Optimization of Candida tropicalis Fermentation Conditions to Produce Longchain Dibasic Acids

Xiang XIU Junqing WANG

Abstract [Objectives]The fermentation conditions of Candida tropicalis 1798pxa1 were optimized to further improve its yield of longchain dibasic acids.[Methods]The strains used in the study were C. tropicalis 1798， C. tropicalis 1798pxa1 and C. tropicalis 1798pxa1p2. First， through single factor experiments， the activated three fungi were cultured in different carbon sources， and the absorbance was measured every 2 h. The growth curve was drawn by software， and finally the most suitable substance for the substrate was selected， i.e.， dodecane. Then， the composition of the fermentation medium and the fermentation process parameters were determined through the PB experiment of the singledeletion C. tropicalis 1798pxa1.[Results]The experiment determined the important components affecting the synthesis of longchain dibasic acids， namely （NH4）2SO4， NaCl and dodecane. After optimization of the culture conditions， the yield of longchain dibasic acids of C. tropicalis 1798pxa1 increased from 5.6 to 10.1 g/L.[Conclusions]The scheme has been verified to be capable of greatly increasing the yield of the corresponding longchain dibasic acids of the C. tropicalis engineering strain.

Key words Synthesis of longchain dibasic acid; Candida tropicalis engineering fungus; Single factor experiment; PB experiment; Condition optimization

Candida tropicalis is a highyielding microorganism that can produce longchain dibasic acids （DCAs） by fermenting fatty acids and alkanes. It can be separated from fruits， vegetables， dairy products， soil， etc.， and can be used in the production of longchain dibasic acids. Longchain dibasic acids are an important raw material in the chemical process. They have important and extensive industrial applications and have been widely used in industrial raw materials. They play an important role in the production of highperformance engineering plastics， highgrade perfumes， highgrade paints and powder coatings， hightemperature electrolytes， lubricating oils， coldresistant plasticizers， resins， liquid crystals， and pesticides and pharmaceuticals. They can be used in the synthesis of aviation lubricating oil， advanced nylon engineering plastics， perfumes and other substances， and are also an important material for pharmaceuticals[1]. The materials synthesized using DCAs as raw materials are not only economical and practical with good performance， but also save energy and reduce environmental pollution. There is also a great research space in the application of medicine[2-7]. Since longchain dibasic acids do not exist in nature， we need to adopt a series of methods for synthesis. At present， the annual output of longchain dibasic acids is about 2 300 t， which is mainly used as an important raw material for the production of nylon， engineering plastics， paints and perfumes.

DCAs are a kind of general chemical intermediate used in the preparation of perfumes， polymers or binder raw materials[8-12]， containing 10 to 18 carbon atoms in the carbon chain and a carboxyl group at the  and ┺ positions， respectively. As a common industrial raw material， they play an important role in fields of paint and perfume synthesis， pharmaceutical production， coldresistant plasticizers， etc.[13].

Nowadays， industrial longchain dibasic acid production is mainly divided into three types： chemical synthesis， vegetable oil cracking and biological fermentation. Among them， the biological fermentation method has the advantages of low production cost， high production intensity， high specificity and low environmental pollution， and has become the most widely used method for industrial production of longchain dibasic acids[7].

With the rapid development of automobile manufacturing and electronic communication industries at home and abroad， Chinas demand for polyamide engineering plastics is increasing， and China has become the largest importer of polyamide engineering plastics in the world. In addition， in the development and application of medicines， polyamide engineering plastics have been flourishing for several years. They have shown special effects in the synthesis of pharmaceutical intermediates， breast cancer detection reagents， drugs for the treatment of skin cancer and AIDS， and synthesis of new hypoglycemic drugs. Therefore， longchain dibasic acid production has broad prospects， the domestic demand will continue to surge， and the market potential is enormous. It is even more important to develop new methods to increase their production.

The strains used in this study were C. tropicalis 1798pxa1 with pxa1 gene single copy deletion and C. tropicalis 1798pxa1p2 with double copy deletion， which were constructed by modifying the metabolic pathway of C. tropicalis 1798 at the early stage of this study. The fermentation conditions were optimized to further increase the yield of longchain dibasic acids.

