Immobilization of trophic anaerobic acetogen for semi-continuous syngas fermentation

Lijuan Zhang,Peng Hu,Jiang Pan,*,Huilei Yu,*,Jianhe Xu

1 State Key Laboratory of Bioreactor Engineering,Shanghai Collaborative Innovation Centre for Biomanufacturing,East China University of Science and Technology,Shanghai 200237,China

2 Shanghai GTL Biotech Co.Ltd.,Shanghai 200438,China

Keywords:Anaerobic Syngas Acetic acid Immobilization Bioreactors Semi-continuous fermentation

ABSTRACT The massive consumption of fossil energy forces people to find new sources of energy.Syngas fermentation has become a hot research field as its high potential in renewable energy production and sustainable development.In this study,trophic anaerobic acetogen Morella thermoacetica was successfully immobilized by calcium alginate embedding method.The ability of the immobilized cells on production of acetic acid through syngas fermentation was compared in both airlift and bubble column bioreactors.The bubble column bioreactor was selected as the better type of bioreactor.The production of acetic acid reached 32.3 g·L?1 in bubble column bioreactor with a space–time yield of 2.13 g·L?1·d?1.The immobilized acetogen could be efficiently reused without significant lag period,even if exposed to air for a short time.A semi-continuous syngas fermentation was performed using immobilized cells,with an average space–time acetic acid yield of 3.20 g·L?1·d?1.After 30 days of fermentation,no significant decrease of the acetic acid production rate was observed.

1.Introduction

The diminishing of fossil energy and the environmental problems caused by greenhouse gas emissions lead to a sharp increase in demand for renewable and clean energy[1].Syngas fermentation,which utilizes anaerobic acetogen to convert syngas into various useful biofuels and biochemical products under mild conditions[2],without strict requirement for a fixed ratio of H2to CO,and less sensitive to syngas contaminants such as sulfur and chlorine[3–5].Therefore,syngas fermentation has become a hot research field in clean energy production and waste utilization [2].Many companies,including NEOS Bio,Coskata and LanzaTech have been trying to commercialize syngas fermentation technology.Among them,LanzaTech and its partner Shougang,a leading steel mill of China,have announced the successful start-up of the world's first commercial facility converting industrial waste gas into 46,000 tons of ethanol per year[6].

In spite of increased interest,syngas fermentation is still in the exploratory stage and faces various challenges to realize commercialization.One bottleneck is the low transformation efficiency of syngas due to the mass transfer limitation of sparingly soluble gaseous substrates especially CO and H2in aqueous phase[7].Many studies tried to increase the gas mass transfer via improving the bioreactor design and operation [8–13].In continuous stirred bioreactors,the most common approach is to accelerate the agitation speed to increase the gas–liquid interfacial area and then improve the mass transfer between gas and aqueous phases[7].However,the high energy consumption of these systems greatly reduces its economic feasibility in industrial scale application of syngas fermentation.Compared with stirred bioreactor,the airlift and bubble column bioreactors are widely used in syngas fermentation due to simple structure,low energy consumption and good gas mass transfer efficiency.The airlift bioreactor promotes gas circulation through the draft tube,which increases the residence time of syngas in the bioreactor,while the bubble column bioreactor reduces the diameter of bubbles through the air distributor,increasing the gas–liquid interfacial area [2].Airlift bioreactor combined with a 20-μm bubble distributor will be the ideal bioreactor configuration for efficient mass transfer of syngas[2].In a bubble column bioreactor,the effect of different gas flow rates on syngas fermentation was studied,when gas composition was 70%CO,30%CO2and the gas flow rate increased from 0.2 to 1 air volume/culture volume/min(vvm),the maximum optical density(OD)increased from 4.3 to 11.3,and the space–time yield of acetic acid increased from 0.30 g·L?1·h?1to 0.55 g·L?1·h?1[14].

