Production of butanol by Clostridium puniceum in batch and continuous culture

Summary The pink-pigmented, amylolytic and pectinolytic bacterium Clostridium puniceum in anaerobic batch culture at pH 5.5 and 25–30°C produced butan-1-ol as the major product of fermentation of glucose or starch. The alcohol was formed throughout the exponential phase of growth and surprisingly little acetone was simultaneously produced. Furthermore, acetic and butyric acids were only accumulated in low concentrations, and under optimal conditions were completely re-utilised before the fermentation ceased. Thus, in a minimal medium containing 4% w/v glucose as sole source of carbon and energy, after 65 h at 25°C, pH 5.5 all of the glucose had been consumed to yield (g product/100 g glucose utilised) butanol 32, acetone 3 and ethanol 2. Butanol was again the major product of glucose fermentation during phosphate-limited chemostat culture wherein, although the organism eventually lost its capacity to sporulate and to synthesize granulose, production of butanol continued for at least 100 volume changes. Under no growth condition was the organism capable of producing more than 13.3 g l^-1 of butanol. At pH 5.5, growth on pectin was slow and yielded a markedly lesser biomass concentration than when growth was on glucose or starch; acetic acid was the major fermentation product with lower concentrations of methanol, acetone, butanol and butyric acid. At pH 7, growth on all substrates produced virtually no solvents but high concentrations of both acetic and butyric acids.     

Improvements of GC and HPLC analyses in solvent (acetone-butanol-ethanol) fermentation byClostridium saccharobutylicum using a mixture of starch and glycerol as carbon source

A study on the feasibility of using improved computer-controlled HPLC and GC systems was carried out to shorten the time needed for measuring levels of the substrates (glucose, maltose, and glycerol) and products (acetone, butanol ethanol, acetic acid, and butyric acid) produced byClostridium saccharobutylicum DSM 13864 during direct fermentation of sago starch to solvent. The use of HPLC system with a single injection to analyse the composition of culture broth (substrates and products) during solvent fermentation was achieved by raising the column temperature to 80°C. Although good separation of the components in the mixture was achieved, a slight overlap was observed in the peaks for butyric acid and acetone. The shape of the peak obtained and the analysis time of 26.66 min were satisfactory at a fixed flow rate of 0.8 mL/min. An improved GC system was developed, that was able to measure the products of solvent fermentation (acetone, butanol, ethanol, acetic acid, and butyric acid) within 19.28 min. Excellent resolution for each peak was achieved by adjusting the oven temperature to 65°C.     

Triphasic esterification of butanol and butyric acid in fermentation media

Butanol and butyric acid produced from acetone-butanol-ethanol (ABE) fermentation can be used to produce butyl butyrate, an important fragrance ester. However, low levels of butanol and butyric acid need to be purified from culture media first with energy-intensive distillation processes. In this study, a triphasic (organic/aqueous/fluorous) system is developed to esterify butanol and butyric acid in spent culture media into butyl butyrate directly without purification. The produced butyl butyrate forms a distinct organic phase floating on top and can then be separated easily. In a model system containing 37.1 g/L of butanol and 44.1 g/L of butyric acid, 57% of the butanol is converted to butyl butyrate after 8 h of esterification. With multiple cycles of esterification and product removal, butanol conversion can be further increased to 86%. When spent culture medium containing 7.12 g/L of butanol and 4.81 g/L of butyric acid is used for esterification, 38% of butanol (0.36 mmol) is consumed and 0.33 mmol of butyl butyrate is produced. However, when ABE fermentation and esterification are carried out simultaneously, only 0.042 mmol of butyl butyrate is produced, probably due to the incompatible pH requirements for cell growth (pH 5–7) and esterification (pH 2–3).     

