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. 2020 Jul 23;10(8):352. doi: 10.1007/s13205-020-02347-6

A four-microorganism three-step fermentation process for producing medium-chain-length polyhydroxyalkanoate from starch

Xiaohui Yang 1,#, Suhang Li 1,#, Xiaoqiang Jia 1,2,3,✉
PMCID: PMC7378139  PMID: 32766094

Abstract

In this study, a four-microorganism three-step fermentation process was established for producing medium-chain-length polyhydroxyalkanoate (mcl-PHA) from starch, which was used as the sole carbon source. The four microorganisms used for this process were Aspergillus niger, Saccharomyces cerevisiae L2612, Acetobacter orientalis, and Pseudomonas putida KT2440-acs. The initial carbon source starch concentration was set to 30 g/L, the maximum glucose concentration reached 17.66 g/L at 48 h after starch hydrolysis, and then, 2.36 g/L of acetic acid was obtained at 96 h. The final output of mcl-PHA was 0.5 g/L at 144 h, overall productivity for mcl-PHA was 3.47 mg/(L·h) and the total starch to mcl-PHA yield for the process was 16.67 mg/g. Although the overall yield and conversion rate of this process were not high, this is the first attempt to produce mcl-PHA using starch as a substrate, and it provides a feasible strategy for producing PHA from kitchen waste. The production process of mcl-PHA with a clear flora structure and short fermentation cycle was realized.

Keywords: Medium-chain-length polyhydroxyalkanoate, Starch, Acetic acid, Pseudomonas putida KT2440-acs

Introduction

Among several developing biodegradable polymers, polyhydroxyalkanoate (PHA) has received considerable attention owing to its properties and similarity to conventional petrochemical-derived plastics (Huang 2006). The chemical structures of different PHAs are currently being investigated for sustained drug release. Furthermore, there are potential applications in bone plates, wound dressings, medical aids, and treatment devices. They have been mainly divided into three categories based on their structural characteristics (Chen 2002; Sudesh 2000). One type of PHAs is short-chain-length PHA (scl-PHA); the monomers of scl-PHA have 3–5 carbon atoms; and they have high crystallinity, brittleness, and hardness (Lee 2000). Then, medium-chain-length PHA (mcl-PHA), which has a higher thermoplasticity and flexibility than scl-PHA, and its monomer has 6–14 carbon atoms (Volker 1998). The last type is long-chain-length PHA (lcl-PHA), and these polyesters contain building blocks with more than 14 carbon atoms; they have a highly amorphous character and low polydispersity and high thermal stability, but they have been scarcely described (Koller 2018).

Mcl-PHA is a polyester that is mainly synthesized by gram-negative bacteria, and Pseudomonas putida is an excellent mcl-PHA-producing strain that can utilize a variety of substrates, such as fatty acids. Thus, most research on mcl-PHA synthesis has focused on using Pseudomonas sp. as the microbial cell factory, increasing the production of mcl-PHA by regulating its metabolism (O'Leary 2005; Zinn 2011). However, there are still problems in the mcl-PHA production process, such as unclear types of microorganisms and long fermentation cycle, and it usually requires 15 days or even months (Fei et al. 2008; Chinwetkitvanich et al. 2004). Moreover, the high production cost is one of the main problems of producing mcl-PHA; the price cannot compete with petrochemical plastics whose cost is lower. Thus, many efforts have been made to reduce the cost of mcl-PHA production, such as recombinant bacteria and improving the efficiency of the fermentation and recovery processes (Choi 1999). Moreover, the utilization of low-cost carbon sources is a current topic of interest.

Starch is a polyhydroxy polymer compound. Starch production in China is second only after that of the United States. It can be applied as a low-cost carbon source in a variety of materials, such as starch sugar, amino acids, ethanol, and other raw materials, for fermentation. In recent years, many researchers have used starch wastewater as a carbon source and mixed it with activated sludge to produce PHA by anaerobic fermentation (Chinwetkitvanich et al. 2004; Chua 2003). This is currently a common method for producing PHA using low-cost carbon sources, but its shortcomings are that the cycle is too long (requires 15–30 days), and most PHA produced by anaerobic fermentation are scl-PHA, which cannot efficiently produce mcl-PHA (Xiaojie Zhou 2010). Additionally, the proportion of volatile fatty acid (VFA) content produced by the anaerobic fermentation process is unstable, and it cannot produce a single targeted class of PHAs. In this study, we developed a four-microorganism three-step fermentation process to achieve targeted production of mcl-PHA.