Furthermore,serious product inhibition by the high concentration of end product,is also one of the reasons that limit the productivity of syngas fermentation.Immobilization of cells can not only increase the cell density and facilitate the cell reuse,but also reduce the sensitivity of cells to environmental change and product inhibition [15–18].Steger et al.immobilized Acetobacterium woodii on the linen membrane to prevent them being washed out from the bioreactor,in which using immobilized cell,acetic acid was produced from H2and CO2at high productivity of 1.21 g·L?1·d?1[19].Cells immobilized by granular activated carbon (GAC) adsorption and polyvinyl alcohol (PVA) embedment were also applied for syngas fermentation.The production of acetic acid by GAC immobilized Clostridium ljungdahlii reached 28 mmol·L?1·d?1at a gas flow rate of 300 ml·min?1in anaerobic fluidized membrane bioreactor(AFMBR)[20].

Acetic acid,the main product in syngas fermentation by Morella thermoacetica(M.theramoacetica),can be further used as the substrate for Yarrowia lipolytica to synthesize lipids[21].However,syngas fermentation was a rate-limiting step in this cascaded process,and the yield of acetic acid was relatively low.Besides,a hollow fiber membrane device was required to avoid cells from the first syngas fermentation bioreactor into the second lipid synthesis bioreactor,which made additional steps and costs.In this study,the M.theramoacetica was successfully immobilized by cells embedding technology and applied in syngas fermentation for the first time,which not only increased the reusability of anaerobic bacteria,but also simplified the process of cell-product separation.Furthermore,the feasibility of immobilized cells was verified in airlift bioreactors for semi-continuous syngas fermentation.

2.Materials and Methods

2.1.Bacterium and cultivation medium

The anaerobic acetogenic bacterium M.theramoacetica DSM 6867 used in this study was purchased from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH.The culture medium(per liter)contained 1.4 g KH2PO4,1.4 g K2HPO4?3H2O,2 g(NH4)2SO4,0.5 g MgSO4?7H2O,5 g yeast extract,10 g 2-morpholinoethanesulfonic acid,0.3 g cysteine,and 20 mL trace elements solution (2.0 g·L?1Nitrilotriacetic acid,1.0 g·L?1MnSO4·H2O,0.8 g·L?1Fe(SO4)2(NH4)2·6H2O,0.2 g·L?1CoCl2·6H2O,0.002 g·L?1ZnSO4·7H2O,0.02 g·L?1CuCl2·2H2O,0.02 g·L?1NiCl2·6H2O,0.02 g·L?1Na2MoO4,0.02 g·L?1Na2SeO4,0.02 g·L?1Na2WO4).In addition,0.5 mL per liter of 0.2%resazurin,a redox potential indicator,was added to indicate the presence or absence of oxygen.After the reagent dissolves,the medium pH was adjusted to 6.0 or 6.8 with KOH.

2.2.Free cells culture in anaerobic bottles

The medium was divided into 25 mL for each 100-mL anaerobic bottle in the anaerobic box,and it was sterilized at 121°C for 20 min after sealing.When the medium was cooled to room temperature,the gas in the anaerobic bottle was replaced with syngas.Single colony of M.thermoacetica on plate was activated in 4 mL anaerobic medium in test tube,then inoculated into the anaerobic bottle and cultured with the syngas till the OD reached about 1.0.The culture was inoculated into fresh medium with 10%(v/v)inoculation for further culture.The acetogen was cultured at 60°C and 200 r·min?1.The syngas was replaced and the culture broth was sampled every 24 h.

2.3.Preparation of immobilized cells

The carriers tested by adsorption method include granular activated carbon,diatomite,molecular sieve 5A(φ 3–5 mm),and silica.The carriers were washed with deionized water,soaked overnight,and then baked to constant weight.The carriers were put into anaerobic bottles,and placed in the anaerobic box for 1–2 days to remove the residual air inside the anaerobic bottle and carriers.Then,the culture medium was packed and sterilized according to the method described above,and the inoculation volume and culture method was the same as that for the free cells.

