Butanol production from corn fiber xylan using Clostridium acetobutylicum

Acetone, butanol, and ethanol (ABE) were produced from corn fiber arabinoxylan (CFAX) and CFAX sugars (glucose, xylose, galactose, and arabinose) using Clostridium acetobutylicum P260. In mixed sugar (glucose, xylose, galactose, and arabinose) fermentation, the culture preferred glucose and arabinose over galactose and xylose. Under the experimental conditions, CFAX (60 g/L) was not fermented until either 5 g/L xylose or glucose plus xylanase enzyme were added to support initial growth and fermentation. In this system, C. acetobutylicum produced 9.60 g/L ABE from CFAX and xylose. This experiment resulted in a yield and productivity of 0.41 and 0.20 g/L x h, respectively. In the integrated hydrolysis, fermentation, and recovery process, 60 g/L CFAX and 5 g/L xylose produced 24.67 g/L ABE and resulted in a higher yield (0.44) and a higher productivity (0.47 g/L x h). CFAX was hydrolyzed by xylan-hydrolyzing enzymes, and ABE were recovered by gas stripping. This investigation demonstrated that integration of hydrolysis of CFAX, fermentation to ABE, and recovery of ABE in a single system is an economically attractive process. It is suggested that the culture be further developed to hydrolyze CFAX and utilize all xylan sugars simultaneously. This would further increase productivity of the reactor.     

Butanol production by Clostridium beijerinckii. Part I: use of acid and enzyme hydrolyzed corn fiber

Fermentation of sulfuric acid treated corn fiber hydrolysate (SACFH) inhibited cell growth and butanol production (1.7 ± 0.2 g/L acetone butanol ethanol or ABE) by Clostridium beijerinckii BA101. Treatment of SACFH with XAD-4 resin removed some of the inhibitors resulting in the production of 9.3 ± 0.5 g/L ABE and a yield of 0.39 ± 0.015. Fermentation of enzyme treated corn fiber hydrolysate (ETCFH) did not reveal any cell inhibition and resulted in the production of 8.6 ± 1.0 g/L ABE and used 24.6 g/L total sugars. ABE production from fermentation of 25 g/L glucose and 25 g/L xylose was 9.9 ± 0.4 and 9.6 ± 0.4 g/L, respectively, suggesting that the culture was able to utilize xylose as efficiently as glucose. Production of only 9.3 ± 0.5 g/L ABE (compared with 17.7 g/L ABE from fermentation of 55 g/L glucose-control) from the XAD-4 treated SACFH suggested that some fermentation inhibitors may still be present following treatment. It is suggested that inhibitory components be completely removed from the SACFH prior to fermentation with C. beijerinckii BA101. In our fermentations, an ABE yield ranging from 0.35 to 0.39 was obtained, which is higher than reported by the other investigators.     

High-titer n-butanol production by clostridium acetobutylicum JB200 in fed-batch fermentation with intermittent gas stripping

Acetone–butanol–ethanol (ABE) fermentation with a hyper‐butanol producing Clostridium acetobutylicum JB200 was studied for its potential to produce a high titer of butanol that can be readily recovered with gas stripping. In batch fermentation without gas stripping, a final butanol concentration of 19.1 g/L was produced from 86.4 g/L glucose consumed in 78 h, and butanol productivity and yield were 0.24 g/L h and 0.21 g/g, respectively. In contrast, when gas stripping was applied intermittently in fed‐batch fermentation, 172 g/L ABE (113.3 g/L butanol, 49.2 g/L acetone, 9.7 g/L ethanol) were produced from 474.9 g/L glucose in six feeding cycles over 326 h. The overall productivity and yield were 0.53 g/L h and 0.36 g/g for ABE and 0.35 g/L h and 0.24 g/g for butanol, respectively. The higher productivity was attributed to the reduced butanol concentration in the fermentation broth by gas stripping that alleviated butanol inhibition, whereas the increased butanol yield could be attributed to the reduced acids accumulation as most acids produced in acidogenesis were reassimilated by cells for ABE production. The intermittent gas stripping produced a highly concentrated condensate containing 195.9 g/L ABE or 150.5 g/L butanol that far exceeded butanol solubility in water. After liquid–liquid demixing or phase separation, a final product containing ～610 g/L butanol, ～40 g/L acetone, ～10 g/L ethanol, and no acids was obtained. Compared to conventional ABE fermentation, the fed‐batch fermentation with intermittent gas stripping has the potential to reduce at least 90% of energy consumption and water usage in n‐butanol production from glucose. Biotechnol. Bioeng. 2012; 109: 2746–2756. © 2012 Wiley Periodicals, Inc.     