Production of carboxylic acids from hydrolyzed corn meal by immobilized cell fermentation in a fibrous-bed bioreactor

Corn meal hydrolyzed with amylases was used as the carbon source for producing acetic, propionic, and butyric acids via anaerobic fermentations. In this study, corn meal, containing 75% (w/w) starch, 20% (w/w) fibers, and 1.5% (w/w) protein, was first hydrolyzed using amylases at 60 degrees C. The hydrolysis yielded approximately 100% recovery of starch converted to glucose and 17.9% recovery of protein. The resulting corn meal hydrolyzate was then used, after sterilization, for fermentation studies. A co-culture of Lactococcus lactis and Clostridium formicoaceticum was used to produce acetic acid from glucose. Propionibacterium acidipropionici was used for propionic acid fermentation, and Clostridium tyrobutylicum was used for butyric acid production. These cells were immobilized on a spirally wound fibrous matrix packed in a fibrous-bed bioreactor (FBB) developed for multi-phase biological reactions or fermentation. The bioreactor was connected to a stirred-tank fermentor that provided pH and temperature controls via medium circulation. The fermentation system was operated at the recycle batch mode. Temperature and pH were controlled at 37 degrees C and 7.6, respectively, for acetic acid fermentation, 32 degrees C and 6.0, respectively, for propionic acid fermentation, and 37 degrees C and 6.0, respectively, for butyric acid production. The fermentation demonstrated a yield of approximately 100% and a volumetric productivity of approximately 1 g/(1 h) for acetic acid production. The propionic acid fermentation achieved an approximately 60% yield and a productivity of 2.12 g/(1 h), whereas the butyric acid fermentation obtained an approximately 50% yield and a productivity of 6.78 g/(1 h). These results were comparable to, or better than those fermentations using chemically defined media containing glucose as the substrate, suggesting that these carboxylic acids can be efficiently produced from direct fermentation of corn meal hydrolyzate. The corn fiber present as suspended solids in the corn meal hydrolyzate did not cause operating problem to the immobilized cell bioreactor as is usually encountered by conventional immobilized cell bioreactor systems. It is concluded that the FBB technology is suitable for producing value-added biochemicals directly from agricultural residues or commodities such as corn meal.     

Bioconversion of Butyric Acid to Butanol by Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564) in a Limited Nutrient Medium

This study was designed to investigate the ability of Clostridium saccharoperbutylacetonicum N1-4 to produce butanol in a limited nutrient medium using mixtures of glucose and butyric acid as substrates. Specific combinations of glucose and butyric acid were found to influence the enhancement and retardation of butanol production as well as the reduction and modulation of the number of bacterial cells. Increasing the butyric acid concentration leads to the inhibition of bacterial growth, whereas the presence of (0?C5?g/L) butyric acid and (0?C10?g/L) glucose enhances the butanol production. The combination of 5?g/L butyric acid with 5 and 10?g/L of glucose was found to be the most suitable, but the use of glucose at concentrations greater than 10?g/L shifted the optimal butyric acid concentrations to 10 and 15?g/L for maximum butanol production signifying the requirement of a specific combination of glucose and butyric acid for enhanced butanol production in the fermentation process. C. saccharoperbutylacetonicum N1-4 demonstrated the ability to produce butanol in the absence of glucose, but no acetone or ethanol was produced under these conditions, reflecting the nature of the pathways involved in the production of butanol using only butyric acid. Ten grams per litre of butyric acid was found able to produce 13?g/L of butanol in the presence of 20?g/L of glucose, and 0.7?g/L butanol was produced in the absence of glucose. This study indicates the importance of the glucose to butyric acid ratio to the enhancement of butanol production.     