Calcium alginate embedding method was also used to immobilize cells.When the OD of the free cells reached 1.0,they were centrifuged at 4 °C,12000 r·min?1for 5 min,resuspended with 10% molecular sieve powder in medium,and then the suspension mixed with sodium alginate gel,with a final concentration of cells,sodium alginate and molecular sieve powder at 0.6 g cell·L?1,3% (w/w) and 3% (w/w),respectively.Subsequently,the mixed bacterial suspension was added drop by drop to 4%(w/w)CaCl2solution,and the resulting calcium alginate gel particles were solidified for 24 h at 60°C.The preparation of gel particles was completed in anaerobic box.When the gel particles were prepared,they were washed with sterile deionized water for 3 times and then added to the culture medium.The culture method was the same as that for free cells.

2.4.Physical properties of the calcium alginate gel particles

When determining the mechanical strength of calcium alginate gel particles,100 randomly sized gel particles with uniform size and regular shape were placed in a shake bottle and added to 10 mL medium,and shaken for 100 min at 200 r·min?1and 60°C.The mechanical strength was represented by the breakage rate of gel particles,i.e.,the higher breakage rate of gel particles,the worse the mechanical strength.

When measuring the mass transfer performance of gel particles,30 randomly sized gel particles of uniform size and regular shape were placed in 10 mL of 50 times diluted red ink,and samples were taken to observe the penetration of red ink into the gel particles every 30 s.The shorter permeation time of gel particles in the red ink indicates a faster mass transfer rate of them.

2.5.Syngas fermentation in bioreactor

Syngas fermentations were performed in an airlift bioreactor and a bubble column bioreactor,respectively.The airlift reactor has a diameter and height of φ 6.5 cm×65 cm with a working volume of 1.5 L,containing an inner draft tube of φ 3.8 cm × 40 cm.The bubble column bioreactor has a diameter and height of φ 8 cm×45 cm with a working volume of 1.0 L,and a 20-μm bubble distributor was fixed at the bottom of the bioreactor.

The bioreactor was sterilized overnight with 0.5 mol·L?1NaOH before use,washed with deionized water until neutral,wiped with 75%ethanol inside and outside of the bioreactor,and then sterilized by ultraviolet radiation for 20 min.The culture medium was sterilized at 121°C for 20 min and then transferred to the bioreactor.Before inoculation,the medium was purged with syngas for 2 h to remove the residual oxygen.The free cells were inoculated in 10%(v/v).When the immobilized cells were cultured in bioreactor,the prepared gel particles were put into the bioreactor,added with the medium,and then purged with syngas.The gas was composed of 40% CO,30% CO2and 30% H2and the gas flow rate of syngas was set at 0.1 vvm or 0.2 vvm during the syngas fermentation.The operation temperature was maintained at 60°C through an external heating belt.The pH of the culture broth was maintained at 6.0 by feeding 3 mol·L?1NaOH during the fermentation.

2.6.Analytical methods

Cell density of free cells was measured by a spectrophotometer(Shimadzu UV1800,Japan)with reading at 600 nm,when the absorbance value was greater than 0.6,the sample was diluted to a certain multiple before measurement.

The concentration of acetic acid produced was analyzed using an HPLC(Agilent 1200,USA)equipped with an Aminex HPX-87H column(Bio-Rad,USA)and a refraction index detector.Before detection,the sample was centrifuged at 12000 r·min?1for 10 min,and the supernatant was taken and filtered with 0.22 μm filter membrane.When the sample was from the immobilized cells embedded in calcium alginate,excessiveshould be added to remove the free Ca2+in the culture broth to prevent the formation of calcium sulfate precipitation in the mobile phase.The sample was centrifuged at 12000 r·min?1for 5 min,and 800 μL supernatant was mixed with 20 μL 5 mmol·L?1H2SO4,centrifuged and filtrated after standing overnight.The eluent used was 5 mmol·L?1sulfuric acid at a flow rate of 0.6 ml·min?1and the column temperature was kept constant at 35°C.The retention time of acetic acid was about 15.4 min.

