بهینه سازی تولید روغن از گونه Wickerhamomyces Siamensis SAKSG جدا شده از ماهی قزل آلا

Introduction

Nowadays, many people and researchers are paying more attention to the use of functional foods as a diet due to the healthy effect of them. PUFAs as functional food have an important effect in human health. These essential fatty acids such as α-linolenic acid (ALA), arachidonic acid (ARA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) cannot be produced on human body so scientists have paid high attention to using them in the daily diets (1).The Food and Drug Administration has stated that it is essential for human to consume0.3-0.5 g of EPA and DHA mixture. Because of the importance and shortage of PUFAs, scientists are searching the appropriate sources to industrially produce them (2,3). Plants and animals are unable to produce LCPUFAs. The main sources of LCPUFAs are the fish lipid, but it requires several difficult and complicated extraction and purification steps. Numerous researches showed that the lipids of some oleaginous species are a rich source of LCPUFAs and so, the commercial exploitation of oleaginous species began to engage for human consumption (1,4,5). Low growth rate and sensitivity of marine bacteria to mechanical stresses are the main problems that are hindering their application for industrial production of LCPUFAs (3). Other important sources of LCPUFAs are algal species including Nitzschia, Nannochloropsis, Navicula, Phaeodactylum, and Porphyridiu. Many kinds of the problems like high production cost, sensitivity to shear stress, and high oxygen demand can get in the way to use the algal species for industrial purposes (2). Alternatively, fungi and yeasts have been considered for PUFAs production because of rapid growth rate and high tolerance to a wide pH range (6). The effect of physical and chemical conditions of fermentation medium in LCPUFAs production has been studied in extensive researches. Several studies have indicated that incubation temperature (7), carbon source, and the ratio of carbon to nitrogen (8) affect the yield of lipid and LCPUFA production in oleaginous species. According to the previous studies, the isolation of oleaginous species that have good ability to produce LCPUFAs and lipid is an effective step to industrialize a wider range of micro-organisms for commercial proposes. Thus in this study, an oleaginous yeast species was isolated from fish and then was identified by using the molecular method. Medium conditions were screened by the one factor at a time method and then optimized by using response surface method.

Materials and Methods

Choosing the Appropriate Fish Species:In the present study, 5 fish species including Common Carp, Mullet, Zander, Trout, and Caspian Kutum fish were chosen. The surface of fish body was washed with alcohol 70%. Then, 1 g of fish gills was removed by a sterile knife. The samples were incubated at 25ºC in a YGC. The single-yeast-colony was isolated, after the growth in PDA, and was kept in the refrigerator at 4ºC temperature (9).

Seed and Fermentation Media: Seed culture (100 ml) containing glucose mono-hydrate (30 g/liter) and soybean (10 g/liter) was prepared in a 500 ml Erlenmeyer and then sterilized at 121ºC for 15 min. Afterward, the yeasts were inoculated at 26ºC temperature and 170 rpm for 72 h. 3-10% of seed cultures were added to the fermentation medium.  (2 g/liter),  (3 g/liter), and (1.5 g/liter) were used as mineral elements in fermentation media (10); moreover, carbon and nitrogen sources as well as pH were used as variables. In order to produce lipid and fatty acid profile, the fermentation medium was incubated at 28ºC and 180 rpm for 5 days.

Analytical Methods: The dry weight of biomass, total lipids and profiles of fatty acids were examined by using methods that were published in previous researches (10,11,12,13).

Species Identification: In order to identify the yeast species which produce the maximum amount of lipid, firstly the yeast sample was incubated in seed culture medium for 48 h; and then, the DNA was extracted using the DNA extraction kit (K721, Thermo, USA). The D1/D2 26S rDNA was amplified using the forward (5’-GCATATCAATAAGCGGAGG AAAAG -3) and reverse primer (5’GGTCCGTGTTTCAAGACGG-3’) in the PCR (14). The PCR product (680 base pairs (bp) length nucleotides) was extracted from the gel and sent to the Takapu-Zist Company to determine the nucleotide sequence. After sequence determination, they were blasted using NCBI website and the phylogenetic tree was drawn by using the Mega5 software.

Results

Oleaginous Species Isolation from Fish: The results showed that three isolated yeast species produced more than 20% w/w lipid and so, they were considered as oleaginous species. Because of the highest ability of the yeast species isolated from the trout fish to produce lipid (Figure 1) these species were selected for more analysis.

Li et al (9) reported that glycerol as a carbon source had a significant effect on the lipid production in some yeast species. Therefore, glucose was replaced by glycerol (30%). The results indicated that glycerol had a negligible effect on the biomass production, while glycerol supported better condition for the production of lipid in isolated yeast from Common Carp and Zander. However, due to the lowest biomass production, glucose, instead of glycerol, was used as the carbon source for lipid production (Figures 1 and 2).

The results showed that yeast which was isolated from zandar species produced higher amount of biomass than glucose (Figure 2).

Fig. 1- The effect of the glycerol and glucose carbon source on the lipid percentage of yeast samples isolated from fish gills.

Fig. 2- The effect of the glycerol and glucose carbon source on the dry weight biomass of yeast isolated from fish gills.

