.


Introduction
Drainage from dairy parlors and milk factories produced in the process of cleaning transport pipes and milking tanks pollute rivers and groundwater with detergents, bactericides, mucus and milk fat are contaminating rivers and underground water [1]. In low temperature conditions, the wastewater is treated by bio-filters [2] and a reed bed system [3,4]. However, the system is not used widely because of the high running cost and the necessity of a large space. Instead, an activated sludge system is now widely used for industrial treatment of dairy parlor wastewater [5] due to its advantages in maintenance and running cost. However, there is a problem in this system of low temperature conditions in winter having adverse effects on microbial functions.
The use of microorganisms living in polar regions for the purpose of removing nitrogen and phosphorus compounds from wastewater under low temperature conditions has been reported by Chevalier et al. [6] and Hirayama-katayama et al. [7], but it has not yet been applied for milk fat.
In our previous work, we examined 305 isolates of fungi including eight Ascomycetous and six Basidiomycetous species collected from Antarctica and found that they included fungi of the genus Mrakia, in psychrophilic Basidiomycetous yeast, suggesting that Mrakia is a major mycoflora highly adapted to the Antarctic environment (Fujiu, 2010; master's thesis in Graduate School of Science, Hokkaido University). Mrakia spp. and Mrakiella spp. are also common fungal species frequently found in cold climate areas such as Arctic, Siberia, Central Russia, the Alps and Antarctica [8][9][10]. Therefore, we screened our Mrakia isolates for their ability to decompose milk fat under low temperature conditions and evaluated their potential for application to an active sludge system in a region with a cold climate. The results showed that 56 Mrakia spp. exhibited a clear zone according to fat decomposition. Antarctic yeast strain SK-4 had physiological characteristics similar to those of Mrakia blollopis [11].
Here we report that activated sludge containing yeast strain SK-4 has the potential to remove milk fat BOD 5 . We also describe identification of yeast strain SK-4 and the purification and characterization of the lipase, considered as a major enzyme to degrade milk fat in wastewater.

Ethics statement
All necessary permits were obtained for the described field studies. Permission required for field studies was obtained from the Ministry of the Environment of Japan. Sample collection in Antarctica was performed with the permission of the Ministry of the Environment of Japan.

Sample isolation
Algal mat samples were collected from sediments of Naga-ike, a lake in Skarvsnes, located near Syowa station, East Antarctica. The isolate was inoculated on potato dextrose agar (PDA) (Difco TM , BD Japan, Tokyo, Japan) at 4uC for 1 week. Yeast strain SK-4 was selectively picked for isolation on the basis of its morphology. Yeast strain SK-4 was maintained on PDA plates at 4uC and long-term storage was performed in 40% (w/v) glycerol at 280uC.

Phylogenetic analysis
Phylogenetic analysis was done by sequencing the ITS region including 5.8S rRNA and D1/D2 domain of 26S rRNA. Cells were harvested from 2-weeks-old cultures. DNA was extracted with an ISOPLANT II kit (Wako Pure Chemical Industries, Osaka, Japan) according to the manufacturer's protocol. Extracted DNA was amplified by PCR using KOD-plus DNA polymerase (TOYOBO, Osaka, Japan). The ITS region was amplified by using the following primers: ITS1F (59-GTA ACA AGG TTT CCG T) and ITS4 (59-TCC TCC GCT TAT TGA TAT GC). The D1/D2 domain was amplified using the following primers: NL1 (59-GCA TAT CAA TAA GCG GAG GAA AAG) and NL4 (59-GGT CCG TGT TTC AAG ACG G). Sequences were obtained with an ABI prism 3100 Sequencer (Applied Biosystems, Life Technologies Japan, Tokyo, Japan) using an ABI standard protocol. The ITS region and D1/D2 domain sequences of yeast strain SK-4 are deposited in DNA Data Bank of Japan (BBDJ) (Accession numbers AB630315 and AB691134). Alignment was

Physiological characterization
Assimilation of carbon was performed at 15uC on modified Czapek-Dox agar composed by 6.7 g/L of yeast nitrogen base without amino acids (Difco TM , BD Japan, Tokyo, Japan), 2.0 g/L of sodium nitrate (Wako Pure Chemical Industries, Osaka, Japan), 30 g/L of carbon source and 15.0 g/L of Agar (Difco TM , BD Japan, Tokyo, Japan). Assimilation of nitrogen and other physiological tests were carried out according to the protocols described by Yarrow [13]. All tests were performed at 15uC after 2 and 4 weeks of inoculation.