3.Results and Discussions

3.1.Acetic acid production by carrier adsorbed cells

Adsorption is a kind of important method for cells immobilization.Granular activated carbon,diatomite,molecular sieve and silica gel were selected as carriers for adsorption immobilization of M.theramoacetica,and the production of acetic acid by the free or immobilized cells was compared.When cells were adsorbed by activated carbon,diatomite or silica-gel,the acetic acid production was nearly the same as that produced by free cells.The titer of acetic acid increased fast in the 4th day of culture,reaching a plateau with 4.5 g·L?1at maximum.While in the culture of cells adsorbed on the molecular sieve,the titer of acetic acid increased continuously,reaching 9.9 g·L?1after 10 days culture(Supplementary Fig.S1).The pH of the culture broth was measured.It was observed that pH of the culture medium added with molecular sieves was always higher than that added with other carriers during the culture process.When the production of acetic acid reached a plateau,the pH of fermentation broths with other carriers has decreased to 5.0,while the lowest pH of fermentation broth containing molecular sieves was maintained at pH 5.5(Supplementary Fig.S2).Hence,it was speculated that the molecular sieve powder may maintain the pH of culture broth within a suitable range for the healthy growth of acetogen,thereby promoting the better production of acetic acid.This hypothesis was verified by the result that the acetic acid titers of free and adsorbed cells were all increased continuously in a consistent trend,with the maximum acetic acid production of approximately 25 g·L?1,which was achieved through maintaining the pH of the broth at 6.0 by addition of NaOH solution(Supplementary Fig.S3).Although the molecular sieve immobilized cells afford the better acetic acid production,the particles of molecular sieve were easily broken into powders during the culture process,which was caused probably by the acetic acid dissolution and mechanical collision,making the cell immobilization method unsuitable for large-scale application.

In previous studies,acetogen have successfully immobilized on the linen or granular activated carbon for syngas fermentation [19,20].However,in this study,the adsorption between these carriers and M.theramoacetica was unsuccessful since these carriers were not suitable for the immobilization of M.theramoacetica,or the higher culture temperature was not conducive to adsorption.

3.2.Optimization of cell immobilization with alginate gel entrapment

For the adsorption method,cells are physically adsorbed or attached to the material,with the advantages of simple operation,low cost,high cell activity and low mass transfer limitation.However,the cells are easy to fall off from the carrier material since the adsorption interaction between cells and carriers is too weak.In the embedding method,cells are trapped in the carrier,allowing the substrate and products to enter and leave.Calcium alginate embedding is one of the most commonly used immobilization methods.Alginate gel is a very effective natural polymer and nontoxic to microorganism which is available with low cost.For the cell immobilization with alginate embedding method,3%(w/w)sodium alginate and 4%(w/w)CaCl2solution were initially chosen to the prepare gel particles.The resultant gel particles were solidified for 12 h at 4°C.Using the immobilized cells,the highest production of acetic acid reached 3.1 g·L?1.Afterwards,the concentrations of sodium alginate and CaCl2,loading of free acetogen cells and embedding temperature were optimized.When the sodium alginate concentration exceeded 4%(w/w),the gel particles were irregular and tailed.With the increase of sodium alginate concentration from 2%to 4%,the mechanical strength of gel particles increased gradually,with the breakage rate decreased from 100%to 30%.The concentration of sodium alginate also affected the mass transfer of syngas and production of acetic acid.When the immobilized cells were cultured on the 4th day,the acetic acid yield by the immobilized cells with 4%sodium alginate concentration was only 0.8 g·L?1,while with the immobilized cells prepared with 2%and 3%(w/w)sodium alginate concentrations,the acetic acid yields reached 1.2 g·L?1(Fig.1a).In the later stage of shaking bottle culture,the production of acetic acid of the three immobilized cells was about 4.2 g·L?1,due to pH limit.Considering the mass transfer,mechanical strength and productivity of acetic acid,3%(w/w)sodium alginate concentration was selected for the cell entrapment and then the other parameters were further optimized.

