Microbial production of a biofuel (acetone–butanol–ethanol) in a continuous bioreactor: impact of bleed and simultaneous product removal

Acetone butanol ethanol (ABE) was produced in an integrated continuous one-stage fermentation and gas stripping product recovery system using Clostridium beijerinckii BA101 and fermentation gases (CO₂ and H₂). In this system, the bioreactor was fed with a concentrated sugar solution (250–500 g L^?1 glucose). The bioreactor was bled semi-continuously to avoid accumulation of inhibitory chemicals and products. The continuous system was operated for 504 h (21 days) after which the fermentation was intentionally terminated. The bioreactor produced 461.3 g ABE from 1,125.0 g total sugar in 1 L culture volume as compared to a control batch process in which 18.4 g ABE was produced from 47.3 g sugar. These results demonstrate that ABE fermentation can be operated in an integrated continuous one-stage fermentation and product recovery system for a long period of time, if butanol and other microbial metabolites in the bioreactor are kept below threshold of toxicity.     

Saccharomyces cerevisiae and Oenococcus oeni immobilized in different layers of a cellulose/starch gel composite for simultaneous alcoholic and malolactic wine fermentations

The production of a two-layer composite biocatalyst for immobilization of two different microorganisms for simultaneous alcoholic and malolactic fermentation (MLF) of wine in the same bioreactor is reported. The biocatalyst consisted of a tubular delignified cellulosic material (DCM) with entrapped Oenococcus oeni cells, covered with starch gel containing the alcohol resistant and cryotolerant strain Saccharomyces cerevisiae AXAZ-1. The biocatalyst was found effective for simultaneous low temperature alcoholic fermentation resulting to conversion of malic acid to lactic acid in 5 days at 10 °C. Improvement of wine quality compared with wine fermented with S. cerevisiae AXAZ-1 immobilized on DCM was attributed to MLF as well as to increased ester formation and lower higher alcohols produced at low fermentation temperatures (10 °C) as shown by GC and headspace SPME GC/MS analysis. Scanning electron microscopy showed that the preparation of a three-layer composite biocatalyst is also possible. The significance of such composite biocatalysts is the feasibility of two or three bioprocesses in the same bioreactor, thus reducing production cost in the food industry     

Production of ethanol in repeated-batch fermentation with membrane-type bioreactor

The average ethanol content in sake is 14 wt%; continuous production of such a high ethanol content was found not to be stably maintained in a packed-bed bioreactor with immobilized yeast cells, used normally for production of an ethanol content of up to 10 wt%. However, use of repeated-batch ethanol fermentation incorporating a membrane filter for product separation enabled a high ethanol content and improved productivity to be achieved. In this bioreactor, the yeast cells were retained within the bioreactor and a high yeast concentration was possible. A filtrate containing 14 wt% ethanol was obtained steadily after each batchwise operation. At a yeast concentration of 110 g/l, an ethanol productivity of 3.5 g/l/h was attained, which is 9 times higher than that in conventional batch fermentation. A mathematical model is proposed for assessment of the repeated-batch fermentation process. The estimated results agreed well with the observed ones. With a view to the application of this system to sake production, the aroma components of the filtrate were assayed and compared with those of a commercial-grade sake.     

Pilot-scale solid-state ethanol fermentation by inert gas circulation using moderately thermophilic yeast

A pilot-scale solid-ethanol fermentation system was experimented on, using corn grits. The ethanol produced was simultaneously stripped in the fermentor by circulating CO₂ gas. Using a moderately thermophilic yeast (Saccharomyces cerevisiae 1031R) at a fermentation temeperature of 40°C, the stripping efficiency was improved and running time shortened. In this case, 467 g-ethanol/kg-initial dry mass was produced during 15 d of running. Fermentation efficiency was 87%, which is twice as much as conventional solid-state ethanol fermentation. The total recovery of ethanol produced was 96%, and the average ethanol concentration in the condensate was 223 g/l.     

Ethanol production by whole cell immobilization using lignocellulosic materials as solid matrix

Amongst four carriers used, rice-straw was found to be superior in terms of ethanol production. The maximum productivity (17.84 gl⁻¹ h⁻¹) corresponded to a dilution rate of 0.39 h⁻¹, the ethanol concentration being 45.80 gl⁻¹. A multistage rhomboidal bioreactor was found to partially overcome the disruption effect caused by the generation of a large volume of carbon dioxide in the column. Increases in productivity of about 12.55% and 3.6%, respectively, were achieved using rhomboidal and tapered bioreactors as compared to the cylindrical bioreactor. It was observed that the generation time of cells, in both the immobilized and free states, was around 2.5 h. The ethanol yield (Y_p/s) in the lower part of the reactor was less in comparison with other zones, where the substrate utilization efficiency was relatively higher.     