萃取耦合技术对玉米秸秆水解液发酵产丁醇的影响

本研究以玉米秸秆水解液为原料,通过萃取发酵技术生产燃料丁醇,以提高丁醇产量,降低生产成本。通过对萃取剂的筛选与条件优化,确定纤维丁醇发酵的萃取剂为油醇,添加时间为发酵0 h,添加比例为1:1 (V/V)。该条件下发酵32 g/L糖浓度的玉米秸秆水解液,丁醇和总溶剂产量分别为3.28 g/L和4.72 g/L,比对照分别提高958.1%和742.9%。以D301树脂脱毒后5%总糖浓度的玉米秸秆水解液进行丁醇萃取发酵,丁醇和总溶剂产量分别达到10.34 g/L和14.72 g/L,发酵得率为0.31 g/g,与混合糖发酵结果相当。研究结果表明萃取发酵技术能够显著提高原料的利用率和丁醇产量,为纤维丁醇工业化生产提供了技术支撑。     

Enhancement of ABE fermentation through regulation of ammonium acetate and D–xylose uptake from acid-pretreated corncobs

Clostridium acetobutylicum TISTR 1462 and Clostridium beijerinckii TISTR 1461 were chosen to optimize acetone–butanol–ethanol (ABE) fermentation by using glucose as a carbon source. The enhancement in its productivity by adding various concentrations of ammonium acetate was studied. Then, the variation of glucose/xylose ratios in the pre-grown medium was investigated. The results showed that both increased ammonium acetate in the production medium and D–xylose in the pre-grown medium could produce more ABE. With these conditions, using corncob hydrolysate as a substrate, 20.58 g/L ABE was produced from C. beijerinckii TISTR 1461 with 0.44 g/L/h and 0.45 of ABE productivity and yield, respectively.     

Screening and characteristics of a butanol-tolerant strain and butanol production from enzymatic hydrolysate of NaOH-pretreated corn stover

As a gasoline substitute, butanol has advantages over traditional fuel ethanol in terms of energy density and hydroscopicity. However, solvent production appeared limited by butanol toxicity. The strain of Clostridium acetobutylicum was subjected to mutation by mutagen of N-methyl-N'-nitro-N-nitrosoguanidine for 0.5?h. Screening of mutants was done according to the individual resistance to butanol. A selected butanol-resistant mutant, strain 206, produced 50?% higher solvent concentrations than the wild-type strain when 60?g glucose/l was employed as substrate. The strain was also able to produce solvents of 23.47?g/l in 80?g/l glucose P2 medium after 70?h fermentation, including 5.41?g acetone/l, 15.05?g butanol/l and 3.02?g ethanol/l, resulting in an ABE yield and productivity of 0.32?g/g and 0.34?g/(l?h). Subsequently, Acetone-butanol-ethanol (ABE) production from enzymatic hydrolysate of NaOH-pretreated corn stover was investigated in this study. An ABE yield of 0.41 and a productivity of 0.21?g/(l?h) was obtained, compared to the yield of 0.33 and the productivity of 0.20?g/(l?h) in the control medium containing 52.47 mixed sugars. However, it is important to note that although strain 206 was able to utilize all the glucose rapidly in the hydrolysate, only 32.9?% xylose in the hydrolysate was used after fermentation stopped compared to 91.4?% xylose in the control medium. Strain 206 was shown to be a robust strain for ABE production from lignocellulosic materials and has a great potential for industrial application.     