When CaCl2concentration was increased from 2%to 6%(w/w),the breakage rate decreased from 100%to 0,indicating that increasing of CaCl2concentration could enhance the mechanical strength of gel particles.When the CaCl2concentration exceeded 4% (w/w),the highest yield of acetic acid reached 4.3 g·L?1,while in the case of 2%(w/w)CaCl2,the highest acetic acid yield was 2.7 g·L?1(Fig.1b).Highconcentration CaCl2cross-linked gel particles had a denser internal structure and higher mechanical strength,which can reduce the influence on anaerobic bacteria by the surrounding environment.But the shape of gel particles was elliptical when the CaCl2concentration was 6%.Considering the mechanical strength,acetic acid production and cost of the gel particles,4%CaCl2concentration was chosen for further optimization.

Effect of free cell loading on the activity of immobilized acetogen bacterium was examined.Different cell dosages,including 0.3 g cell·L?1,0.6 g cell·L?1,and 0.9 g cell·L?1,were used to prepare the immobilized cell particles.When the cell loading was 0.3 g cell·L?1,the acetic acid did not increase substantially on the 4th day in the process of immobilized cell culture(Fig.1c).It can be seen that there was a certain positive correlation between the activity of acetogen and the embedded cell amount,but when the embedded cell amount of acetogen exceeded 0.6 g·L?1,there was little effect on production of acetic acid by the loading of acetogen cells due to the severe pH restriction.

The solidified temperature of alginate gel particles with Ca2+also affected its mechanical strength and the activity of entrapped cells.The breakage rates of gel particles prepared at 4°C,25°C and 60°C were 100%,80%and 10%respectively,indicating that the mechanical strength of gel particles increased with the increase of embedding temperature.The yield of acetic acid increased significantly until the 6th day,and the yields of acetic acid all reached over 3.0 g·L?1(Fig.1d).Although the activity of acetogen cells was the highest when the gel solidified temperature was 25 °C,the gel particles were finally solidified with Ca2+ions at 60°C considering the effect of temperature on the mechanical strength of gel particles.

In the culture process,the phosphate in the medium can easily erode the calcium alginate gel,which greatly reduced the stability and mechanical strength of immobilized particles,and not conducive to the subsequent application in bioreactors.Different concentrations of glutaraldehyde(0.0%,0.05%,0.1%,0.2%and 0.5%)were selected to further crosslink the gel particles.The gel particles crosslinked without glutaraldehyde were completely saturated with red ink in 2 min,while the gel particles crosslinked with glutaraldehyde could be completely saturated in 5 min,indicating that the surface or inside of gel particles crosslinked with glutaraldehyde formed more compact structure.The breakage rate of glutaraldehyde crosslinked gel particles was 0 in the medium containing 1.0 g·L?1phosphate after shaking 24 h at 60 °C and 200 r·min?1,indicating that glutaraldehyde crosslinking could improve the mechanical strength of gel particles.However,it was found that the acetic acid production of glutaraldehyde crosslinked immobilized particles was very low on 4th day,and even the concentration of glutaraldehyde was reduced to as low as 0.05%,the production of acetic acid was much still lower than that of the uncrosslinked immobilized cells(Supplementary Fig.S4).This might be due to the toxic effect of glutaraldehyde on the acetogen cells.Therefore,this method was not suitable for the modification of immobilized cells of the acetogen.

