Introduction

Oleaginous microorganism such as bacteria, mold, yeast, algae, etc. can accumulate and synthesize a large amount of lipids in cells using carbohydrates under certain conditions, and the microbial lipids become an important alternative raw material for biodiesel production1,2,3,4. The fatty acid composition of microbial lipids is similar to that of common vegetable oil, and some also contain rich polyunsaturated fatty acids, such as arachidonic acid (AA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), etc.5,6,7,8. Although the development prospects of microbial lipids are broad, high cost is still the main factor restricting their industrial production.

Cellulosic waste is one of the most abundant and cheapest renewable raw materials in nature2,9,10. For example, crop straw is the main agricultural cellulosic waste, which is mostly used as feed, fuel, and organic fertilizer11,12,13. Using crop straw to produce microbial lipids can effectively reduce the production cost and realize the sustainable development of agriculture1,14.

The oleaginous fungus Mucor circinelloides, as the first commercially lipid producing strain in the world, can synthesize high level of γ-linolenic acid (GLA) with various important physiological functions15,16,17. Previously, we presented a novel approach by co-expression of cellobiohydrolase and delta-6 desaturase in M. circinelloides to facilitate the GLA production from cellulose in one step15,18. In the present study, an in-depth analysis of lipid production by recombinant M. circinelloides strains Mc-C2TD6 and Mc-C2PD6 using corn straw under solid state fermentation was to be explored. The optimal addition amount of the cellulase and the inoculum of engineered M. circinelloides strains by using corn straw for lipid production were investigated. In addition, we developed a two-stage temperature control strategy for lipid production from corn straw under solid state fermentation. Our study provides a certain foundation for the further application of the engineered M. circinelloides strains in industrial production of microbial lipids from crop straw.

Materials and methods

Strains and media

The original M. circinelloides strain WJ11 and recombinant strains Mc-2075 (as a control, with empty plasmid), Mc-C2TD6 and Mc-C2PD6 (cellobiohydrolase and delta-6 desaturase co-expression) were previously constructed and stocked in our lab. The strains used in this work are summarized in Table 115,19.

Table 1 Strains used in this work.
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The seed medium is composed of glucose 20 g/L, peptone 20 g/L and yeast extract 10 g/L. The basic nutrient solution consists of: glucose 5 g/L, ammonium tartrate 2 g/L, KH2PO4 7 g/L, Na2HPO4 2 g/L, MgSO4·7H2O 1.5 g/L, yeast 1.5 g/L, CaCl2·2H2O 0.1 g/L, FeCl3·6H2O 8 mg/L, ZnSO4·7H2O 1 mg/L, CuSO4·5H2O 0.1 mg/L, Co(NO3)2·6H2O 0.1 mg/L, and MnSO4·5H2O 0.1 mg/L. The cellulase (5000 IFPU/g) is produced by Jinan Sansheng Biological Products Co., LTD.

The corn straw was collected from straw recycling center of Dongping, Tai’an, Shandong Province. The corn straw was baked in the oven at 80 °C until the quality remained unchanged, crushed, sifted 40 mesh for use. The corn straw solid fermentation medium is prepared by adding 80 g corn straw powder into 100 mL basic nutrient solution and then sterilizing.

Culture conditions

The recombinant strains of M. circinelloides stored in glycerol tube at – 80 ℃ were added into the seed medium and cultured in a shaker with a controlled rotational speed of 200 rpm/min, culture temperature of 28 °C, initial pH value of 5.0, and culture time of 24 h to obtain the seed liquid.

The corn straw solid fermentation medium was prepared and sterilized by high-pressure steam at 121 °C for 20 min. And then 0.0–2.0% (w/w) cellulase powder (protein content was 1 mg/mL) and above cultured seed liquid was added at 5.0–20.0% (w/w) of the inoculated amount. The fermentation culture was placed in a constant temperature incubator at 28 °C, pH 4.5 for 192 h. The two-stage temperature control strategy was 32 °C for the first 48 h and 28 °C for the last 144 h. It should be noted that among the above fermentation parameters, the temperature, the addition amount of cellulase and the inoculum of strains depend on the experimental design. All experiments were carried out in triplicate.

Determination of lipid yield

The fermentation mixture residue obtained after filtration was treated for 30 min by ultrasonic grinder, which promoted the cell fragmentation of microbial strain and the release of lipid in the cell. Centrifuge was used to separate the fermentation mixture, and the upper liquid was added to ether-petroleum ether, fully shaken and mixed, and left for 30 min to extract the lipid in the water phase. The lipid layer was sucked out, and then the ether-petroleum ether was steamed in a water bath (90 °C), and dried to constant weight in a constant temperature drying oven (105 °C). The lipid quality was obtained by using the difference method, and the amount of lipid produced per 100 g dry material was calculated. The fatty acid composition in the mixture of corn straw fermented by microbial strains was determined by gas chromatography (GC) analysis15.