It has been reported that Aspergillus niger has a good ability to hydrolyze starch (Kay 1996; Sorimachi 1997). The α-amylase produced by A. niger is typically used for liquefaction and saccharification of starch (Henkin 1991). Namely, starch is liquefied by α-amylase into a low-molecular-weight product, dextrin, which is saccharified to glucose via an enzymatic reaction. Yang et al. (1983) also isolated and sequenced the gene encoding α-amylase from B. subtilis. Therefore, A. niger and B. subtilis were selected because they have better hydrolytic capacity. In this study, a starch hydrolysis capability test was performed for these two strains, and the strain that had the higher capability of hydrolyzing starch was selected as the experimental strain in the first step of the fermentation process.

Although P. putida can directly use glucose as a substrate to produce mcl-PHA, the efficiency is poor (Munir 2018). Our laboratory completed the construction of a mutant called P. putida KT2440-acs that can efficiently use acetic acid to produce mcl-PHA. Therefore, it is necessary to further convert glucose into a substrate that can be efficiently used by P. putida KT2440-acs during this process. We used S. cerevisiae L2612 and A. orientalis to produce acetic acid using glucose in the second step. However, there are other fatty acids, such as glycerol and lactic acid, which can be used by P. putida KT2440-acs to efficiently produce mcl-PHA. The flow chart of the whole process is shown in Fig. 1.

Fig. 1.

Fig. 1

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Four-microorganism three-step process established in this study

Materials and methods

Bacterial strain and growth conditions

The four-microorganism three-step fermentation process used in this study was independently developed and optimized by our laboratory. The A. niger used in the first step of this study was from the China General Microbiological Culture Collection Center, No 3.3928. Bacillus subtilis 168 was from the Chen Tao laboratory at Tianjin University. A. niger was streaked on a Modified Martin solid medium and placed in a 30 °C biochemical incubator. The mycelium matured from initial white to black after approximately three weeks. The Modified Martin solid medium included peptone (5 g/L), KH2PO4⋅2H2O (1 g/L), MgSO4⋅7H2O (0.5 g/L), yeast powder (2 g/L), agar powder (15 g/L), and glucose (20 g/L) at a pH of approximately 6.4. All ingredients except glucose were dissolved at 25 °C, and the pH was adjusted to 6.8; then, glucose was added after boiling the above solution. Then, it was shaken well, the pH was adjusted to 6.4, and it was sterilized at 115 °C. Bacillus subtilis 168 was streaked on a solid LB medium and placed in a 30 °C biochemical incubator. The LB solid medium included peptone (10 g/L), NaCl (10 g/L), yeast powder (5 g/L), and agar powder (15 g/L) at a pH of approximately 7.

The S. cerevisiae L2612 used in the second step was from the Yuan Yingjin laboratory at Tianjin University (Jian 2012) and was cultured in a liquid shake flask containing Yeast Peptone Dextrose (YPD) medium for 12 h. The YPD medium included glucose (20 g/L), yeast powder (10 g/L), and peptone (20 g/L), and the pH was approximately 7. Acetobacter orientalis was obtained from the pulp of rotten fruits and screened on Acetobacter selection medium. After 16SrDNA identification and strain morphology analysis, it was confirmed that the bacterium was Acetobacter orientalis. The Acetobacter sp. selection medium included 10 g/L of yeast powder and 30 g/L of glucose. After sterilizing the Acetobacter sp. selection medium at 115 °C, 40 mL/L of absolute ethanol was added, and the pH was adjusted to approximately 5.5.

P. putida KT2440-acs was maintained and recombined in our laboratory. The production of acetyl-CoA through acetic acid requires the enzyme encoded by the acs gene. To enhance this process, the acs gene was overexpressed to obtain the mutant strain, P. putida KT2440-acs, which can efficiently use acetic acid (Yang 2019). The acetic acid was used after culturing for 24 h in LB broth. The initial starch culture medium composition was starch (30 g/L), casein peptone (3 g/L), NaCl (5 g/L), NH4Cl (5 g/L), and KCl (3 g/L) at a pH of 7.

Co-culture and continuous fermentation

The first step of this process is taking the A. niger culture into Modified Martin medium; we added starch as the sole carbon source. The fermentation process was performed in triplicate, and a control experiment was performed (subsequent experiments used the same method). The fermentation conditions included shaking the flask culture at 30 °C and at a rotation speed of 220 rpm. Samples were taken every 6 h after initial fermentation, and continuous fermentation was performed for 48 h for the first fermentation step. After completion of the first fermentation step, the suspended mycelium was removed via centrifugation, and the supernatant was collected, sterilization is performed after that.