Solvent screening methodology for in situ ABE extractive fermentation

Solvent screening for in situ liquid extraction of products from acetone-butanol-ethanol (ABE) fermentation was carried out, taking into account biological parameters (biocompatibility, bioavailability, and product yield) and extraction performance (partition coefficient and selectivity) determined in real fermentation broth. On the basis of different solvent characteristics obtained from literature, 16 compounds from different chemical families were selected and experimentally evaluated for their extraction capabilities in a real ABE fermentation broth system. From these compounds, nine potential solvents were also tested for their biocompatibility towards Clostridium acetobutylicum. Moreover, bioavailability and differences in substrate consumption and total n-butanol production with respect to solvent-free fermentations were quantified for each biocompatible solvent. Product yield was enhanced in the presence of organic solvents having higher affinity for butanol and butyric acid. Applying this methodology, it was found that the Guerbet alcohol 2-butyl-1-octanol presented the best extracting characteristics (the highest partition coefficient (6.76) and the third highest selectivity (644)), the highest butanol yield (27.4 %), and maintained biocompatibility with C. acetobutylicum.     

Extractive fermentation for butyric acid production from glucose by Clostridium tyrobutyricum

A novel extractive fermentation for butyric acid production from glucose, using immobilized cells of Clostridium tyrobutyricum in a fibrous bed bioreactor, was developed by using 10% (v/v) Alamine 336 in oleyl alcohol as the extractant contained in a hollow-fiber membrane extractor for selective removal of butyric acid from the fermentation broth. The extractant was simultaneously regenerated by stripping with NaOH in a second membrane extractor. The fermentation pH was self-regulated by a balance between acid production and removal by extraction, and was kept at approximately pH 5.5 throughout the study. Compared with conventional fermentation, extractive fermentation resulted in a much higher product concentration (>300 g/L) and product purity (91%). It also resulted in higher reactor productivity (7.37 g/L. h) and butyric acid yield (0.45 g/g). Without on-line extraction to remove the acid products, at the optimal pH of 6.0, the final butyric acid concentration was only approximately 43.4 g/L, butyric acid yield was 0.423 g/g, and reactor productivity was 6.77 g/L. h. These values were much lower at pH 5.5: 20.4 g/L, 0.38 g/g, and 5.11 g/L. h, respectively. The improved performance for extractive fermentation can be attributed to the reduced product inhibition by selective removal of butyric acid from the fermentation broth. The solvent was found to be toxic to free cells in suspension, but not harmful to cells immobilized in the fibrous bed. The process was stable and provided consistent long-term performance for the entire 2-week period of study.     

Enhanced Butanol Production by Clostridium beijerinckii BA101 Grown in Semidefined P2 Medium Containing 6 Percent Maltodextrin or Glucose

Dramatically elevated levels of butanol and acetone resulted in higher butanol and total solvent yields for hyperamylolytic Clostridium beijerinckii BA101 relative to the NCIMB 8052 parent strain grown in semidefined P2 medium containing either 6% glucose or STAR-DRI 5 maltodextrin. C. beijerinckii BA101 consistently produced on the order of 19 g of butanol per liter in 20-liter batch fermentations. This represents a greater than 100% increase in butanol concentration by the BA101 strain compared to the parent NCIMB 8052 strain. The kinetics of butanol production over time also indicate a more rapid rate of butanol production by BA101 in semidefined P2 medium containing glucose or maltodextrin. The lower levels of butyric and acetic acids produced over the course of the fermentation carried out by BA101 are consistent with an enhanced capacity for uptake and recycling of these acids. C. beijerinckii BA101 appears to more completely utilize carbohydrate compared to the 8052 strain. Carbon balance following fermentation by C. beijerinckii 8052 and BA101 indicates that sufficient carbon is available for the twofold increase in butanol concentration observed during BA101 fermentations. C. beijerinckii BA101 also has superior solvent production capacity during continuous culture fermentation in P2 medium containing 6% glucose. Volumetric solvent yields of 0.78 and 1.74 g/liter/h for BA101 and 0.34 and 1.17 g/liter/h for NCIMB 8052 were obtained at dilution rates of 0.05 and 0.20 h(sup-1), respectively. No drift towards acid synthesis (strain degeneration) was observed for up to 200 h (d = 0.05 h(sup-1)) and 100 h (d = 0.20 h(sup-1)).     