Parameter oscillation attenuation and mechanism exploration for continuous VHG ethanol fermentation

A bioreactor system composed of a stirred tank and three tubular bioreactors in series was established, and continuous ethanol fermentation was carried out using a general Saccharomyces cerevisiae strain and a very high gravity medium containing 280 g L(-1) glucose, supplemented with 5 g L(-1) yeast extract and 3 g L(-1) peptone. Sustainable oscillations of glucose, ethanol, and biomass were observed when the tank was operated at the dilution rate of 0.027 h(-1), which significantly affected ethanol fermentation performance of the system. After the tubular bioreactors were packed with 1/2' Intalox ceramic saddles, the oscillations were attenuated and quasi-steady states were achieved. Residence time distributions were studied for the packed bioreactors by the step input response technique using xylose as a tracer, which was added into the medium at a concentration of 20 g L(-1), indicating that the backmixing alleviation assumed for the packed tubular bioreactors could not be established, and its contribution to the oscillation attenuation could not be verified. Furthermore, the role of the packing's yeast cell immobilization in the oscillation attenuation was investigated by packing the tubular bioreactors with packings with significant difference in yeast cell immobilization effects, and the experimental results revealed that only the Intalox ceramic saddles and wood chips with moderate yeast cell immobilization effects could attenuate the oscillations, and correspondingly, improved the ethanol fermentation performance of the system, while the porous polyurethane particles with good yeast cell immobilization effect could not. And the viability analysis for the immobilized yeast cells illustrated that the extremely lower yeast cell viability within the tubular bioreactors packed with the porous polyurethane particles could be the reason for their inefficiency, while the yeast cells loosely immobilized onto the surfaces of the Intalox ceramic saddles and wood chips could be renewed during the fermentation, guaranteeing their viability and making them more efficient in attenuating the oscillations. The packing Raschig rings without yeast cell immobilization effect did not affect the oscillatory behavior of the tubular bioreactors, further supporting the role of the yeast cell immobilization in the oscillation attenuation.     

Ethanol from Sugar Cane: Flask Experiments Using the EX-FERM Technique

Alcohol production at the laboratory scale from sugar cane pieces by the EX-FERM technique was studied with 37 strains of Saccharomyces spp. The EX-FERM process is novel in that it employs the simultaneous extraction and fermentation of the sucrose in a cane-water suspension. Two types of cane treatments were used: chips and shredded pith, either fresh or dried. A mother culture of the yeast was prepared in enriched cane juice and then added to the cane-water mixture. After static fermentation for 40 h at 30°C, the cane was removed, and fresh cane was added to the yeast-alcohol broth. After an additional 24 h, the cane was again removed and the liquor was analyzed. After the first 40-h cycle, sugar consumption was above 99% with 10 of the 37 yeast strains tested, and ethanol reached levels of 1.29 to 4.00 g per 100 ml, depending on the yeast strain. The final ethanol concentration reached 4.27 to 5.37 g per 100 ml, and sugar consumption was above 98% in three cases during a second EX-FERM cycle employing previously air-dried chips and pith. Product yields were within accepted values. Cane treatment did not appear to affect the results at this level.     

Hollow fibre bioreactor for ethanol production: Application to the conversion of lactose by Kluyveromyces fragilis

Kluyveromyces fragilis cells have been packed into the shell side of an industrial size hollow fibre module. The feed was pumped through the tube side under pressure. During continuous, single-pass operation with a synthetic lactose medium containing 50 g l^?1lactose, ethanol productivity was 30–60 g l^?1h^?1at dilution rates of 1–4 h^?1. With 150 g l^?1lactose concentration, the productivity was 100–135 g l^?1h^?1. Productivity was generally lower when cottage cheese whey permeate (45 g l^?1lactose) was used as the feed. Long-term stability of the hollow fibre bioreactor was good, provided adequate care was taken to bleed the gas generated and restrict cell concentration in the shell side.     