The second step involved a S. cerevisiae L2612–A. orientalis co-culture in which S. cerevisiae L2612 and A. orientalis were inoculated in test tubes separately and cultured for 24 h in a shaker at 220 rpm and 30 °C as the second fermentation seed liquid. Then, the S. cerevisiae L2612 and A. orientalis seed liquids were inoculated into the supernatant collected from the first step, and then, the culture was shaken in a shaker at 120 rpm and 30 °C. Continuous fermentation was maintained under 12-h anaerobic and 12-h aerobic conditions, and the fermentation product was sampled every 6 h for 48 h. An ultrasonic homogenizer was used to extract the microbial consortium, and the cell debris were removed via centrifugation. Sterilization is also performed after the new substrate is produced, which is used as carbon sources for the next step.

The last step was the fermentation of P. putida KT2440-acs. We used the products of the second fermentation steps as carbon sources for P. putida KT2440-acs to synthesize mcl-PHA.

Analysis methods

The growth of P. putida KT2440-acs was monitored by measuring the optical density at 600 nm (OD600) using a UV-1200 spectrophotometer (Mapada, China). Accurate, quantitative analysis of glycerol, lactic acid, acetic acid, and glucose in the culture supernatant was performed using an Ultimate 3000 high-performance liquid chromatography system (Dionex, Sunnyvale, CA, USA) equipped with an Aminex HPX-87H ion exchange column using a refractive index detector at 65 °C and at a flow rate of 0.6 mL/min of 5 mM H2SO4. The retention times of glucose, lactic acid, glycerol, and acetic acid were 8.75 min, 12.25 min, 12.98 min, and 14.69 min, respectively (Fig. 2). PHA was extracted and detected as previously described. The microbial cells were collected by centrifugation at 10,000 g for 10 min and thoroughly washed with distilled water, frozen at  – 8 0 °C, and then lyophilized in a FD-1–50 vacuum freeze dryer (Beijing Boyi Kang Experimental Instrument Co., Ltd.), at 65 MPa and  – 50 °C for 24 h.

Fig. 2.

Fig. 2

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The retention times of glucose, lactic acid, glycerol and acetic acid which measured by HPLC. Peak 2 is glucose, peak 3 is lactic acid, peak 4 is glycerol, and peak 5 is acetic acid

The obtained lyophilized product was subjected to methanol esterification at 100 °C for 4 h in an esterification solution (0.5 g of benzoic acid dissolved in 475 mL of pure methanol; then, we added 15 mL of concentrated sulfuric acid and stored it at 4 °C), and benzoic acid was used as the internal standard. The lyophilized product was washed again with distilled water to ensure that all the intracellular fatty acids were dissolved in the organic solvent and the sample was subjected to membrane treatment. The building blocks of PHA, including volatile methyl esters of 3-hydroxyalkanoates, could be detected by gas chromatography (BRUKER 456-GC). The column temperature was started at 80 °C for 1 min, then was increased to 250 °C at a rate of 20 °C/min, and finally was held for 1.5 min; the detector is flame ionization detector (FID). The final mcl-PHA products consisted of two monomers, 3-hydroxydecanoate (C10) and 3-hydroxyoctanoate (C8). The internal standard method was used to determine the retention time of each monomer in the mcl-PHA, and the content of each monomer substance was calculated using the area ratio method using benzoic acid as the internal standard (Yang 2019). The formula for mcl-PHA production is as follows:

ABAS=CBCP,

where AB refers to the peak area of benzoic acid and AS refers to the peak area sum of the two monomers; CB refers to the concentration of benzoic acid, and CP refers to the total concentration of the two monomers.

Results and discussion

Four-microorganism three-step reaction process

Relevant literature has shown that the growth of bacterial cells can be inhibited in a medium containing acetic acid with concentrations greater than 5 g/L (Lee 2016). Furthermore, the degree of utilization of acetic acid is different for different microbial cells. Directional conversion of starch to acetic acid and then from acetic acid to mcl-PHA was demonstrated in our study. Many studies have shown that both A. niger and Bacillus subtilis have good starch hydrolysis ability (Williamson 1997; Asgher 2007). Therefore, we needed to evaluate the ability of A. niger and B. subtilis 168 to hydrolyze starch. The presence of glucose was not detected in the starch fermentation medium containing B. subtilis 168, but it could be detected in the fermentation medium containing A. niger (Fig. 3). Therefore, A. niger was selected as the fermentation strain for the first process. Additionally, the amount of accumulated glucose increased with a prolonged hydrolysis time. At 48 h of fermentation, the maximum amount of glucose accumulated was 17.66 g/L; thus, this time point was selected as the best starting point for the second fermentation step as glucose was used as the substrate in the second fermentation process.