Reverse osmosis processing of organic model compounds and fermentation broths

Post-treatment of an anaerobic fermentation broth was evaluated using a 150 gal/day, single cartridge prototype reverse osmosis (RO) system. Baseline tests were conducted at 25 degrees C using six organic model compounds representing key species found in the fermentation broth: ethanol, butanol, acetic acid, oxalic acid, lactic acid, and butyric acid. Correlations of the rejection and recovery efficiencies for these organic species, individually and in simulated mixtures, were obtained as a function of feed pressure with and without recirculation of the retentate. The actual fermentation broth obtained from a continuous-flow biohydrogen process was treated by the RO system under the operating conditions similar to those used in the baseline tests, resulting in greater than 95% removal of total organic carbon. These results are encouraging and useful for further studies on the feasibility of incorporating the RO technology into an integrated and field deployable wastewater management and water recovery system.     

Characterization of recombinant strains of the Clostridium acetobutylicum butyrate kinase inactivation mutant: need for new phenomenological models for solventogenesis and butanol inhibition?

Two metabolic engineering tools, namely gene inactivation and gene overexpression, were employed to examine the effects of two genetic modifications on the fermentation characteristics of Clostridium acetobutylicum. Inactivation of the butyrate kinase gene (buk) was examined using strain PJC4BK, while the combined effect of buk inactivation and overexpression of the aad gene-encoding the alcohol aldehyde dehydrogense (AAD) used in butanol formation-was examined using strain PJC4BK(pTAAD). The two strains were characterized in controlled pH > or = 5.0 fermentations, and by a recently enhanced method of metabolic flux analysis. Strain PJC4BK was previously genetically characterized, and fermentation experiments at pH > or = 5.5 demonstrated good, but not exceptional, solvent-production capabilities. Here, we show that this strain is a solvent superproducer in pH > or = 5.0 fermentations producing 225 mM (16.7 g/L) of butanol, 76 mM of acetone (4.4 g/L), and 57 mM (2.6 g/L) of ethanol. Strain PJC4BK(pTAAD) produced similar amounts of butanol and acetone but 98 mM (4.5 g/L) of ethanol. Both strains overcame the 180 mM (13 g/L) butanol toxicity limit, without any selection for butanol tolerance. Work with strain PJC4BK(pTAAD) is the first reported use of dual antibiotic selection in C. acetobutylicum. One antibiotic was used for selection of strain PJC4BK while the second antibiotic selected for the pTAAD presence. Overexpression of aad from pTAAD resulted in increased ethanol production but did not increase butanol titers, thus indicating that AAD did not limit butanol production under these fermentation conditions. Metabolic flux analysis showed a decrease in butyrate formation fluxes by up to 75% and an increase in acetate formation fluxes of up to 100% during early growth. The mean specific butanol and ethanol formation fluxes increased significantly in these recombinant strains, up to 300% and 400%, respectively. Onset of solvent production occurred during the exponential-growth phase when the culture optical density was very low and when total and undissociated butyric acid levels were <1 mM. Butyrate levels were low throughout all fermentations, never exceeding 20 mM. Thus, threshold butyrate concentrations are not necessary for solvent production in these stains, suggesting the need for a new phenomenological model to explain solvent formation.     

Agitation and pressure effects on acetone-butanol fermentation

Batch fermentations were run at varying agitation rates and were either pressurized to 1 bar (15.2 psig) or nonpressurized. Agitation and pressure both affect the level of dissolved hydrogen gas in the media, which in turn influences solvent production. In nonpressurized fermentations volumetric productivity of butanol increased as the agitation rate decreased. While agitation had no significant effect on butanol productivity under pressurized conditions, overall butanol productivity was increased over that obtained in the nonpressurized runs. Maximum butyric acid productivity, however, was found to occur earlier and increased as agitation increased. Peak hydrogen productivity occurred simultaneously with peak butyric acid productivity. The proporation of reducing equivalents used in forming the above products was determined using a redox balance based on the fermentation stoichiometry. An inverse relationship between the final concentrations of acetone and acetoin was found in all fermentations studied. The results show that agitation and pressure are important parameters for solvent productivity in acetone-butanol fermentation.     