Preparation of active sludge and measurement of biochemical oxygen demand
Activated sludge (AS) was cultivated at room temperature with aeration by using cow's milk as the substrate. After one month, the sludge was divided into two parts. One part of the activated sludge was mixed with M. blollopis SK-4 (1.4 g/L, dry weight), and the other part was used as a control. Separated activated sludge was prepared with Mixed Liquor Suspended Solids (MLSS, 3000 mg/ L) and cow's milk at 10uC with aeration. One week later, prepared activate sludge was added to cow's milk, and biochemical oxygen demand (BOD 5 ) of waste-treated water was measured after 24 hours. BOD 5 assay was carried out using a coulometer (Ohkura Electric, Saitama, Japan).

Assay of lipase activity
Lipase activity was measured by a colorimetric method using pnitrophenyl-palmitate as a substrate [14]. Forty mL of 50 mM sodium phosphate buffer (pH 7.0) containing 50 mg gum arabic and 0.2 g TritonX-100 was mixed with 3 mL 2-propanol containing 1 mM p-nitrophenyl-palmitate. Eight hundred mL of prepared substrate was added to 200 mL of enzyme solution. The enzyme reaction was carried out at 30uC for 30 min. The released p-nitrophenol was measured at A 410 . One unit of lipase activity was defined as the activity required to release 1 mmol of free fatty acids per minute at 30uC.

Measurement of protein concentration
Protein concentration was measured by BCA protein assay reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions using bovine serum albumin as a standard.
Purification of lipase M. blollopis SK-4 lipase was purified by ultrafiltration and Toyopearl-butyl 650 M (Tosho, Tokyo, Japan) hydrophobic interaction chromatography. Four hundred mL of lipase production liquid medium was centrifuged at 4uC for 15 min at 30006g. The supernatant was filtered through a 0.45-mm of membrane filter (Advantec, Tokyo, Japan). The filtered medium was concentrated by ultrafiltration using an ultracel YM-30 membrane (Millipore, Billerica, MA, USA). The concentrated sample was adsorbed to a Toyopearl butyl 650 M column (2.5620 cm) containing 1 M sodium chloride and eluted with a linear gradient from 750 mM to 100 mM sodium chloride in 20 mM Tris-HCl buffer (pH 8.5) at a flow rate of 60 mL/h. Fractions of high lipase activity was pooled and concentrated and then stored at 4uC until use. Protein molecular weight was estimated by SDS-PAGE according to Laemmli [15] and stained with CBB R-250. Precision plus protein unstained standards (Bio-Rad Laboratories Japan, Tokyo, Japan) were used as protein molecular weight makers.

Characterization of lipase
Substrate specificity was determined by using substrate as different p-nitrophenyl esters (C 4 -C 18 ). For determining the effects of metal ions and EDTA on lipase activity, residual lipase activity assays were carried out under standard assay conditions with final concentrations of 1 mM of various bivalent metal ions and EDTA. Lipase activity assay in the absence of metal ions and EDTA was carried out as a control. Optimum pH was measured at 30uC for 30 min and determined at various pH values of 50 mM buffer as follows: sodium citrate (pH 3.0-5.0), sodium phosphate (pH 6.0, 7.0 and 8.0), Tris-HCl (pH 7.5 and 8.5), glycine-NaOH (pH 9.0) and sodium carbonate (pH 9.3, 9.5 and 10.0).
Optimum temperature was measured in 50 mM sodium phosphate buffer (pH 7.0) for 30 min. To determine the pH stability of lipase, the enzyme was preincubated in various buffers for 15 h at 30uC and then adjusted to pH 8.5. The residual enzyme activity was measured by using p-nitrophenyl-palmitate as a substrate at 65uC for 30 min. For determining thermo-stability, lipase was preincubated for 30 min at different temperatures and the residual activity was measured at 65uC for 30 min in 50 mM Tris-HCl (pH 8.5). Effects of organic solvents on lipase activity were determined at 65uC for 30 min in 50 mM Tris-HCl (pH 8.5) containing various organic solvents at final concentration of 5% (v/v). Lipase activity assay in the absence of organic solvents was carried out as a control.