限制葡萄糖、葡萄糖/乙酸双底物条件下自由控制丙丁梭菌ABE发酵丙酮浓度和丙酮/丁醇比

提出一种可以提高和自由控制丙丁梭菌ABE发酵丙酮浓度与丙酮/丁醇比的方法。（1）通过控制糖化酶用量、反应时间和温度调节玉米培养基初始葡萄糖浓度,使发酵进入到产溶剂期后,残留葡萄糖浓度降至接近于0 g/L的水平;（2）在葡萄糖受限的条件下,诱导丙丁梭菌合成分泌糖化酶,分解寡糖,将葡萄糖维持于低浓度,进而限制梭菌胞内糖酵解途径的碳代谢和NADH生成速度。与此同时,外添乙酸形成葡萄糖/乙酸双底物环境。在能量代谢基本不受破坏、丁醇未达到抑制浓度的条件下,适度抑制丁醇生产,有效地利用外添乙酸强化丙酮合成;（3）在外添乙酸的基础上,添加适量酿酒酵母,形成丙丁梭菌/酿酒酵母混合发酵体系,提高梭菌对高丁醇浓度的耐受能力。整个发酵体系可以将丙酮浓度和丙酮/丁醇比自由控制在5~12 g/L和0.5~1.0的水平,最大丙酮浓度和丙酮/丁醇比达到11.74 g/L和1.02,并可维持丁醇浓度于10~14 g/L的正常水平,充分满足工业ABE发酵对于丙酮和丁醇产品的不同需求。     

Continuous fermentation of feed streams containing D-glucose and D-xylose in a two-stage process utilizing immobilized Saccharomyces cerevisiae and Pachysolen tannophilus

In the U.S., forest and crop residues contain enough glucose and xylose to supply 10 times the country's usage of ethanol and ethylene, but an efficient fermentation scheme is lacking,(1,2,3) To develop a strategy for process design, specific ethanol productivities and yields of Pachysolen tannophilus NRRL Y-2460 and Saccharomyces cerevisiae NRRL Y-2235 were compared. Batch cultures and continuous stirred reactors (CSTR) loaded with immobilized cells were fed glucose and xylose. As expected from previous reports, Y-2235 fermented glucose but not xylose. Y-2460 consumed both sugars but fermented glucose inefficiently relative to Y-2235, and it suffered a diauxic lag lasting 10-20 h when given a sugar mixture. Immobilized Y-2235 exhibited increasing productivity but constant yield with in creasing glucose concentration. In contrast, Y-2460 exhibited an optimum productivity at 30-40 g/L xylose and a declining yield with increasing xylose concentration. Immobilized Y-2235 tolerated more than 100 g/L ethanol while the productivity and yield of Y-2460 fell by 80 and 58%, respectively, as ethanol reached 50 g/L. A 38.8-g/L ethanol stream could be produced as 103 g/L xylose was continuously fed to Y-2460. If it was blended with a 274 g/L glucose stream to give a composite of 23.7 g/L ethanol and 107 g/L glucose, Y-2235 could en rich the ethanol to 75 g/L. Taken together these results suggest use of a two-stage continuous reactor for pro cessing xylose and glucose from lignocellulose. An immobilized Y-2460 CSTR (or cascade) would convert the hemicellulose hydrolyzate. Then downstream, an immobilized Y-2235 plug flow reactor would enrich the hemicellulose-derived ethanol to more than 70 g/L upon addition of cellulose hydrolyzate.     