Materials and Methods

Materials

Strains

C. tropicalis 1798; C. tropicalis 1798pxa1; C. tropicalis 1798pxa1p2.

Reagents

Peptone; sucrose; glucose; yeast extract; （NH4）2SO4; VB1; sodium chloride; KH2PO4; NaHPO4·12H2O; urea; MgSO4·7H2O; dodecane; hexadecane; peanut oil; NaOH.

Media

The used YEPD yeast medium contained glucose 20 g/L， peptone 20 g/L and yeast extract powder 10 g/L. In this experiment， 1 g of peptone， 1 g of glucose and 0.5 g of yeast extract powder were used to prepare six parts of 50 ml of the medium.

The used seed medium contained glucose 20 g/L， peptone 20 g/L and yeast extract powder 10 g/L. In this experiment， 1 g of peptone was used to prepare two parts of 100ml of the seed medium.

The used fermentation medium contained glucose 62 g/L， （NH4）2SO4 1 g/L， VB1 0.2 g/L， NaCl 2 g/L， KH2PO4 8 g/L， Na2HPO4·12H2O 10.08 g/L， urea 3 g/L and MgSO4·7H2O 6.15 g/L. In this experiment， 18.6 g of glucose， 1.5 g of （NH4）2SO4， 0.3 g of VB1， 3 g of NaCl， 12 g of KH2PO4 and 15.12 g of NaOPO4·12H2O were used to prepare 1.2 L of the medium; 100 ml of 3 g/L urea solution was prepared with 6 g of urea; and 6.15 g/L MgSO4·7H2O was prepared using 12.3 g of MgSO4·7H2O. The fermentation medium was sterilized separately with the urea and MgSO4·7H2O solutions in a steam sterilization pot. Specifically， the fermentation medium was sterilized at 115 for 20 min， and the remaining two solutions were sterilized at 105 for 20 min.

Methods

Single factor experiments of C. tropicalis 1798， C. tropicalis 1798pxa1 and C. tropicalis 1798pxa1p2

The experimental strains were taken out from the preservation freezing chamber and thawed， which was performed in a clean bench. 1 ml of the fungal solutions were inoculated into the YEPD yeast medium， obtaining three liquids， which were placed in a shaker， incubated at 30 for 12 h at 220 r/min， and labeled as C， C1 and C2， respectively.

The various operations were performed in the ultraclean workbench to avoid affecting the experimental results. 5 ml of the activated experimental fungi were taken out and added to the fermentation medium， and different carbon sources were added， which were glucose， dodecane， hexadecane and oil， respectively. Each group included three bottles， and there are 12 bottles in total. Thereafter， the mixtures were placed in a shaker at 30， and the fermentation was carried out at 220 r/min. The bottles were taken out once every 2 h for the determination of absorbance， and the fermentation was carried out for at least 14 h.

The absorbance was measured with a spectrophotometer， and the result was maintained between 0.2 and 0.8. If the test result was too large， the mixtures were diluted with distilled water， and the distilled water was used as a control to measure the OD600.

Determination of optimal C. tropicalis fermentation conditions

Through the fermentation experiment， engineering fungi grown better were selected for the optimization of fermentation conditions.

Optimization of fermentation conditions

（1） The experiment was carried out according to the PlackettBurman （PB） method. The experimental data were analyzed by the software DesignExpert V8.0.5b for the main effects of various factors.

（2） According to the results of the PlackettBurman （PB） experiment， the optimum amounts of carbon source， nitrogen source and inorganic salts suitable for the synthesis of twelvecarbon dibasic acids were screened by single factor method. Unless otherwise specified， three parallel experiments were set for each group of single factor experiments.

（3） On the basis of single factor experiments， glucose， urea， yeast extract and MgSO4·7H2O were used for the fourfactor threelevel orthogonal experiment. The L9（34） orthogonal experimental design table is shown in Table 1.

Strain fermentation experiment

The experimental strains were taken out from the preservation freezing chamber and thawed， which was performed in a clean bench. 1 ml of the fungal solutions were inoculated into the YEPD yeast medium， obtaining two liquids， which were placed in a shaker， incubated at 30 for 12 h at 220 r/min， and labeled as C1 and C2， respectively.