In addition to the cross-linking method,the addition of activated carbon powder,silica,and other materials with high surface porosity to the calcium alginate gel can also enhance the stability of the gel particles.In this study,molecular sieve powder was added to alginate gel to enhance its mechanical strength.There was little difference between the mass transfer of composite gel particles added with molecular sieve powder and without addition.After shaking 24 h,the breakage rate of gel particles was only 2%with 2%(w/w)molecular sieve powder addition,and when the molecular sieve powder addition was more than 3%(w/w),the breakage rate decreased to 0,which showed that the addition of molecular sieve powder greatly enhanced the resistance of the gel particles to phosphate and mechanical collision.When the addition amount of molecular sieve powder was at 3%–5%(w/w),the acetic acid production did not change significantly,all above 4.5 g·L?1(Fig.1e).

Fig.2.Time courses of acetic acid production by immobilized cells in airlift bioreactor or bubble column bioreactor,under different gas flow rates in batch fermentation.(The immobilized acetogen was cultured in airlift and bubble bioreactors at gas flow rate of 0.1 vvm (Phase I) and 0.2 vvm (Phase II).The gas was composed of 40% CO,30% CO2 and 30% H2 ,the culture temperature and pH was maintained at 60 °C and 6.0,respectively.In phase I,when the acetic acid production no longer increased,the gas flow was changed to 0.2 vvm and the immobilized cells were reused.)

Furthermore,the concentration of phosphate in the medium was optimized to further enhance the mechanical strength of the gel particles.The proper reduction of the phosphate content in the medium could enhance the mechanical strength of the gel particles,and did not affect the metabolism of acetogen.When the phosphate content was 1.0 g·L?1and 1.4 g·L?1,the breakage rate of gel particles without molecular sieve powder reached 57%and 70%,respectively.While the content of phosphate was decreased to less than 0.5 g·L?1,the breakage rate of the gel particle decreased to 0.When the phosphate content was 0 g·L?1and 0.2 g·L?1,the acetic acid production increased significantly,giving a final acetic acid production of 4.4 g·L?1.While the phosphate content was increased to higher than 0.5 g·L?1,the production of acetic acid decreased(Fig.1f).When the gel particles were less damaged,the lowest pH that the immobilized cells could tolerate was 4.5,and when the structure of gel particles was destroyed,the minimum pH became 4.7,indicating that the intact gel particles had a certain protective effect on the acetogen.The higher phosphate content,the higher erosion degree of gel particles,and the metabolism of acetogen is more susceptible to the product inhibition.In addition,it was found that when the phosphate concentration was as low as 0 g·L?1,the acetic acid production of free cells also decreased.Finally,0.2 g·L?1phosphate in the medium was used for further investigation.

Previous study has used PVA to immobilized Clostridium ljungdahlii,the production of both acetate and ethanol was lower because of relatively poor mass transfer efficiency[20].In this study,the gel particles crosslinked with glutaraldehyde was applied to increase mechanical strength,however,mass transfer efficiency and cell activity greatly reduced.The calcium alginate gel provides support for the growth and reproduction of M.theramoacetica and maintains cell viability.In the process of culture,pH tolerated by calcium alginate immobilized cells was lower than free cells,indicating that calcium alginate gel had a certain protective effect on the acetogen.

3.3.Bioreactor selection for syngas fermentation with immobilized acetogen cells

Syngas fermentations by calcium alginate immobilized cells were performed in airlift and bubble column bioreactors,respectively.At first,the cultures were conducted at a gas flow rate of 0.1 vvm(vvm means air volume/culture volume/min).The production of acetic acid reached 22.0 g·L?1in the bubble column bioreactor when the acetic acid increased no longer,with a space–time yield of 1.46 g·L?1·d?1,while in the airlift bioreactor,the acetic acid was 11.9 g·L?1with a space–time yield of 1.08 g·L?1·d?1,significantly lower than that in bubble column bioreactor(Fig.2).

Fig.4.The time courses of acetic acid production and space–time yield during semicontinuous syngas fermentation with immobilized cells at 0.2 vvm gas flow rate in the bubble column bioreactor.(The immobilized acetogen was cultured in bubble bioreactors at 0.2 vvm gas flow rate.The gas was composed of 40%CO,30%CO2 and 30%H2 ,the culture temperature and pH was maintained at 60°C and 6.0,respectively.After the sixth day,300 ml fermentation broth was discharged,and the same volume fresh medium was added every day.)