Regulation and butanol inhibition of D-xylose and D-glucose uptake in Clostridium acetobutylicum

Clostridium acetobutylicum exhibited diauxie growth in the presence of mixtures of glucose and xylose. Both glucose- and xylose-grown cells had a glucose uptake activity. On the other hand, growth on xylose was associated with the induction of a xylose permease activity, which was repressed by glucose in xylose-induced cells. The rate of sugar uptake with increasing sugar concentrations showed saturation kinetics with an apparent Km of 1.25 X 10(-5) M for glucose and 5 X 10(-3) M for xylose. Concomitant with the production of solvents, the activities of the glucose and xylose transport systems decreased. Among the main products of fermentation, butanol was shown to be a potent inhibitor of the growth of the organism and of the rate of sugar uptake as well as of sugar incorporation into cell materials. These inhibitory effects of butanol were more pronounced in xylose-grown cells than in glucose-grown cells. Butanol completely inhibited growth at a concentration of 14 g/liter for cultures growing on glucose and 8 g/liter for cultures growing on xylose. Concentrations of 7 and 10.5 g/liter of butanol caused a 50% inhibition of the xylose and glucose incorporations into cell materials. These inhibitory levels of butanol were found in typical glucose or xylose fermentation.     

Regulation and butanol inhibition of D-xylose and D-glucose uptake in Clostridium acetobutylicum.

Clostridium acetobutylicum exhibited diauxie growth in the presence of mixtures of glucose and xylose. Both glucose- and xylose-grown cells had a glucose uptake activity. On the other hand, growth on xylose was associated with the induction of a xylose permease activity, which was repressed by glucose in xylose-induced cells. The rate of sugar uptake with increasing sugar concentrations showed saturation kinetics with an apparent Km of 1.25 X 10(-5) M for glucose and 5 X 10(-3) M for xylose. Concomitant with the production of solvents, the activities of the glucose and xylose transport systems decreased. Among the main products of fermentation, butanol was shown to be a potent inhibitor of the growth of the organism and of the rate of sugar uptake as well as of sugar incorporation into cell materials. These inhibitory effects of butanol were more pronounced in xylose-grown cells than in glucose-grown cells. Butanol completely inhibited growth at a concentration of 14 g/liter for cultures growing on glucose and 8 g/liter for cultures growing on xylose. Concentrations of 7 and 10.5 g/liter of butanol caused a 50% inhibition of the xylose and glucose incorporations into cell materials. These inhibitory levels of butanol were found in typical glucose or xylose fermentation.     

Characterization of an immobilized cell, trickle bed reactor during long term butanol (ABE) fermentation

Acetone-butanol-ethanol (ABE) fermentation was performed continuously in an immobilized cell, trickle bed reactor for 54 days without, degeneration by maintaining the pH above 4.3. Column clogging was minimized by structured packing of immobilization matrix. The reactor contained two serial glass columns packed with Clostridium acetobutylicum adsorbed on 12- and 20-in.-long polyester sponge strips at total flow rates between 38 and 98.7 mL/h. Cells were initially grown at 20 g/L glucose resulting in low butanol (1.15 g/L) production encouraging cell growth. After the initial cell growth phase a higher glucose concentration (38.7 g/L) improved solvent yield from 13.2 to 24.1 wt%, and butanol production rate was the best. Further improvement in solvent yield and butanol production rate was not observed with 60 g/L of glucose. However, when the fresh nutrient supply was limited to only the first column, solvent yield increased to 27.3 wt% and butanol selectivity was improved to 0.592 as compared to 0.541 when fresh feed was fed to both columns. The highest butanol concentration of 5.2 g/L occurred at 55% conversion of the feed with 60 g/L glucose. Liquid product yield of immobilized cells approached the theoretical value reported in the literature. Glucose and product concentration profiles along the column showed that the columns can be divided into production and inhibition regions. The length of each zone was dependent upon the feed glucose concentration and feed pattern. Unlike batch fermentation, there was no clear distinction between acid and solvent production regions. The pH dropped, from 6.18-6.43 to 4.50-4.90 in the first inch of the reactor. The pH dropped further to 4.36-4.65 by the exit of the column. The results indicate that the strategy for long term stable operation with high solvent yield requires a structured packing of biologically stable porous matrix such as polyester sponge, a pH maintenance above 4.3, glucose concentrations up to 60 g/L and nutrient supply only to the inlet of the reactor.     