The various operations were performed in the ultraclean workbench to avoid affecting the experimental results. 5 ml of the activated experimental fungi were taken out and added to the fermentation medium. Thereafter， the mixtures were placed in a shaker at 30， and the fermentation was carried out at 220 r/min.

Adjustment of pH and determination of dibasic acids

After 12 h of fermentation， the pH was adjusted to about 7.5 with NaOH， and 4 ml of dodecane was added to the fermentation medium. Thereafter， pH was adjusted every 24 h.

After adjusting the pH for about 6 d， the fermentation can be stopped， and the fermentation liquid was taken out from the fermentation medium and added into a centrifuge tube.

The pH of each fermentation liquid in the centrifuge tube was adjusted with NaOH to 12. Then， the fermentation liquid was heated in a constant temperature water bath at 80 for 3 h. After the heating was completed， the centrifuge tube was taken out and centrifuged at 7 000 r/min for 10 min. After centrifugation， 25 ml of the middle liquid in the tube was taken out and adjusted to pH 2-3 with NaOH， followed by standing for 1-3 h.

Centrifugation was performed again properly， followed by air pump filtration. During the filtration process， the centrifuge tube was rinsed for 3 times with deionized water to ensure that all precipitate was taken out.

After the air pump filtration was completed， the filter paper was placed in a conical flask， and 30 ml of 95% ethanol was added to dissolve the precipitate. The flask was sealed with a sealing film and heated in a constant temperature water bath at 50 for 10 min. Three drops of bromothymol blue were then added， and then， titration was performed with NaOH and stopped when discoloration just happened followed by internal fading within half a minute. Data of used NaOH were recorded， based on which the amount of dibasic acid produced was calculated：

Results and Analysis

Growth curve of the C. tropicalis strains under single factor conditions

Using the obtained absorbance data， the growth curves of the strains in different carbon sources can be plotted.

It can be seen from Fig. 1， Fig. 2 and Fig. 4 that C. tropicalis 1798 grew fastest when dodecane， hexadecane and peanut oil were used as the substrate， respectively， followed by C. tropicalis 1798pxa1， and the slowest one was C. tropicalis 1798pxa1p2 with double copy deletion. It can be seen that knocking out the pxa1 transporter gene has a certain effect on the growth rate of the strain， indicating that the gene is one of key genes in the fatty acid metabolism pathway. It can be seen from Fig. 3 that the growth curve of C. tropicalis 1798 was almost the same as that of the engineering fungi when glucose was used as the fermentation substrate， indicating that knocking out the single copy and the double copy of pxa1 gene have no large adverse effect on the growth of the strain.

Determination of optimal fermentation conditions

Through the above fermentation experiment， it is concluded that the growth rate of the engineering fungus with single gene deletion was faster than that of the engineering fungus with double gene deletion. Therefore， C. tropicalis 1798pxa1 was selected for PB experiment optimization.

According to the literature， the fermentation medium of C. tropicalis using dodecane to produce twelvecarbon dibasic acids mainly includes following substances： glucose， ammonium sulfate， yeast extract， NaCl， Na2HPO4， KH2PO4， urea， MgSO4， and vitamin B1. The above substances were used as the factors to be investigated by the PlackettBurman method， and the four factors most important for the production of twelvecarbon dibasic acids were screened. At the same time， in order to calculate the experimental error， three virtual factors （X3， X6， X9） were designed in this experiment. In the PB method， +1 and -1 were used to indicate the high and low levels of each factor. The true values of the high and low levels of each factor are shown in Table 2.

Table 3 was designed according to the high and low levels of each factor set in Table 2. Different concentrations of fermentation media were prepared according to Table 3， and fermentation was carried out for 144 h. The yield of twelvecarbon dibasic acids in each medium was measured. The results are shown in Table 3.