Fig. 3.

Fig. 3

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Glucose accumulation of A. niger and B. subtilis 168 (no glucose accumulation) using starch as the initial substrate from 0 to 60 h in the first step

The S. cerevisiae L2612–A. orientalis combined fermentation process used in the second step of this study is one of the crucial features of this process. An artificial microbial consortium was constructed using S. cerevisiae L2612 and A. orientalis as chassis microorganism cells and using optimized fermentation conditions for the consortium. Based on metabolic engineering theory, S. cerevisiae L2612 uses glucose to produce ethanol via glycolysis under anaerobic fermentation conditions (Saur 1968; Ge 2012). The NADH associated with other metabolites, such as pyruvate (a metabolic intermediate), causes a dynamic imbalance in the cellular redox system. This usually oxidizes excess NADH in the cell to NAD+ via the glycerol biosynthetic pathway to maintaining the redox balance in the cell. Furthermore, glycerol maintains osmotic pressure balance inside and outside S. cerevisiae L2612 cells. In addition to glycerol, lactic acid is an important by-product of yeast cellular fermentation (Porro 1995; Ishida 2006).

Many microorganism cells can mutually benefit from each other. In this study, S. cerevisiae L2612 was found to use glucose to produce ethanol, and then A. orientalis could convert ethanol to acetic acid. Therefore, we attempted to produce acetic acid by mixed fermentation to shorten the fermentation time and to simplify the process equipment. Analysis of the results of response surface experiments showed that the acetic acid yield reached 44 g/L in the 240 g/L glucose medium. The optimal fermentation conditions were a pH of 5.5, a rotation speed of 150 rpm, and an inoculation ratio of A. orientalis to S. cerevisiae L2612 of 40:1, and the second fermentation step was maintained under 12-h anaerobic and 12-h aerobic conditions.

The use of glycerol as the sole carbon source to produce PHA has been extensively studied. Teeka et al. (2010) used AIK7 as a PHA-producing strain; the PHA content of AIK7 reached 35% dry cell weight in 72 h in waste glycerol and 33% dry cell weight in 120 h in pure glycerol. Recently, mcl-PHA was synthesized from crude glycerol by P. putida (Kenny 2012; Fu 2014). P. putida KT2440 has a tightly regulated metabolic glycerol system. Escapa et al. (2012) adopted a coordinated metabolic approach that combines glycerol catabolism and PHA synthesis to form specific mutations that regulate the global network. A glpR knockout mutant of P. putida KT2440 was created, which increased the production of PHA. In this study, S. cerevisiae L2612 accumulated a certain amount of glycerol and organic acids in the cell under anaerobic fermentation, and it provided a mixed carbon source for mcl-PHA production using P. putida KT2440-acs.

This study also describes the changes of glycerol, lactic acid, the main products acetic acid, and glucose in this four-microbial system. The experimental results showed that when using the starch hydrolysate from the first fermentation process and when the mixed fermentation time reached 48 h, the maximum acetic acid yield was 2.36 g/L. Similarly, the glycerol and lactic acid contents in the fermentation broth reached a maximum at 48 h (Fig. 4).

Fig. 4.

Fig. 4

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Acetic acid, glycerol, and lactic acid accumulation of S. cerevisiae L2612–A. orientalis consortium using glucose as the main substrate from 0 to 60 h in the second step

Analysis of product changes and process optimization

Starch hydrolysis was achieved via continuous fermentation for 48 h. A. niger plays a key role in accumulating glucose in the first step; thus, it is a key strain, and the maximum amounts of glucose accumulated were obtained at 48 h. At this time, the second step of the fermentation process was started, and glucose was continuously consumed as the main carbon source in the substrate. With the continuous decrease of glucose, the accumulation of acetic acid, which is the main products of the primary metabolism, continues to increase. Furthermore, the contents of glycerol and lactic acid, as secondary metabolites, increased.