Statistical analysis

A statistical analysis was carried out using SPSS 16.0 for Windows (SPSS Inc., Chicago, IL). The mean values and the standard error of the mean were calculated from the data obtained from three independent experiments. Student’s t test was used to evaluate the differences between the means of the test, and P < 0.05 was considered as significantly different.

Results

Effect of cellulase concentration on lipid production of engineered M. circinelloides strains from corn straw

The effect of cellulase concentration on lipid production of engineered M. circinelloides strains from corn straw was investigated as shown in Fig. 1. The results showed that when cellulase is not added, the amounts of lipids synthesized by the strains are little. With the increase of cellulase supplemented, lipid production gradually increased. When cellulase content reached 1.5%, the lipid yield reached the maximum. In view of the effect and cost savings of cellulase, the optimal amount of cellulase was determined to be 1.5%. When cellulase was added to 1.5%, the maximum lipid yields of the engineered strains were improved significantly by 58% for Mc-C2TD6 and 80% for Mc-C2PD6 compared with that of the control strain, respectively.

Figure 1
figure 1

Lipid production of engineered M. circinelloides strains under corn straw solid medium containing different concentrations (0.0–2.0%, w/w) of cellulase. Strains were cultured in solid medium of corn straw during 192 h at 28 °C and pH 4.5 with an inoculum of 10%. Values were mean of three independent fermentation experiments. Error bars represent the standard error of the mean.

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Effect of inoculum amount on lipid production of engineered M. circinelloides strains from corn straw

The lipid yield of engineered M. circinelloides strains under corn straw solid medium with different inoculum concentrations (5.0–20.0%, w/w) were shown in Fig. 2. The results showed that with the increase of inoculation amount, lipid production gradually increased. When inoculation content reached 15%, the lipid yield reached the maximum. After that, the lipid yield did not increase significantly when the quantity of inoculation continued to increase. The optimal amount of inoculation was determined to be 15%. Similarly, under the optimal inoculation content, the lipid yields of the engineered strains were increased by 53% in Mc-C2TD6 and 72% in Mc-C2PD6 higher than that of the control strain, respectively.

Figure 2
figure 2

Lipid production of engineered M. circinelloides strains under corn straw solid medium with different inoculum concentrations (5.0–20.0%, w/w). Strains were cultured in solid medium of corn straw during 192 h at 28 °C, pH 4.5 and supplemented with 1.5% cellulase. Values were mean of three independent fermentation experiments. Error bars represent the standard error of the mean.

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Lipid production of engineered M. circinelloides strains from corn straw during a temperature two-stage strategy

A two-stage temperature control strategy was adopted to investigate the lipid production of engineered M. circinelloides strains from corn straw under the condition of adding 1.5% cellulase and 15% inoculum. As shown in Fig. 3, the lipid accumulation patterns in different strains were similar: the lipid accumulation increased gradually with fermentation time until the highest lipid yield reached at 168 h, and then it slowed down and leveled off. Notably, the maximum lipid yields of engineered strains were 1.35 g for Mc-C2TD6 and 1.56 g for Mc-C2PD6 per 100 g dry medium, which were significantly enhanced by 1.5- and 1.8-fold, respectively, compared with the control strain. This indicated that the engineered M. circinelloides strains were more likely to cooperate with cellulase to synthesize lipids from corn straw.

Figure 3
figure 3

Lipid production of engineered M. circinelloides strains under corn straw solid medium during a temperature two-stage strategy. Strains were cultured in solid medium of corn straw during 192 h at pH 4.5, inoculated at 15% and supplemented with 1.5% cellulase. The two-stage temperature control strategy was 32 °C for the first 48 h and 28 °C for the last 144 h. Values were mean of three independent fermentation experiments. Error bars represent the standard error of the mean.

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GLA production of engineered M. circinelloides strains from corn straw

The properties of fatty acids produced by engineered M. circinelloides strains using corn straw were shown in Table 2. The maximum GLA yields of engineered strains were 216 mg for Mc-C2TD6 and 274 mg for Mc-C2PD6 per 100 g dry medium. Compared with the control strain, the intracellular GLA yields of Mc-C2TD6 and Mc-C2PD6 were increased by 1.8- and 2.3-fold, respectively, which were consistent with our previous results on microcrystalline cellulose and carboxymethyl cellulose15,18. Therefore, the engineered M. circinelloides strains, especially for Mc-C2PD6, showed advantages in using corn straw to produce GLA.

Table 2 The fatty acid composition in engineered M. circinelloides strains cultured with corn straw.
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Discussion

Although microbial lipids become an important alternative resource for biodiesel production, and some functional lipids beneficial to people's health are also the main production objects of the food and pharmaceutical industry, high cost is still the main factor restricting their industrial production1,3,6,13. The use of cheap alternative raw materials (such as crop straw) is one of the important problems to solve the industrialization and scale of microbial lipids11,13. The recent researches focus on the strategies of raw material treatment, strain screening and optimization of fermentation to improve the microbial lipid production from cellulosic raw materials4,10,13. However, the adoption of efficient strains that could use cheap cellulosic feedstock is an important way to achieve high microbial lipid production.