Phylogenetic analysis
As a result of phylogenetic analysis of the ITS region and D1/ D2 domain, yeast strain SK-4 was grouped with the clade of Mrakia blollopis CBS 8921 T (Fig. 1 A and B). By comparison of the ITS region sequence containing 5.8 S rRNA, yeast strain SK-4 showed high homologies (.99.6%) with M. blollopis CBS8921 T . The D1/D2 domain sequence showed no variation with M. blollopis CBS 8921 T .  Results of assimilation of carbon compounds and other physiological tests of M. blollopis SK-4 are shown in Table 1 with its type strain and related species. Test data for M. blollopis SK-4 are compared with those for M. blollopis CBS8921 T [8], M. psychrophila AS2.1971 T [16], and M. frigida CBS5270 T [17]. Maximum growth temperature of M. blollopis SK-4 was 22uC. Maximum growth temperatures of other related species were lower than 20uC. M. blollopis SK-4 differed from the other strains in substrate utilization as well. The strain could thrive well on lactose, D-arabinose, and inositol medium. Unlike other strains, this strain also grew on vitamin-free medium. A comparison of fermentabilities showed that M. blollopis SK-4 could ferment typical sugars such as glucose, sucrose, galactose, maltose, lactose, raffinose, trehalose and melibiose, while other related species were not able to strongly ferment such as various sugars ( Table 1).

Assessment of milk fat decomposition in model wastewater
Model wastewater containing cow's milk as a substrate of milk fat was prepared as an equivalent of BOD sludge loading in standard waste water treatment (0.35 kg-BOD/kg-MLSS?day). Activated sludge containing M. blollopis SK-4 had a BOD removal rate of 83.1%, higher than that in the control (63.8%, Fig. 2A). When BOD volume load was adjusted to 1.5 fold of standard wastewater treatment (0.52 kg-BOD/Kg-MLSS?day), BOD removal rate by activated sludge containing M. blollopis SK-4 was 80.1%, higher that in the control (65.2%, Fig. 2B). Regardless of BOD volume load, activated sludge containing M. blollopis SK-4 had a 1.25-fold higher BOD removal rate than that of the control.

Production of lipase from Mrakia blollopis SK-4
Many microorganisms are known to produce lipase using Tween 80 as a substrate [18,19]. Basidiomycetous yeast M. blollopis SK-4 also produced the enzyme. It is known that the production of lipase from Candida rugosa increased when yeast extract was used as a nitrogen source [20]. The same results as those for M. blollopis SK-4 were obtained. When 0.5% (w/v) yeast extract was used as a nitrogen source in the medium, lipase production by M. blollopis SK-4 lipase increased dramatically after 180 h. Maximum lipase  activity of 0.695 U/mL was obtained with 0.5% (w/v) yeast extract, as compared to 0.059U/mL in its absence, after 324 h of inoculation, after which the accumulation of lipase markedly decreased (Spp. Fig. S1). M. blollopis SK-4 morphology was observed by the fluorescence in situ hybridization (FISH) method during secretion of lipase. Therefore, all of the morphology of M. blollopis SK-4 during secretion of lipase was yeast form (data not shown).

Purification of lipase
Lipase production medium was centrifuged at 30006for 15 min at 4uC. The supernatant was filtered through a 0.45-mm membrane filter. The filtered solution was concentrated by ultrafiltration. After ultrafiltration, 72.3% of total lipase activities were recovered and 2.2-fold lipase specific activity was obtained. Then, enzyme solution was applied on a Toyopearl butyl-650 M column (2.5620 cm) and purified by single-step hydrophobic interaction chromatography. Finally, 9.4% of the enzyme was recovered and 20.1-fold of specific activity, compared to crude sample, was obtained with a specific activity of 51.7 U/mg ( Table 2). The purified enzyme showed a single band on SDS-PAGE with a molecular mass of 60 kDa (Supp. Fig. S2).