Acetone–butanol–ethanol production from corn stover pretreated by alkaline twin-screw extrusion pretreatment

In this study, the alkaline twin-screw extrusion pretreated corn stover was subjected to enzymatic hydrolysis after washing. The impact of solid loading and enzyme dose on enzymatic hydrolysis was investigated. It was found that 68.2 g/L of total fermentable sugar could be obtained after enzymatic hydrolysis with the solid loading of 10 %, while the highest sugar recovery of 91.07 % was achieved when the solid loading was 2 % with the cellulase dose of 24 FPU/g substrate. Subsequently, the hydrolyzate was fermented by Clostridium acetobutylicum ATCC 824. The acetone–butanol–ethanol (ABE) production of the hydrolyzate was compared with the glucose, xylose and simulated hydrolyzate medium which have the same reducing sugar concentration. It was shown that 7.1 g/L butanol and 11.2 g/L ABE could be produced after 72 h fermentation for the hydrolyzate obtained from enzymatic hydrolysis with 6 % solid loading. This is comparable to the glucose and simulated hydrozate medium, and the overall ABE yield could reach 0.112 g/g raw corn stover.     

Acetone-butanol-ethanol (ABE) fermentation in an immobilized cell trickle bed reactor

Acetone-butanol-ethanol (ABE) fermentation was successfully carried out in an immobilized cell trickle bed reactor. The reactor was composed of two serial columns packed with Clostridium acetobutylicum ATCC 824 entrapped on the surface of natural sponge segments at a cell loading in the range of 2.03-5.56 g dry cells/g sponge. The average cell loading was 3.58 g dry cells/g sponge. Batch experiments indicated that a critical pH above 4.2 is necessary for the initiation of cell growth. One of the media used during continuous experiments consisted of a salt mixture alone and the other a nutrient medium containing a salt mixture with yeast extract and peptone. Effluent pH was controlled by supplying various fractions of the two different types of media. A nutrient medium fraction above 0.6 was crucial for successful fermentation in a trickle bed reactor. The nutrient medium fraction is the ratio of the volume of the nutrient medium to the total volume of nutrient plus salt medium. Supplying nutrient medium to both columns continuously was an effective way to meet both pH and nutrient requirement. A 257-mL reactor could ferment 45 g/L glucose from an initial concentration of 60 g/L glucose at a rate of 70 mL/h. Butanol, acetone, and ethanol concentrations were 8.82, 5.22, and 1.45 g/L, respectively, with a butanol and total solvent yield of 19.4 and 34.1 wt %. Solvent productivity in an immobilized cell trickle bed reactor was 4.2 g/L h, which was 10 times higher than that obtained in a batch fermentation using free cells and 2.76 times higher than that of an immobilized CSTR. If the nutrient medium fraction was below 0.6 and the pH was below 4.2, the system degenerated. Oxygen also contributed to the system degeneration. Upon degeneration, glucose consumption and solvent yield decreased to 30.9 g/L and 23.0 wt %, respectively. The yield of total liquid product (40.0 wt %) and butanol selectivity (60.0 wt %) remained almost constant. Once the cells were degenerated, they could not be recovered.     

Improvements of metabolites tolerance in <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> by micronutrient zinc supplementation

Micronutrient zinc is of great importance for acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum. The effect of zinc supplementation on toxic metabolites (formic, acetic, butyric acid and butanol) tolerance during ABE fermentation was investigated under various stress-shock conditions without pH control. Great improvements on cell growth, glucose utilization and butanol production were achieved. In the presence of 0.45 g/L formic acid, zinc contributed to 11.28 g/L butanol produced from 55.24 g/L glucose compared to only 5.27 g/L butanol from 29.49 g/L glucose in the control without zinc supplementation. More importantly, relatively higher levels of 7.5 g/L acetic acid, 5.5 g/L butyric acid and 18 g/L butanol could be tolerated by C. acetobutylicum with zinc supplementation while no fermentation was observed under the same stress-shock condition respectively, suggesting that the acids and butanol tolerance in C. acetobutylicum could be significantly facilitated by pleiotropic regulation of micronutrient zinc. Thus, this paper provides an efficient bioprocess engineering strategy for improving stress tolerance in Clostridium species.     