It is believed that the oleaginous fungus M. circinelloides has the following specific advantages and potential over other oleaginous microorganisms in lipid production. Firstly, M. circinelloides was the first commercially lipid producing strain in the world, and its synthesized lipids are rich in GLA, which has the role of preventing inflammation, softening blood vessels, and lowering blood sugar and lipids16,19. Secondly, M. circinelloides can absorb and metabolize multiple sugars including glucose, xylose, and cellobiose, so it could make full use of cellulose and hemicellulose in crop straw, which has superiority in reducing production cost1,17,18. Thirdly, M. circinelloides has become a model organism to study the lipid mechanism in fungi with its successfully analyzed genome sequence and perfect gene manipulation system15,20,21. Finally, the ability of GLA synthesis from cellulose in M. circinelloides was enhanced through the genetic engineering, and the simultaneous conversion of cellulose-monosaccharide-lipids was realized in one step, which could not be achieved in other oleaginous microorganisms15,18. In view of this, the lipid production conditions from corn straw by engineered M. circinelloides strains Mc-C2TD6 and Mc-C2PD6 were investigated.

The complex structure of corn straw needs a series of pretreatments to be effectively degraded22,23. In this study, in order to improve the utilization efficiency of corn straw, cellulase was added in the fermentation process to promote the degradation of corn straw. The addition of cellulase is too little, resulting in poor degradation effect of corn straw. Excessive addition of cellulase can cause excessive cost. The amount of cellulase was added according to its production instructions, and the addition range is 0.5–2.0% (w/w), with 0.0% as a control. The results showed that the optimum concentration of cellulase was 1.5%. However, the lipid yield did not increase significantly when the quantity of cellulase continued to increase. The reason might be that cellulase can assist the engineered M. circinelloides strains to degrade cellulose in corn straw and produce reducing sugars, and meanwhile the stains can further use reducing sugars to synthesize lipids. Thus, with the increase of cellulase addition, the production of synthesized lipids also increased. When the amount of cellulase added reaches a certain value, the final lipid production does not increase or increases slightly due to the limited cellulose that can be degraded in the raw material. In addition, it could also be seen that with the assistance of cellulase, the engineered strains might be more efficient in the decomposition of corn straws, and the resultant lipid yields are much higher than that of the control strain (Mc-2075) and the wild strain (WT).

The amount of inoculation is an important factor affecting the growth and lipid production of engineered M. circinelloides strains from corn straw1. A larger amount of inoculation can shorten the time when mycelium propagation reaches its peak and make the formation of products arrive in advance24. However, too much inoculation will cause insufficient oxygen supply and affect product synthesis, and it is not economical. Generally, the inoculation amount of mold is usually 5–20% (w/w). Therefore, in order to determine the optimal inoculum, we set the inoculum range at dose of 5–20%. The results showed that the optimum inoculation amount was 15%.

Generally, the production of lipids from corn straw requires first degradation of corn straw into small molecules of available monosaccharides, and then these monosaccharides are converted into lipids by microbial fermentation1,25. In the early stage, the engineered M. circinelloides strains cooperated with cellulase to degrade corn straws, and then in the middle and late stages, the strains further used the decomposed small molecules of sugars to grow and synthesize lipids. However, the temperature of enzymatic hydrolysis of cellulose by cellulase was relatively high, while the temperature of growth and lipid accumulation of M. circinelloides was low. Thus, a two-stage temperature control strategy was adopted to investigate the lipid production of engineered M. circinelloides strains from corn straws under the condition of adding 1.5% cellulase and 15% inoculum. The maximum lipid and GLA yields of engineered strains were 1.56 g and 274 mg per 100 g dry medium (for Mc-C2PD6), which were significantly enhanced by 1.8- and 2.3-fold, respectively, compared with the control strain. These results indicated that the engineered M. circinelloides strains could cooperate with cellulase well to synthesize lipids and GLA from corn straw.

It should be noted that the pure biological treatment method of microbial and enzyme co-fermentation adopted in this study has incomparable advantages over traditional chemical treatment methods, such as non-toxic by-product inhibition, simple steps, and no environmental pollution, etc. However, it is undeniable that this study also has some limitations, such as long degradation process, incomplete degradation, few available monosaccharide and slow utilization of corn straw.

In conclusion, one of the current research hotspots of bioenergy is the production of biodiesel by microbial fermentation using lignocellulosic biomass such as crop straw. In the present study, the effects of cellulase concentration and inoculum amount on lipid production of engineered M. circinelloides strains from corn straw were analyzed. Furthermore, a two-stage temperature control strategy was developed to facilitate the lipid and GLA synthesis from corn straws by engineered M. circinelloides strains. This study laid a foundation for the direct microbial transformation from corn straw to functional GLA in one step, which might be helpful for reducing the production cost.