Continuous production of butanol from starch-based packing peanuts

Acetone, butanol, ethanol (ABE, or solvents) were produced from starch-based packing peanuts in batch and continuous reactors. In a batch reactor, 18.9 g/L of total ABE was produced from 80 g/L packing peanuts in 110 h of fermentation. The initial and final starch concentrations were 69.6 and 11.1 g/L, respectively. In this fermentation, ABE yield and productivity of 0.32 and 0.17 g/(L·h) were obtained, respectively. Compared to the batch fermentation, continuous fermentation of 40 g/L of starch-based packing peanuts in P2 medium resulted in a maximum solvent production of 8.4 g/L at a dilution rate of 0.033 h⁻¹. This resulted in a productivity of 0.27 g/(L·h). However, the reactor was not stable and fermentation deteriorated with time. Continuous fermentation of 35 g/L of starch solution resulted in a similar performance. These studies were performed in a vertical column reactor using Clostridium beijerinckii BA101 and P2 medium. It is anticipated that prolonged exposure of culture to acrylamide, which is formed during boiling/autoclaving of starch, affects the fermentation negatively.     

Oil Palm Empty Fruit Bunch as Alternative Substrate for Acetone–Butanol–Ethanol Production by <Emphasis Type="Italic">Clostridium butyricum</Emphasis> EB6

Acetone–butanol–ethanol (ABE) production from renewable resources has been widely reported. In this study, Clostridium butyricum EB6 was employed for ABE fermentation using fermentable sugar derived from treated oil palm empty fruit bunch (OPEFB). A higher amount of ABE (2.61 g/l) was produced in a fermentation using treated OPEFB as the substrate when compared to a glucose based medium that produced 0.24 g/l at pH 5.5. ABE production was increased to 3.47 g/l with a yield of 0.24 g/g at pH 6.0. The fermentation using limited nitrogen concentration of 3 g/l improved the ABE yield by 64%. The study showed that OPEFB has the potential to be applied for renewable ABE production by C. butyricum EB6.     

Effectively Converting Cane Molasses into 2,3-Butanediol Using Clostridium ljungdahlii by an Integrated Fermentation and Membrane Separation Process

Firstly, 2,3-butanediol (2,3-BDO) is a chemical platform used in several applications. However, the pathogenic nature of its producers and the expensive feedstocks used limit its scale production. In this study, cane molasses was used for 2,3-BDO production by a nonpathogenic Clostridium ljungdahlii. It was found that cane molasses alone, without the addition of other ingredients, was favorable for use as the culture medium for 2,3-BDO production. Compared with the control (i.e., the modified DSMZ 879 medium), the differential genes are mainly involved in the pathways of carbohydrate metabolism, membrane transport, and amino acid metabolism in the case of the cane molasses alone. However, when cane molasses alone was used, cell growth was significantly inhibited by KCl in cane molasses. Similarly, a high concentration of sugars (i.e., above 35 g/L) can inhibit cell growth and 2,3-BDO production. More seriously, 2,3-BDO production was inhibited by itself. As a result, cane molasses alone with an initial 35 g/L total sugars was suitable for 2,3-BDO production in batch culture. Finally, an integrated fermentation and membrane separation process was developed to maintain high 2,3-BDO productivity of 0.46 g·L⁻¹·h⁻¹. Meanwhile, the varied fouling mechanism indicated that the fermentation properties changed significantly, especially for the cell properties. Therefore, the integrated fermentation and membrane separation process was favorable for 2,3-BDO production by C. ljungdahlii using cane molasses.     

Ethanol fermentation in a tower fermenter using self-aggregatingSaccharomyces uvarum

A self-aggregating strain ofSaccharomyces uvarum (U4) was used as a biocatalyst to carry out continuous ethanol fermentation in a tower fermentor equipped with a cell separator. Cell aggregates (2–3 mm) formed a stable packed bed in the fermentor, and the cell separator retained yeast cells effectively. Corn steep liquor was used as a nitrogen source for the fermentation of corn syrup and black strap molasses. An ethanol productivity of 54 g/L/h was reached using corn syrup at a dilution rate of 0.7/h, and sugar concentration in the feed was 15% (w/v). For molasses fermentation, an ethanol productivity of 22 g/L/h was obtained at a dilution rate of 0.7/h, and sugar concentration in the feed was 12.5% (w/v). Ethanol yields obtained from tower fermentation are higher than those obtained from flask fermentation (96% for corn syrup fermentation and 92% for molasses fermentation). No significant loss in fermentation activity was observed after 3 mo of operation.     