Effects of extractive fermentation on butyric acid production byClostridium acetobutylicum

Summary The addition of an oleyl alcohol extractant to a batch fermentation of glucose byClostridium acetobutylicum resulted in a concentration profile that was distinctly different from the non-extractive control fermentation. The concentration of butyric acid increased and subsequently decreased in the control fermentation. The concentration of butyric acid increased but did not subsequently decrease in the oleyl alcohol extractive fermentation. The production of butyric acid was found to have been prolonged into the solventogenic phase in the oleyl alcohol extractive fermentation. Butyric acid was continually replenished from glucose while it was being converted to butanol. Supplementation of exogenous acetic and butyric acids, the metabolic uncoupler carbonyl cyanide 3-chlorophenylhydrazone, or decanol to the oleyl alcohol extractive fermentation helped to reinstate the normal butyric acid concentration profile. These findings are discussed with respect to the effects of these additives on the pH ofC. acetobutylicum and its importance with regard to the production of butyric acid.     

Clostridium thermobutyricum: growth studies and stimulation of butyrate formation by acetate supplementation

Clostridium thermobutyricum produces butyrate as the main fermentation product from glucose, and from yeast extract, which is required for substantial growth. After sequential transfer in the presence of increasing butyrate concentrations, strain JW 171 K grew in the presence of up to 350 mM butyrate either at pH 5.5 or at pH 8.0 and at 40 degrees C as well as at 60 degrees C. This result indicated that butyrate-dependent growth inhibition was independent from the concentration of undissociated butyric acid. Increased butyrate concentration decreased the level of tolerated glucose from above 15% to below 10%. At 0.05 and 2.0% (wt/vol) yeast extract, the Y(Glucose) was 30 and 55 g dry weight cells per mole glucose, respectively. Y(ATP) values between 18 and 21 g weight cells per mole ATP, obtained after growth in the presence of 2% yeast extract, indicate that the butyrate fermentation under thermophilic growth conditions is as energy efficient as it is under mesophilic conditions. Externally added acetate stimulated the production of butyrate. Supplemented 14C-acetate was converted to butyrate, resulting in the formation of 44% labeled butyrate (i.e. formed from 14C-acetate) and 56% unlabeled butyrate (formed from glucose and yeast extract). Continuous removal of H2 in batch cultures led to a shift in the fermentation products from more butyrate to the more oxidized and more energy yielding acetate.     

Production of butyric acid by batch fermentation of cheese whey withClostridium beijerinckii

Summary The effect of pH on the fermentation of butyric acid byClostridium beijerinckii using cheese whey as a substrate was studied. Maximum concentrations of the acid were produced when the pH was controlled at 5.5. Raising or lowering of pH was found to reduce the total acid formation. This particular strain ofC. beijerinckii produced insignificant amounts of butanol in all the pure culture cases investigated. A comparative study of the fermentation in a synthetic glucose medium and in cheese whey showed the whey to produce more butyric acid.     

Intracellular Conditions Required for Initiation of Solvent Production by Clostridium acetobutylicum