According to the PlackettBurman （PB） method， four of the 11 factors， which had the most obvious effects on the accumulation of twelvecarbon dibasic acids， were selected： glucose （X1）， yeast extract （X4）， Na2HPO4 （ X8） and urea （X10）. According to the above tables， the effects of various factors on longchain twelvecarbon dibasic acids when the fermentation culture was carried out in the fermentation medium can be obtained. Only when the confidence is greater than 95%， i.e.， P<0.05， it can be considered that the factor and its impact are significant. Therefore， the factors that had a significant effect on the production of twelvecarbon dibasic acids were as follows： ammonium sulfate， sodium chloride and dodecane， and the effects of these three factors were positive， indicating that a high concentration is beneficial to the improvement of dibasic acid yield， while other factors that have a positive effect can be selected at a high concentration level when optimizing the medium， and those with negative values should be selected at a low level when formulating the medium.

Through the PB experiment， factors that had the most significant effect on the production of twelvecarbon dibasic acids were screened as follows： （NH4）2SO4， NaCl and dodecane， so the latter experiments will further optimize these three components. In addition， MgSO4·7H2O can be added to the subsequent optimization scheme because of the large impact of MgSO4·7H2O on the yield.

The relationship between the response value and each variable existed in the following equation：

Y=12.99-1.57X1-3.98X2-0.48X3+0.55X4+1.8X1X2+0.2X1X3+0.29X1X4+0.15X2X3-0.37X2X4-0.04X3X4-1.16X21+0.66X22+0.03X23+0.02X24， wherein Y represents the yield of twelvecarbon dibasic acid.

From the above Table 4， the optimal combination of these factors in the medium was： （NH4）2SO4 1 g/L， NaCl 2 g/L， dodecane 5 g/L， and MgSO4·7H2O 1.5 g/L.

Yield of dibasic acid produced by fermentation of C. tropicalis 1798pxa1

The engineering fungus that had undergone gene knocking was cultured in the optimized fermentation medium for 6 d. According to the above figure， the final yield of twelvecarbon dibasic acids of C. tropicalis 1798pxa1 was 10.1 g/L， and compared with the original C. tropicalis 1798， its acid production increased by 80.4%.

Conclusions and Discussion

Through the single factor experiments of the C. tropicalis engineering fungi， it can be concluded that the growth curve of C. tropicalis 1798 was almost the same as the strains with gene deletion when glucose was used as the unified carbon source， which proves that the missing genes do not affect the growth of the strain. However， among dodecane， hexadecane and peanut oil， the fungi with gene deletion grew slower than that of the original fungus， with lower OD600 value， and the effect of dodecane was particularly obvious. Therefore， dodecane was used as the substrate for the next experiment of the amount of the dibasic acid produced.

With the DesignExpert software as a tool， a specific PB experiment was designed to optimize the medium to increase the yield of dibasic acid. The two C. tropicalis engineering strains with gene deletion were used as the fermentation microorganisms to ferment in the fermentation medium. Through the above experiment and results analysis， the optimized fermentation medium was finally obtained： peptone 4 g/L， sucrose 55 g/L， yeast extract powder 3 g/L， （NH4）2SO4 1 g/L， VB1 0.4 g/L， NaOH 2 g/L， KH2PO4 8 g/L， Na2HPO4·12H2O 1 g/L， urea 3 g/L， MgSO4·7H2O 1.5 g/L， dodecane 5 g/L， and NaOH 2 g/L， by which the highest yield of twelvecarbon dibasic acid reached 10.1 g/L， which was significantly higher than the original dibasic acid yield.

In this study， the two constructed C. tropicalis strains with gene deletion， C. tropicalis 1798pxa1 and C. tropicalis 1798pxa1p2， were cultured in the fermentation medium to produce corresponding longchain dibasic acids. The strains used have been reconstructed， and on this basis， it is necessary to optimize the fermentation conditions so that the longchain dibasic acid yield is higher. The most important thing to optimize the fermentation conditions is to optimize the fermentation medium. DesignExpert was used to establish an experimental model， and then， the acid amount obtained by the experiment was substituted into DesignExpert for analysis， finally obtaining the optimized fermentation medium desired. The fermentation medium was verified to be capable of greatly increasing the yield of corresponding longchain dibasic acids.

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