Characterization of lipase
Optimum temperature of lipase activity was 60265uC (k cat = 3.93 and 4.04 S 21 ). At temperatures of 80uC and 95uC, 30.5% (k cat = 1.23 S 21 ) and 6.8% (k cat = 0.27 S 21 ) of enzyme activity were retained (Fig. 3A). The enzyme showed thermo-stability up to 65uC with 98.3% (k cat = 5.12 S 21 ) of residual enzyme activity even after 30-min preincubation. At 75uC, 80uC and 85uC, 48.2% (k cat = 2.51 S 21 ), 41.8% (kcat = 2.18 S 21 ) and 28.03% (k cat = 1.46 S 21 ) of enzyme activity remained after 30-min preincubation (Fig. 3A). Optimum pH range of lipase activity was between pH 8.0 and pH 9.0 (kcat =1.43 and 1.51 S 21 ), whereas 57.0% (k cat = 0.86 S 21 ),63.5%(kcat = 0.96 S 21 ) and 27.8% (kcat = 0.42 S 21 ) of the enzyme activity was retained at pH 7.0, pH 9.3 and pH 9.7 compared to 100% at pH 8.5 (Fig. 3B). The enzyme was quite stable over a wide pH range (4.0210.0) and 45.5% (k cat = 2.38 S 21 ) of the enzyme activity remained at pH 3.0 even after preincubation for 15 h at 30uC (Fig. 3B). Enzyme activity was affected by metal ions at a concentration of 1 mM, retaining relative activity higher than 80%. There was little inhabitation of lipase activity in the presence of Cu 2+ and Pb 2+ ions. The metal-chelating agent EDTA did not affect lipase activity ( Table 3). One of the most important characteristics of lipase was substrate specificity toward various p-nitrophenyl esters (C 4 2C 18 ). The substrate specificity was determined in the presence of 1 mM p-nitrophenyl esters as a substrate with 50 mM sodium phosphate (pH 7.0) at 30uC for 30 min. Relative activity toward C 4 2C 14 was more than 100% compared to 100% toward p-nitrophenyl-palmitate. C 18 had relative activity of 71.6% (Fig. 4). Various organic solvents (ethanol, methanol, diethyl ether, dimethyl sulfoxide, hexane and N, Ndimethyl formamide) enhanced SK-4 lipase acidity. Solvents such as acetone and chloroform however, had only a slight inhibitory effect in the activity, with relative activities of 77.9% and 70.4%, respectively (Table 3). Organic solvents are known to be severely toxic to most enzymes including lipase [21].

Discussion
The Antarctic yeast strain SK-4, which we identified as M. blollopis, showed several uniques characteristics; for example, it can use various carbon sources as nutrition, it prefers relatively high temperature conditions among allied species and it can be activated even in vitamin-free conditions. These results suggested that strain SK-4 is a potential candidate for a biological agent to decompose various sugars in milk parlor wastewater under low temperature conditions. This expectation was further reconfirmed by the application of strain SK-4 in AS; namely, addition of SK-4 to AS in the model parlor wastewater improved the BOD removal rate.
Improved BOD removal rate of the activated sludge was attributed to lipase activity of SK-4 added. As expected, the lipase purified from M. blollopis SK-4 showed clearly weaker activity at lower temperatures but stronger activity in middle to high temperature conditions compared to activities of those from the other psychrophilic fungi [22]. M. blollopis SK-4 lipase, in addition, was quite stable in wide ranges of temperature and pH conditions and was not affected by the existence of EDTA, various metals ions, or organic solvents. M. blollopis SK-4 is thought to have acquired stable lipases by growing in extreme environments such as the Antarctica.
Comparison of the lipase from M. blollopis SK-4 with that from Cryptococcus sp. S-2, which has actually been used in wastewater treatment [23,24], revealed that the former was superior to the latter both in thermo-stability and pH stability (Table 4); i.e., the lipase from M. blollopis SK-4 retained 71.1% of the enzyme activity after 30 min at 60uC and was stable for 6 hrs at 30uC in the pH range from 5.0 to 9.0.
In conclusion, M. blollopis SK-4 has the ability to assimilate various carbon compounds and to use various sugars for fermentation. Moreover, the lipase is more tolerant in relatively higher temperature conditions and wider pH ranges, less sensitive to various metal ions and organic solvents, and highly reactive to various chain lengths of substrates. SK-4 lipase, therefore, is a promising biological agent for parlor wastewater treatment even in low temperature regions of the world. Figure S1 Effect of yeast extract on lipase production. Mrakia blollopis SK-4 was cultivated by lipase production medium (¤) and lipase production medium without yeast extract (N). (TIF) Figure S2 SDS-PAGE of purified lipase from Mrakia blollopis SK-4. Lane 1. Molecular weight marker; 2. Purified lipase.