When the acetic acid no longer increased,the fermentation broth was discharged,the immobilized particles retained in bioreactor were cleaned three times with deionized water.Then,the fresh medium was added for the second batch culture at a gas flow rate of 0.2 vvm.In the second batch of culture,the highest yield of acetic acid reached 32.3 g·L?1in bubble column bioreactor,with a space–time yield of 2.13 g·L?1·d?1,which was significantly higher than that in the first batch of culture.In addition to the increase in mass transfer due to the increase of gas flow rate,it was also due to the increase of cell density.In the airlift bioreactor,the highest yield of acetic acid was 18.8 g·L?1,with a space–time yield of 1.24 g·L?1·d?1(Fig.2).From these results,one can conclude that bubble column bioreactor was better than airlift bioreactor in the syngas fermentation using calcium alginate immobilized cells,which might be due to the improved mass transfer through decreasing the size of bubbles by air distributor,thus effectively increasing the gas–liquid interfacial area.

When the gas flow rate was increased from 0.1 vvm to 0.2 vvm in syngas fermentation,the mass transfer of CO,H2and CO2could be improved.Previous studies have shown that in syngas fermentation,gas–liquid mass transfer,especially CO mass transfer,is the most restricted factor.The acetic acid production is positively correlated with the mass transfer rate of CO[14].However,when the CO partial pressure and gas flow reached a certain value,the cell density was too low to consume CO,and excessive CO was toxic to cells,the acetic acid production would not increase.

Fig.3.The schematic diagram of semi-continuous fermentation in the bubble column bioreactor.

In the second run of culture,no significant lag period was observed on the immobilized cells reused,no matter in airlift or bubble column bioreactor,even if they were exposed to air for a short time,indicating that cells immobilization can effectively retain cells,shorten the lag phase,maintain cell viability,and reduce the sensitivity of M.thermoacetica to oxygen.

3.4.Semi-continuous syngas fermentation for acetic acid production

To further improve the acetic acid production,a semi-continuous fermentation was performed by using the immobilization cells in the bubble column bioreactor,as shown in Fig.3.The production of acetic acid increased fast in the first 5 days,with a space–time yield of 3.6 g·L?1·d?1acetic acid on the fifth day.While in the sixth day,the space–time yield decreased to 1.2 g·L?1·d?1(Fig.4).From then,300 mL fermentation broth was discharged from the bioreactor directly,and fresh medium of the same volume was uploaded every day.In the phase of semi-continuous fermentation,the average space–time yield of acetic acid was 3.20 g·L?1·d?1(Fig.4),which was the highest result in syngas fermentation by immobilized cells so far.The rate of acetic acid production was maintained during 30 days of semi-continuous fermentation,with a total acetic acid production of 96.1 g·L?1.

4.Conclusions

In this study,calcium alginate embedding immobilization method was selected and optimized for anaerobic syngas fermentation.The abilities of immobilized cells to produce acetic acid were compared in airlift bioreactor and bubble column bioreactor,and the performance in the bubble column bioreactor was superior to that in airlift bioreactor.The cell immobilization not only increases the reusability of the acetogen,but also simplifies the separation process of cells and products and at the same time facilitates the subsequent treatment process.A semicontinuous syngas fermentation with immobilization cells was performed for reuse of the acetogen cells,which showed many advantages and could be further optimized in pilot scales to re-evaluate its industrial scale feasibility.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the National Key Research and Development Program of China(2019YFA0905000),the National Natural Science Foundation of China (21536004,21922804,21776085,and 21871085)and the Fundamental Research Funds for the Central Universities(22221818014).

Supplementary Material

Supplementary data to this article can be found online at https://doi.org/10.1016/j.cjche.2020.07.041.