When the second fermentation process last for 48 h, the contents of acetic acid, glycerol, and lactic acid reached the maximum simultaneously (Fig. 4). At 96 h of whole process, glucose was almost completely consumed; thus, this time point was selected as the starting point for the third step of the fermentation process. The remaining amount of glucose was near 0 g/L at 108 h. Meanwhile, the products acetic acid and glycerol gradually decreased and were completely consumed at 144 h. The product utilization rate reached 100%, and there was no material loss caused by volatilization and extraction (Fig. 5).

Fig. 5.

Fig. 5

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Changes in concentrations of the products in different steps throughout the whole process. The 0-h–48-h period is the first step, when A. niger converted starch to glucose; 48 h–96 h is the second step, when S. cerevisiae L2612–A. orientalis consortium converted glucose to acetic acid, lactic acid, and glycerol; 96 h–144 h is the third step, when mcl-PHA was synthesized by P. putida KT2440-acs

During the microbial fermentation process, the organic acid component measured in the fermentation broth sample only contained the part that the microorganism secreted outside of the cell, and the remaining part was still stored in the cell and could not be released. For example, in S. cerevisiae L2612, the discharge of glycerol is mainly controlled by the channel protein Fps1p embedded in the cell membrane. The channel protein is closed under high osmotic pressure and turned on under low osmotic pressure (Tze-Hsien 2001; Tamás 1999). Additionally, other organic acid components, such as succinic acid, malic acid, and pyruvic acid, in S. cerevisiae L2612 cells have not yet entered the tricarboxylic acid cycle nor have they been consumed and metabolized by S. cerevisiae L2612. Therefore, we chose the cell disruption method to simplify the process and shorten the fermentation time.

We utilized an ultrasonic homogenizer disruption instrument to break the cells in the fermentation broth to use as the experimental group, and unbroken cells were used as a control group. The amounts of glycerol and lactic acid that changed in the experimental group and the control group before and after cell disruption were compared. The glycerol content in the fermentation broth after crushing reached 0.56 g/L, the glycerol content in the unbroken cell fermentation broth reached 0.43 g/L, and the lactic acid content after crushing reached 0.63 g/L; however, the lactic acid content in the unbroken cell fermentation broth was only 0.52 g/L.

The fermentation products in the experimental and control groups in the second step were used as the substrate by P. putida KT2440-acs to perform the third fermentation process, and we measured the OD600 of P. putida KT2440-acs every 6 h. The changes in the contents of each substance in the fermentation products at different time periods were recorded. We found that the number of P. putida KT2440-acs cells increased in the experimental group, which were always higher than in the control group. This result was also consistent with the changes in the substrate of the fermentation broth. During the initial 10 h, the consumption rates of acetic acid in the experimental and control groups were very fast. At 10 h, P. putida KT2440-acs began to grow steadily. From 12 to 42 h, P. putida KT2440-acs entered the logarithmic phase, and the amounts of glycerol, lactic acid, and acetic acid slowly decreased in both groups. They entered the plateau phase when they were fermented to 48 h, and the substrate in the fermentation process was almost consumed (Fig. 6).

Fig. 6.

Fig. 6

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Cell growth of P. putida KT2440-acs measured every 6 h in the third step (a) and concentration changes of acetic acid, glycerol, and lactic acid (b) in the experiment group (cell disruption) and control group (no cell disruption)

We measured the OD600 value of P. putida KT2440-acs in the third step because the first two steps were meant to provide substrates for P. putida KT2440-acs to accumulate PHA. From Fig. 6a and Fig. 7, we know that P. putida KT2440-acs enters the logarithmic growth phase at 12 h after the start of the third step. The mcl-PHA output of P. putida KT2440-acs in the early stage of growth was very low; thus, the accumulation of mcl-PHA was measured at 30 h after the start of the third process. As shown in Fig. 7, as the OD600 of P. putida KT2440-acs gradually increased, the accumulation of PHA also gradually increased and reached a maximum 48 h after the start of the third step. At 144 h, the three products from the second step (acetic acid, glycerol, and lactic acid) were almost completely consumed. Analysis and comparison using gas chromatography showed that mcl-PHA accumulation of P. putida KT2440-acs in the control group at 42 h reached 0.31 g/L; whereas that in the experimental group at 42 h was 0.39 g/L. However, the accumulation amount of mcl-PHA in the control group after fermentation for 48 h reached 0.45 g/L, and that in the experimental group reached 0.5 g/L (Fig. 7). The monomer compositions of mcl-PHA in the experimental and control groups were the same, consisting of 3-hydroxydecanoate (C10) and 3-hydroxyoctanoate (C8). Among the two, 3-hydroxydecanoate accounted for more than 75% of the total mcl-PHA content and was the main component of mcl-PHA. In this study, most glucose from starch hydrolyzing was used for the growth of S. cerevisiae L2612 and A. orientalis, and a small amount was used for acetic acid to produce mcl-PHA. Therefore, the direct carbon source for promoting the accumulation of mcl-PHA in P. putida KT2440-acs was derived from acetic acid. Moreover, a small amount of organic matter, such as glycerol and lactic acid, was produced.