串联填充管式反应系统中高浓度乙醇连续发酵

在一套由搅拌罐和管式反应器串联而成的组合式反应系统中,利用酿酒酵母进行连续发酵生产高浓度乙醇。后续管式反应器内通过装填聚氨酯颗粒和木块对酵母细胞进行吸附固定化,在乙醇抑制造成细胞活性大幅降低的情况下,通过大幅提高细胞浓度保证发酵效率,在稀释速率0.02h^-1和280g/L葡萄糖的条件下,系统的终点乙醇浓度为15.4 % (v/v)。研究表明在一定稀释速率之下,应该通过增加反应器的级数来降低稀释速率,以达到提高终点乙醇浓度,如简单地降低进料速率则可能增加整个系统所受的乙醇抑制,对提高终点乙醇浓度效果不显著。     

Continuous alcohol production from starchy materials with a novel immobilized cell/enzyme bioreactor

In a novel bioreactor system that was packed with both Al alginate biocatalyst A (entrapment of adsorbed-glucoamylase on γ-alumina) and biocatalyst B (entrapment of a mixture of different strains of yeast cells), 60–95 kg/m³ of alcohol solution were continuously produced through parallel operation of saccharifying and fermenting liquefied starchy materials of potato and grains without contamination. This continuous process was anaerobically performed at a pH of 2.8–3.2 and a temperature of 30°C.     

High solids fermentation of hydrolysates of wheat starch B in a continuous dynamic immobilized biocatalyst bioreactor

Summary A simple and efficient method of conversion of wheat starch B to ethanol was investigated. Employing a two-stage enzymatic saccharification process, 95% of the wheat starch was converted to fermentable sugars in 40 h. From 140 g/l total sugars in the feed solution, 63.6 g/l ethanol was produced continuously with a residence time of 3.3 h in a continuous dynamic immobilized biocatalyst bioreactor by immobilized cells ofSaccharomyces cerevisiae. The advantages and the application of this bioreactor to continuous alcoholic fermentation of industrial substrates are presented.     

Production of ethanol from molasses at 45 °C using alginate-immobilized Kluyveromyces marxianus imb3

The thermotolerant, ethanol-producing yeast strain, Kluyveromyces marxianus IMB3, has been immobilized in calcium alginate matrices. The ability of the biocatalyst to produce ethanol from cane molasses originating in Guatemala, Honduras, Senegal, Guyana and the Philippines was examined. In each case the molasses was diluted to yield a sugar concentration of 140?g/l and fermentations were carried out in batch-fed mode at 45?°C. During the first 24 hours, the maximum ethanol concentrations obtained ranged from 43–57?g/l with optimum production on the molasses from Honduras. Ethanol production during subsequent re-feeding of the fermentations at 24-hour intervals over a 120-hour period, decreased steadily to concentrations ranging from 20–36?g/l and it was found that ethanol productivity remained highest in fermentations containing the molasses from Guyana. When each set of fermentations was re-fed at 120?h and allowed to continue for 48?h, ethanol production again increased to a maximum with concentrations ranging from 25–52?g/l. It was also found however, that increasing the time between re-feeding at this stage in fermentation had a detrimental effect on the functionality of the biocatalyst.     

Selective production of acetone during continuous synthesis gas fermentation by engineered biocatalyst Clostridium sp. MAceT113

Aims: To engineer acetogen biocatalyst capable of fermenting synthesis gas blend to acetone as the only liquid carbonaceous product. Methods and Results: The metabolic engineering comprised inactivation of phosphotransacetylase via integration of a cassette comprising synthetic genes erm(B), thiolase and HMG‐CoA synthase. Acetaldehyde dehydrogenase was inactivated via integration of a cassette consisting of synthetic genes cat, HMG‐CoA lyase and acetoacetate decarboxylase. The engineered biocatalyst Clostridum sp. MAceT113 lost production of 253 mmol l^?1 ethanol and 296 mmol l^?1 acetate and started producing 1·8 mol l^?1 acetone in single‐stage continuous syngas fermentation. Conclusions: The acetone concentration in culture broth is economical for bulk manufacture because it is about twenty times of that achieved with known acetone–butanol–ethanol fermentation of sugars. Significance and Impact of the Study: The process shows the opportunity to produce acetone from synthesis gas at concentrations comparable with production of acetone from products of petroleum cracking. This is the first report on elimination of acetate and acetaldehyde production and directing carbon flux from Acetyl‐CoA to acetone via a non‐naturally occurring in acetogen acetone biosynthesis pathway identified in eukaryotic organisms.     