Production of acetone butanol ethanol from degermed corn using Clostridium beijerinckii BA101

In this article we report on acetone butanol ethanol (ABE) fermentation characteristics of degermed corn when using Clostridium beijerinckii BA101. Recent economic studies suggested that recovery of germ from corn and hence corn oil would help to make the ABE fermentation process more economical. C. beijerinckii BA101 ferments corn mash efficiently to produce ABE under appropriate nutritional and environmental conditions. Corn mash contains germ/corn oil that is, possibly, ancillary to the production of butanol during the ABE fermentation process. Since the presence of corn oil is not a critical factor in solvent fermentation, it can be removed and this will allow for byproduct credit. Batch fermentation of degermed corn resulted in 8.93 g/L of total ABE production as compared with 24.80 g/L of total ABE when supplemented with P2 medium nutrients. During the course of the germ separation process, corn steeping is required prior to grinding and removing the germ. It is likely that some nutrients from the corn are leached out during the steeping process. This may reduce the rate of fermentation and impact the final concentration of butanol/ABE that can be achieved. Fermentation of degermed corn with corn steep liquor resulted in the production of 19.28 g/L of ABE.     

Direct Fermentation of Gelatinized Cassava Starch to Acetone,Butanol, and Ethanol Using Clostridium acetobutylicum Mutant Obtained by Atmospheric and Room Temperature Plasma

The mutant strain designated as ART18, obtained from the wild-type strain Clostridium acetobutylicum PW12 treated by atmospheric and room temperature plasma, showed higher solvent tolerance and butanol production than that of the wild-type strain. The production of butanol was 11.3?±?0.5 g/L, 31 % higher than that of the wild-type strain when it was used for acetone, butanol, and ethanol fermentation in P2 medium. Furthermore, the effects of cassava flour concentration, pH regulators, and vitamins on the ABE production were also investigated. The highest butanol production of 15.8?±?0.8 g/L and butanol yield (0.31 g/g) were achieved after the above factors were optimized. When acetone, butanol, and ethanol fermentation by ART18 was carried out in a 15-L bioreactor, the butanol production, the productivity of butanol, and the total solvent were 16.3?±?0.9, 0.19, and 0.28 g/L^/h, respectively. These results indicate that ART18 is a promising industrial producer in ABE fermentation.     

Maximizing the xylitol production from sugar cane bagasse hydrolysate by controlling the aeration rate

Batch fermentations of sugar cane bagasse hemicellulosic hydrolysate treated for removing the inhibitors of the fermentation were performed byCandida guilliermondii FTI20037 for xylitol production. The fermentative parameters agitation and aeration rate were studied aiming the maximization of xylitol production from this agroindustrial residue. The maximal xylitol volumetric productivity (0.87 g/L h) and yield (0.67 g/g) were attained at 400/min and 0.45 v.v.m. (K_La 27/h). According to the results, a suitable control of the oxygen input permitting the xylitol formation from sugar cane bagasse hydrolysate is required for the development of an efficient fermentation process for large-scale applications.     

A Novel Process for Direct Production of Acetone–Butanol–Ethanol from Native Starches Using Granular Starch Hydrolyzing Enzyme by Clostridium saccharoperbutylacetonicum N1-4

In this work, a new approach for acetone–butanol–ethanol (ABE) production has been proposed. Direct fermentation of native starches (uncooked process) was investigated by using granular starch hydrolyzing enzyme (GSHE) and Clostridium saccharoperbutylacetonicum N1-4. Even the process was carried out under suboptimal condition for activity of GSHE, the production of ABE was similar with that observed in conventional process or cooked process in terms of final solvent concentration (21.3?±?0.4 to 22.4?±?0.4 g/L), butanol concentration (17.5?±?0.4 to 17.8?±?0.3 g/L) and butanol yield (0.33 to 0.37 g/g). The production of solvents was significantly dependent on the source of starches. Among investigated starches, corn starch was more susceptible to GSHE while cassava starch was the most resistant to this enzyme. Fermentation using native corn starch resulted in the solvent productivity of 0.47 g/L h, which was about 15 % higher than that achieved in cooked process. On the contrary, uncooked process using cassava and wheat starch resulted in the solvent productivity of 0.30 and 0.37 g/L h, which were respectively about 30 % lower than those obtained in cooked process. No contamination was observed during all trials even fermentation media were prepared without sterilization. During the fermentation using native starches, no formation of foam is observed. This uncooked process does not require cooking starchy material; therefore, the thermal energy consumption for solvent production would remarkably be reduced in comparison with cooked process.     

Statistical Optimization for Succinic Acid Production from E. Coli in a Cost-Effective Medium

Response surface methodology (RSM) was employed for optimization of medium components and cultural parameters in cost effective cane molasses based medium for attaining high yield of succinic acid. The important factors obtained by “one-variable-at-a-time-approach” (cane molasses, corn steep liquor, sodium carbonate, and inoculum density) were further optimized by RSM. The optimum values of the parameters obtained through RSM (cane molasses 12.5%, corn steep liquor 7.5%, and sodium carbonate 25 mM) led to almost double yield of succinic acid (15.2 g/l in 36 h) as against “one-variable-at-a-time-approach” (7.1 g/l in 36 h) in 500-ml anaerobic bottles containing 300-ml cane molasses based medium. Subsequently, in 10-l bioreactor succinic acid production from Escherichia coli was further improved to 26.2 g/l in 30 h under conditions optimized through RSM. This fermentation-derived succinic acid will definitely help in replacing existing environmentally hazardous and cost-intensive chemical methods for the production of succinic acid.     