The acetone butanol fermentation on glucose and xylose. II. Regulation and kinetics in fed-batch cultures

The kinetics in fed-batch cultures of acetone butanol fermentation by Clostridium acetobutylicum is compared on glucose, xylose, and mixtures of both sugars. The final conversion yield of sugars into solvents always increases with the sugar feeding rate. At low feeding rates, the sugar concentration in the medium becomes limiting, which results in a slower cellular growth, a slower metabolic transition from an acid to a solvent fermentation and, thus, a higher accumulation of acids. It is only at sufficiently high feeding rates that fed-batch fermentations yield kinetic results comparable to those of batch fermentations. With mixtures of glucose and xylose, because of a maintained low glucose level, both sugars are taken up at the same rate during a first fermentation period. An earlier accumulation of xylose when the fermentation becomes inhibited suggest that xylose utilization is inhibited when the catabolic flux of glucose alone can satisfy the metabolic activity of the cell. Kinetic results with batch and fed-batch fermentations indicate several important features of the regulation of C. acetobutylicum metabolism: an early inhibition by the produced acids; an initiation of solvent formation between 4 and 6 g/L acetic and butyric acid depending on the metabolic activity of the cell; a metabolic transition from acids to solvents production at a rate closely related to the rate of sugar uptake; during solvent production, a reassimilation of acids above a minimal rate of sugar consumption of 0.2 h(-1); a final inhibition of the fermentation at a total butanol and acids concentration of ca. 20 g/L.     

The acetone butanol fermentation on glucose and xylose. I. Regulation and kinetics in batch cultures

The kinetics in batch culture of the acetone butanol fermentation by Clostridium acetobutylicum is compared on glucose, xylose, and mixtures of both sugars. The fastest initial growth and transition from an acid to a solvent metabolism occurs on glucose, with a final 62 g/L glucose conversion. On xylose, an initial slower growth rate and a longer metabolic transition result in higher cellular and acids concentration, thus in a level of fermented sugar limited to 47 g/L. Batch fermentations on mixtures of glucose and xylose show that both sugars can be fermented, with a higher rate for glucose. However, xylose fermentation is inducible and inhibited at glucose level above 15 g/L. Mixtures of glucose and xylose yield the highest amount of fermented sugars, up to 68 g/L, as a result of both a fast metabolic transition on glucose and a strong acid reconsumption on xylose. In all cases, solvent production is triggered at a total acid concentration between 4 and 5 g/L, whereas the final inhibition of the fermentation takes place at a total butanol and acid concentration between 18 and 20 g/L.     

Acetone–Butanol–Ethanol Production by Clostridium acetobutylicum ATCC 824 Using Sago Pith Residues Hydrolysate

Sago pith residues (58 % starch, 23 % cellulose, 9.2 % hemicellulose, and 4 % lignin) are one of the abundant lignocellulosic residues generated after starch extraction process in sago mill. In this study, fermentable sugars from enzymatic hydrolysis of sago pith residues were converted to acetone–butanol–ethanol (ABE) by Clostridium acetobutylicum ATCC 824. With an initial concentration of 30 g/L of concentrated sago pith residues hydrolysate containing 23 g/L of glucose and 4.58 g/L of cellobiose, 4.22?±?0.17 g/L of ABE were produced after 72 h of fermentation with yield and productivity of 0.20 g/g glucose and 0.06 g/L/h, respectively. Results are in agreement when synthetic glucose was used as a carbon source. Increasing sago pith residue hydrolysate to 50 g/L (containing 40 g/L glucose) and supplementing with 0.5 g/L yeast extract, approximately 8.84?±?0.20 g/L of ABE (5.41?±?0.10 g/L of butanol) were produced with productivity and yield of 0.12 g/L/h and 0.30 g/g glucose respectively, providing a 52 % improvement.     