Ethanol production by Zymomonas mobilis CP4 from sugar cane chips

Summary The fermentation of large sugar cane chips (1.0–1.5 in) to ethanol by Zymomonas mobilis CP4 (Z. mobilis) was studied in two glass fermentors operating with culture circulation for agitation (the EX-FERM type): a. A laboratory scale(2.5 liter) cylindrical vessel; b. A bench scale (8 liter) wide vessel. Z. mobilis cultures consumed 89–96% of the cane sucrose, converting it to ethanol by 90–97% of the theoretical yield in the laboratory scale fermentor and by 83–90% in the bench scale fermentor culture. Comparative Saccharomyces spp. cultures in laboratory fermentor consumed 96–98% of the cane sucrose, with ethanol conversion of only 75–79% of the theoretical yield.These preliminary results indicated that sucrose in agricultural size sugar cane chips was ethanol fermentable as compared to small size sugar cane chips or to sugar cane juice. Z. mobilis CP4 cultures converted sucrose more efficiently to ethanol than Saccharomyces spp. as shown in the laboratory scale fermentor studies.The ethanol yields in a wide bench scale fermentor cultures were slightly lower than in a laboratory fermentor.     

Continuous ethanol production from sago starch using immobilized amyloglucosidase and Zymomonas mobilis

To produce ethanol more economically than in a conventional process, it is necessary to attain high productivity and low production cost. To this end, a continuous ethanol production from sago starch using immobilized amylogucosidase (AMG) and Zymomonas mobilis cells was studied. Chitin was used for immobilization of AMG and Z. mobilis cells were immobilized in the form of sodium alginate beads. Ethanol was produced continuously in an simultaneous saccharification and ethanol fermentation (SSF) mode in a pacekd bed reactor. The maximum ethanol productivity based on the void volume, V_v, was 37 g/l/h with ethanol yield, Y_p/s, 0.43 g/g (84% of the theoretical ethanol yield) in this system. The steady-state concentration of ethanol (46 g/l could be maintained in a stable manner over two weeks at the dilution rate of 0.46 h⁻.     

Butyric acid fermentation in a fibrous bed bioreactor with immobilized Clostridium tyrobutyricum from cane molasses

Butyrate fermentation by immobilized Clostridium tyrobutyricum was successfully carried out in a fibrous bed bioreactor using cane molasses. Batch fermentations were conducted to investigate the influence of pH on the metabolism of the strain, and the results showed that the fermentation gave a highest butyrate production of 26.2 g l⁻¹ with yield of 0.47 g g⁻¹ and reactor productivity up to 4.13 g l⁻¹ h⁻¹ at pH 6.0. When repeated-batch fermentation was carried out, long-term operation with high butyrate yield, volumetric productivity was achieved. Several cane molasses pretreatment techniques were investigated, and it was found that sulfuric acid treatment gave better results regarding butyrate concentration (34.6 ± 0.8 g l⁻¹), yield (0.58 ± 0.01 g g⁻¹), and sugar utilization (90.8 ± 0.9%). Also, fed-batch fermentation from cane molasses pretreated with sulfuric acid was performed to further increase the concentration of butyrate up to 55.2 g l⁻¹.     

Ethanol production from lignocellulosic biomass of energy cane

Ethanol produced from lignocellulosic biomass is a renewable alternative to diminishing petroleum based liquid fuels. The release of many new sugarcane varieties by the United States Department of Agriculture to be used as energy crops is a promising feedstock alternative. Energy cane produces large amounts of biomass that can be easily transported, and production does not compete with food supply and prices because energy cane can be grown on marginal land instead of land for food crops. The purpose of this study was to evaluate energy cane for lignocellulosic ethanol production. Energy cane variety L 79-1002 was pretreated with weak sulfuric acid to remove lignin. In this study, 1.4 M sulfuric acid pretreated type II energy cane had a higher ethanol yield after fermentation by Klebsiella oxytoca without enzymatic saccharification than 0.8 M and 1.6 M sulfuric acid pretreated type II energy cane. Pretreated biomass was inoculated with K. oxytoca for cellulose fermentation and Pichia stipitis for hemicellulose fermentation under simultaneous saccahrification and fermentation (SSF) and separate hydrolysis and fermentation (SHF) conditions. For enzymatic saccharification of cellulose, the cellulase and ??-glucanase cocktail significantly increased ethanol production compared to the ethanol production of fermented acid pretreated energy cane without enzymatic saccharification. The results revealed that energy cane variety L 79-1002 produced maximum cellulosic ethanol under SHF (6995 mg/L) and produced 3624 mg/L ethanol from fermentation of hemicellulosic sugars.     