Fig. 7.

Fig. 7

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The monomer compositions of mcl-PHA in the experimental (cell disruption) and control groups (no cell disruption). Slash gray background represents 3-hydroxyoctanoate (C8) in the experimental groups, slash white background represents 3-hydroxyoctanoate (C8) in the control groups, gray background represents 3-hydroxydecanoate (C10) in the experimental groups, white background represents 3-hydroxydecanoate (C10) in the control groups

This four-microorganism three-step fermentation process is the first demonstration of the aerobic process from starch to mcl-PHA. Optimization of A. niger hydrolysis and further optimization of the mixed microbial system have not yet been completed and will be the target of future studies.

Conclusions

In this study, the initial substrate was a starch solution. By comparing the hydrolysis capabilities of A. niger and B. subtilis 168, A. niger was selected as the crucial strain for the first hydrolysis process. Because A. orientalis cannot directly use glucose as a substrate to produce acetic acid, S. cerevisiae L2612 was added to construct a mixed bacteria fermentation system. The production of acetic acid by Acetobacter sp. is currently the best method for obtaining large amounts of acetic acid. The fermentation method of A. orientalis and S. cerevisiae L2612 considerably shortened the process time. The continuous fermentation of a single strain to produce mcl-PHA required 96 h; whereas, the mixed microbial system shortened the fermentation time to 48 h.

We optimized the fermentation system of the S. cerevisiae L2612 and A. orientalis mixed microorganism system. Finally, we obtained 2.36 g/L of acetic acid. S. cerevisiae L2612 inevitably produces glycerol, lactic acid, and other organic acids during the production of ethanol from glucose. These by-products can also be used by P. putida KT2440-acs to synthesize mcl-PHA. To optimize this reaction process further, we chose to crush S. cerevisiae L2612 and A. orientalis using an ultrasonic cell disruption method. The organic matter in the cells were released and then could be used by P. putida KT2440-acs. The results showed that after 48 h of fermentation, the optimized mcl-PHA accumulation improved by 0.05 g/L compared with that before optimization. Finally, 0.5 g/L of PHA was obtained. We did not measure the change of starch content because it is difficult to determine after its hydrolyzing in in the process.

These three steps can be separated. In this study, we focused mainly on the more efficient use of starch so that the ability of each strain could be maximized under optimal conditions. This fermentation process can be separated if needed, and individual steps can be added in other production processes depending on the substrate (glucose or acetic acid). Because starch is the main ingredient of kitchen wastes, the design and implementation of the fermentation process could be beneficial for advances and future research in managing kitchen waste.

We know that starch is hydrolyzed to amylase first, and then to glucose by maltase. Maltase is a key enzyme that converts maltose into glucose. To reduce the accumulation of intracellular maltose, we can accelerate the conversion of maltose to glucose by overexpression of maltase genes.

Currently, we have only completed preliminary optimization of this process. A few ways to further enhance the potential application ability of this process were summarized here:

  1. Further strengthening the metabolic communication between S. cerevisiae L2612 and A. orientalis, reducing the outflow of by-products and increase the inflow of main products by means of genetic modification.

  2. Blocking some unessential carbon flow metabolism to increase the production rate of acetic acid in the second step.

  3. Designing new bioreactors adopting fed-batch or continuous feeding strategy.

Acknowledgements

The authors wish to acknowledge the financial support provided by the National Key Research and Development Program of China (Project No. 2018YFA0902100), the National Natural Science Foundation of China (No. 21576197), and Tianjin Research Program of Application Foundation and Advanced Technology (No. 18JCYBJC23500).

Author contributions

XY drafted and edited the manuscript. XY, SL collected the background information, SL implemented the experiment. All authors read and approved the final manuscript.

Compliance with ethical standard

Conflict of interest

The authors declare that they have no conflict of interest in the publication.

Footnotes

Xiaohui Yang and Suhang Li contributed equally to this work and should be considered co-first authors.

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