We investigated the intracellular physiological conditions associated with the induction of butanol-producing enzymes in Clostridium acetobutylicum. During the acidogenic phase of growth, the internal pH decreased in parallel with the decrease in the external pH, but the internal pH did not go below 5.5 throughout batch growth. Butanol was found to dissipate the proton motive force of fermenting C. acetobutylicum cells by decreasing the transmembrane pH gradient, whereas the membrane potential was affected only slightly. In growing cells, the switch from acid to solvent production occurred when the internal undissociated butyric acid concentration reached 13 mM and the total intracellular undissociated acid concentration (acetic plus butyric acids) was at least 40 to 45 mM. Similar values were obtained when cultures were supplemented with 50 mM butyric acid initially or when a phosphate-buffered medium was used instead of an acetate-buffered medium. To measure the induction of the enzymes involved in solvent synthesis, we determined the rates of conversion of butyrate to butanol in growing cells. The rate of butanol formation reached a maximum in the mid-solvent phase, when the butanol concentration was 50 mM. Although more solvent accumulated later, de novo enzyme synthesis decreased and then ceased.     

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.     

Selective separation of biobutanol from acetone-butanol-ethanol fermentation broth by means of sorption methodology based on a novel macroporous resin

The traditional distillation method for recovery of butanol from fermentation broth is an energy-intensive process. Separation of butanol based on adsorption methodology has advantages in terms of biocompatibility and stability, as well as economy, and therefore gains much attention. However, the application of the commercial adsorbents in the integrated acetone-butanol-ethanol (ABE) fermentation process is restricted due to the low recovery (less than 85%) and the weak capability of enrichment in the eluent (3-4 times). In this study, we investigated the sorption properties of butanol onto three kinds of adsorbents with different polarities developed in our laboratory, that is, XD-41, H-511, and KA-I resin. The sorption behaviors of single component and ABE ternary mixtures presented in the fermentation broths on KA-I resin were investigated. KA-I resin had higher affinity for butanol than for acetone, ethanol, glucose, acetic acid, and butyric acid. Multicomponent ABE sorption on KA-I resin was modeled using a single site extended Langmuir isotherm model. In a desorption study, all the adsorbed components were desorbed in one bed volume of methanol, and the recovery of butanol from KA-I resin was 99.7%. The concentration of butanol in the eluent was increased by a factor of 6.13. In addition, KA-I resin was successfully regenerated by two bed volumes of water. Because of its quick sorption, high sorption capacity, low cost, and ease of desorption and regeneration, KA-I resin exhibits good potential for compatibility with future ABE fermentation coupled with in situ recovery product removal techniques.     

Effects of butyric and acetic acids on acetone-butanol formation by Clostridium acetobutylicum

The actions of butyric and acetic acids on acetone-butanol fermentation are investigated. Production of butyric and acetic acids are controlled by the extracellular concentrations of both acids: acetic acid added to the medium inhibits its own formation but has no effect on butyric acid formation, and added butyric acid inhibits its own formation but not that of acetic acid. The ratio of end metabolites depends upon acetic and butyric acid quantities excreted during the fermentation. In contrast to acetic acid, which specifically increases acetone formation, butyric acid increases both acetone and butanol formations. Acetate and butyrate kinase activities were also examined. Both increase at the start of fermentation and decrease when solvents appear in the medium. Coenzyme A transferase activity is weak in the acidogenic phase and markedly increases in the solvent phase. Acetic and butyric acids appear to be co-substrates. On the basis of these results, a mechanism of acetic and butyric acid pathways, coupled to solvent formation by C. acetobutylicum glucose fermentation is proposed.     

添加有机酸对Clostridium acetobutylicum合成丙酮和丁醇的影响

为提高丙酮-丁醇梭菌厌氧发酵生产丙酮和丁醇的能力,在发酵过程中添加有机酸（乙酸和丁酸）,考察其对菌体生长、溶剂合成影响。实验表明：当添加1.5 g/L乙酸时能够促进菌体的生长,促进丙酮的合成,在600 nm处的最大OD值比参照值高出18.4%,丙酮的最终质量分数提高了21.05%,但不能促进丁醇的合成;当添加1.0g/L丁酸时能够促进菌体生长,促进丁醇的合成,在600 nm处的最大OD比参照值高22.29%,丁醇的最终质量分数比对照组提高了24.32%,但不能促进丙酮的合成。