Molecular Identification of the Microbial Species: In this step, the similarity of Non Transcribed Spacer 2 sequence of the isolated yeast was compared with the ribosomal sequence of other similar species obtaining from NCBI website. The phylogenetic tree that was drawn with mega5 software (Figure 3) verified that the isolated species was very similar to Wickerhamomy sp and therefore it was named Wickerhamomyces Siamensis SAKSG (Figure 3) which has not been reported as oleaginous species yet. Wickerhamomyces Siamensis which was isolated formphylloplane was so closest to Wickerhamomyces ciferrii (15).

Fig 3- The phylogenetic of the isolated yeast strainunknown with 11 similar species based on the Nearest neighbor interchange analysis of the Non Transcribed Spacer 2 region (The bootstrap level: 1000 pseudo-replications)

The “One Factor at a Time” Method: The “one factor at a time” method was used to screen important variables by changing one factor while keeping other variables constant.

pH:The effect of pH on lipid and biomass production byWickerhamomyces SiamensisSAKSG showed that pH= 6 among the others, it had the greatest impact on biomass and lipid production (data not shown). Thus, pH = 6 was chosen for subsequent experiments.

Carbon Source:Three carbon sources such as glucose, xylose, and sucrose were selected for investigating their effects on lipid and biomass production. The results showed that xylose and glucose had the highest and the lowest effects on the biomass production (Figure 4), respectively; but regarding to the lipid production, it was vice versa (Figure 5).

Fig 4- The effect of Glucose, xylose and Sucrose on dry weight biomass of Wickerhamomyces Siamensis SAKSG

Fig 5- The effect of Glucose, xylose and Sucrose on the lipid of Wickerhamomyces Siamensis SAKSG

It was concluded that glucose, among the others, was the best carbon source for lipid production by these species (Figure 4). In other words, glucose greatly converted into lipid and little amount of it were remained to convert into the biomass

Nitrogen Source:The results showed that ammonium nitrate wasn’t suitable substrate from lipid and biomass production, while the organic nitrogen sources have a significant effect on lipid and biomass production (Figures 6, 7). As can be seen in Figure 7, soybean had the highest effect on lipid production, and consequently it was selected for further analysis.

Fig 6- The effect of Soybean, Peptone, Yeast Extract and NH4NO3 on dry weight biomass

Fig 7- The effect of Soybean, Peptone, Yeast Extract and NH₄NO₃ on the lipid

According to the results of “one factor at a time method”, at the initial pH of 6, glucose and soybean as the carbon and nitrogen sources respectively, were significantly influenced in lipid production by Wickerhamomyces Siamensis SAKSG. Subsequently, response surface methodology (RSM) was used to optimize the amount of glucose and soybean toward lipid and oleic acid production.

Optimization by Response Surface Method (RSM):A face centered central composite design (FCCCD) of RSM was applied to optimize the level of glucose and soybean on the lipid and oleic acid production by Wickerhamomyces Siamensi sSAKSG. Table 1 indicates the actual and coded substrate content used in the FCCCD.

The results calculated by FCCCD were then examined by analysis of variance (ANOVA) and the results were applied to get a second order polynomial equation. The results either predictions or observations are observed in Table 1. The following equation indicated the effect of glucose (A) and soybean (B) on lipid (Y1) and oleic acid (Y2) production

In hence ‘Prob> F’of model is less than 0.05 it were considered significant. Linear effects of soybean and glucose on the lipid and oleic acid production were significant (p < 0.05). The interaction effect of glucose-soybean on the amount of oleic acid production was also significant, but it was insignificant on the lipid production (p < 0.05). Moreover, the quadratic effect of glucose on the oleic acid production was not significant (p < 0.05). ANOVA indicated that R²–values for oleic acid and lipid production were 0.95 and 0.98, respectively (data not shown). Observed and predicted results were so close together that it indicated optimization method was done in appropriate style (Table 1).

The model predicted that the maximum oleic acid (66.5%) and lipid production (27.1%) could be obtained by using (g/L) glucose 80, soybean 15 and glucose 42, soybean 5, respectively. The actual and predicted responses are shown in Table 1. As can be seen in Table 1, increasing in the soybean at the middle and constant levels of glucose had negative effect on oleic acid and lipid production (runs6 and 8), on the other hand, with increasing of soybean at the lowest levels of glucose, lipid was slightly reduced while oleic acid greatly declined (runs3 and 5).At the constant level of soybean with increasing in glucose level lipid production decreased while this condition caused oleic acid extensively to increase (runs 9, 10).

Lipid and Oleic Acid Results:Figure 8 shows effect of different levels of glucose and soybean on the amount of lipid production. The response surfaces indicated that similar to other oleaginous species, lipid and the type of fatty acids were affected by medium conditions. The lipid of Wickerhamomyces Siamensis SAKSG was increased in middle amount of glucose level while increasing in the glucose up to the highest content had negative effect on lipid production.

Table1- The central composite design of the response surface method together with real and predicted data

Fig 8- RSM curve for produced lipid and Oleic acid by Wickerhamomyces Siamensis SAKSG