Ex situ product recovery for enhanced butanol production by <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

In situ butanol recovery fermentation has been intensively studied as an effective alternative to conventional butanol production, which is limited due to the cellular toxicity of butanol. However, the low biocompatibility of adsorbents often leads to failure of in situ recovery fermentations. In this study, Clostridium beijerinckii NCIMB 8052 was cultured in flasks without shaking and in situ recovery fermentation was performed by using an adsorbent L493. The amounts of acetone, butanol, and ethanol (ABE) increased by 34.4 % in the presence of the adsorbent. In contrast, cell growth and production of organic acids and ABE were retarded in the 7-L batch fermentations with in situ butanol recovery. Cell damage occurred in the fermentor upon agitation in the presence of the adsorbent, unlike in static flask cultures with in situ recovery. Ex situ recovery fermentation using circulation of fermentation broth after mid-exponential phase of cell growth was developed to avoid adsorbent-cell incompatibility. No apparent cell damage was observed and 25.7 g/L of ABE was produced from 86.2 g/L glucose in the fed-batch mode using 7 L fermentors. Thus, ex situ recovery fermentation with C. beijerinckii is effective for enhancing butanol fermentation.     

Efficient butanol production under aerobic conditions by coculture of Clostridium acetobutylicum and Nesterenkonia sp. strain F

Clostridium acetobutylicum is widely used for the microbial production of butanol in a process known as acetone–butanol–ethanol (ABE) fermentation. However, this process suffers from several disadvantages including high oxygen sensitivity of the bacterium which makes the process complicated and necessitate oxygen elimination in the culture medium. Nesterenkonia sp. strain F has attracted interests as the only known non-Clostridia microorganism with inherent capability of butanol production even in the presence of oxygen. This bacterium is not delimited by oxygen sensitivity, a challenge in butanol biosynthesis, but the butanol titer was far below Clostridia. In this study, Nesterenkonia sp. strain F was cocultivated with C. acetobutylicum to form a powerful “coculture” for butanol production thereby eliminating the need for oxygen removal before fermentation. The response surface method was used for obtaining optimal inoculation amount/time and media formulation. The highest yield, 0.31 g/g ABE (13.6 g/L butanol), was obtained by a coculture initiated with 1.5 mg/L Nesterenkonia sp. strain F and inoculated with 15 mg/L C. acetobutylicum after 1.5 hr in a medium containing 67 g/L glucose, 2.2 g/L yeast extract, 4 g/L peptone, and 1.4% (vol/vol) P2 solution. After butanol toxicity assessment, where Nesterenkonia sp. strain F showed no butanol toxicity, the coculture was implemented in a 2 L fermenter with continual aeration leading to 20 g/L ABE.     

Production of L(+)-lactic acid from glucose and starch by immobilized cells of Rhizopus oryzae in a rotating fibrous bed bioreactor

A rotating fibrous-bed bioreactor (RFB) was developed for fermentation to produce L(+)-lactic acid from glucose and cornstarch by Rhizopus oryzae. Fungal mycelia were immobilized on cotton cloth in the RFB for a prolonged period to study the fermentation kinetics and process stability. The pH and dissolved oxygen concentration (DO) were found to have significant effects on lactic acid productivity and yield, with pH 6 and 90% DO being the optimal conditions. A high lactic acid yield of 90% (w/w) and productivity of 2.5 g/L.h (467 g/h.m(2)) was obtained from glucose in fed-batch fermentation. When cornstarch was used as the substrate, the lactic acid yield was close to 100% (w/w) and the productivity was 1.65 g/L.h (300 g/h.m(2)). The highest concentration of lactic acid achieved in these fed-batch fermentations was 127 g/L. The immobilized-cells fermentation in the RFB gave a virtually cell-free fermentation broth and provided many advantages over conventional fermentation processes, especially those with freely suspended fungal cells. Without immobilization with the cotton cloth, mycelia grew everywhere in the fermentor and caused serious problems in reactor control and operation and consequently the fermentation was poor in lactic acid production. Oxygen transfer in the RFB was also studied and the volumetric oxygen transfer coefficients under various aeration and agitation conditions were determined and then used to estimate the oxygen transfer rate and uptake rate during the fermentation. The results showed that the oxygen uptake rate increased with increasing DO, indicating that oxygen transfer was limited by the diffusion inside the mycelial layer.