Continuous high-ethanol fermentation from cane molasses by a flocculating yeast

Repeated-batch fermentation by a flocculating fusant, Saccharomyces cerevisiae HA 2, was done in a molasses medium that contained 20% (w/v) total sugar, at 30°C in an automatically controlled fermentor, and the effects of ethanol concentration on the specific growth rate and the specific production rate of ethanol were studied. Both the specific growth rate and the specific production rate of ethanol fell with increase of ethanol concentration, and there was a linear correlation between each rate and the concentration of thanol. The maximum specific growth rate (μ_max) and the maximum specific production rate of ethanol (q_max) were 0.12 h⁻¹ and 0.1 g ethanol/10⁹ cells·h, respectively. The specific growth rate and the specific production rate of ethanol fell to zero at ethanol concentration of 89 g/l and 95 g/l, respectively. The number of viable cells, calculated from the linear inhibition equation, was 1.3 × 10⁹ cells/ml for production of 85 g/l ethanol at a dilution rate (D₁) of 0.2 h⁻¹. Based on this estimation, a laboratory-scale continuous fermentation, using two fermentors in series, was done. In the second fermentor, 85 g/l ethanol was produced at a dilution rate (D₁) of 0.2 h⁻¹ by the active feedig of the fermented mash from the first fermentor into the second fermentor by pumping (hereafter called active feeding). To maintain the number of viable cells above 10⁹ cells/ml in the second fermentor, a active feeding ratio of more than 23% was required. Under these conditions, 81 g/l ethanol was produced in the second fermentor at a dilution rate (D_t) of 0.25 h⁻¹, and the high ethanol productivity of 20.3 g/l·h could be achieved. A bench-scale continuous fermentation, using two fermentors in series, with a active feeding ratio of 25% was done. An ethanol concentration of 84 g/l in the second fermentor at a dilution rate (D_t) of 0.25 h⁻¹ was achieved, just as it was in the laboratory-scale fermentation test.     

Petroleum-contaminated water treatment in a fluidized-bed bioreactor with immobilized Rhodococcus cells

Biotreatment of petroleum-contaminated water in a fluidized-bed bioreactor (FBB) is a relatively new and promising technology, which efficiency is strongly affected by the biocatalyst used. Our laboratory experiments involved biotreatment of the water contaminated with a synthetic petroleum mixture consisting of aliphatic and polyaromatic hydrocarbons (PAHs) using a continuous column bioreactor with recycle. Different type biocatalysts were tested, including Rhodococcus bacteria immobilized in hydrophobized carriers such as sawdust, poly(vinyl alcohol) cryogel (cryoPVA) and poly(acrylamide) cryogel (cryoPAAG). Biocatalyst abilities to oxidize petroleum hydrocarbons were evaluated using the Columbus Micro-Oxymax^® respirometer. The hydrophobized sawdust-supported biocatalyst demonstrated substantially higher metabolic activity than C₁₂-cryoPAAG-based biocatalyst due to larger number of immobilized Rhodococcus cells and, therefore, had benefits for application in FBBs. The obtained results showed that designed FBB process is successful, providing 70–100% removal of n-alkanes (C₁₀–C₁₉) and 66–70% removal of 2–3-ring PAHs from contaminated water after 2–3 weeks.     

Production of high ethanol concentrations from glucose using a vertical rotating immobilized cell reactor of the bacterium zymomonas mobilis

Long-term continuous ethanol production of up to 80 g.l¹ with a volumetric ethanol productivity of 63 g. l^?1. h^?1 was maintained for more than 72 days using a Vertical Rotating Immobilized Cell Reactor of the bacterium Z. mobilis. Continuous production of higher ethanol concentration was unsuccessful due to an inhibition of cell growth by long exposure to high ethanol concentrations. However, ethanol concentration as high as 120g. l^?1 and volumetric ethanol productivity of 13g. l^?1. h^?1 were achieved in a repeated-batch fermentation system using the same bioreactor. By a simple washing operation at the end of each run, immobilized biomass could be effectively regenerated and used to carry out more than 10 successive fermentation cycles.