Continuous Ethanol Production from Cassava Through Simultaneous Saccharification and Fermentation by Self-Flocculating Yeast Saccharomyces Cerevisiae CHFY0321

In this study, a fermentor consisting of four linked stirred towers that can be used for simultaneous saccharification and fermentation (SSF) and for the accumulation of cell mass was applied to the continuous production of ethanol using cassava as the starchy material. For the continuous process with SSF, the pretreated cassava liquor and saccharification enzyme at total sugar concentrations of 175 g/L and 195 g/L were continuously fed to the fermentor with dilution rates of 0.014, 0.021, 0.031, 0.042, and 0.05 h⁻¹. Considering the maximum saccharification time, the highest volumetric productivity and ethanol yield were observed at a dilution rate of 0.042 h⁻¹. At dilution rates in the range of 0.014 h⁻¹ to 0.042 h⁻¹, high production rates were observed, and the yeast in the first to fourth fermentor showed long-term stability for 2 months with good performance. Under the optimal culture conditions with a feed sugar concentration of 195 g/L and dilution rate of 0.042 h⁻¹, the ethanol volumetric productivity and ethanol yield were 3.58 g/L∙h and 86.2%, respectively. The cell concentrations in the first to fourth stirred tower fermentors were 74.3, 71.5, 71.2, and 70.1 g dry cell/L, respectively. The self-flocculating yeast, Saccharomyces cerevisiae CHFY0321, developed by our group showed excellent fermentation results under continuous ethanol production.     

Kinetic and Stoichiometric Parameters in the Production of Carotenoids by <Emphasis Type="Italic">Sporidiobolus salmonicolor</Emphasis> (CBS 2636) in Synthetic and Agroindustrial Media

With the objective of determining the kinetic behavior (growth, substrate, pH, and carotenoid production) and obtain the stoichiometric parameters of the fermentative process by Sporidiobolus salmonicolor in synthetic and agroindustrial media, fermentations were carried out in shaken flasks at 25°C, 180 rpm, and initial pH of 4.0 for 120 h in the dark, sampling every 6 h. The maximum concentrations of total carotenoids in synthetic (913 μg/L) and agroindustrial (502 μg/L) media were attained approximately 100 h after the start of the fermentative process. Carotenoid bioproduction is associated with cell growth and the ratio between carotenoid production and cell growth (Y _P/X) is 176 and 163 μg/g in the synthetic and agroindustrial media, respectively. The pH of the agroindustrial fermentation medium varied from 4.2 to 8.5 during the fermentation. The specific growth rate (μ _X) for S. salmonicolor in synthetic and agroindustrial media was 0.07 and 0.04 h⁻¹, respectively. The synthetic medium allowed for greater productivity, obtaining maximum cell productivity (P _x) of 0.08 g L⁻¹ h⁻¹ and maximum total carotenoid productivity (P _car) of 14.2 μg L⁻¹ h⁻¹. Knowledge of the kinetics of a fermentative process is of extreme importance when transposing a laboratory experiment to an industrial scale, as well as making a quantitative comparison between different culture conditions.     

Production and characterization of pullulan from beet molasses using a nonpigmented strain of Aureobasidium pullulans in batch culture

The production of pullulan from beet molasses by a pigment-free strain of Aureobasidium pullulans on shake-flask culture was investigated. Combined pretreatment of molasses with sulfuric acid and activated carbon to remove potential fermentation inhibitors present in molasses resulted in a maximum pullulan concentration of 24 g/L, a biomass dry wt of 14 g/L, a pullulan yield of 52.5%, and a sugar utilization of 92% with optimum fermentation conditions (initial sugar concentration of 50 g/L and initial pH of 7.0). The addition of other nutrients as carbon and nitrogen supplements (olive oil, ammonium sulfate, yeast extract) did not further improve the production of the exopolysaccharides. Structural characterization of the isolated polysaccharides from the fermentation broths by ¹³C-nuclear magnetic resonance spectroscopy and pullulanase digestion combined with size-exclusion chromatography confirmed the identity of pullulan and the homogeneity (>93% dry basis) of the elaborated polysaccharides by the microorganism. Using multiangle laser light scattering and refractive index detectors in conjunction with high-performance size-exclusion chromatography molecular size distributions and estimates of the molecular weight (M _w =2.1−4.1×10⁵), root mean square of the radius of gyration (R _g =30−38 nm), and polydispersity index (M _w /M _n =1.4−2.4) were obtained. The fermentation products of molasses pretreated with sulfuric acid and/or activated carbon were more homogeneous and free of contaminating proteins. In the concentration range of 2.8−10.0 (w/v), the solution’s rheologic behavior of the isolated pullulans was almost Newtonian (within 1 and 1200 s⁻¹ at 20°C); a slight shear thinning was observed at 10.0 (w/v) for the high molecular weight samples. Overall, beet molasses pretreated with sulfuric acid and activated carbon appears as an attractive fermentation medium for the production of pullulan by A. pullulans.     

Production of Acetone–Butanol–Ethanol (ABE) in Direct Fermentation of Cassava by Clostridium saccharoperbutylacetonicum N1-4

In this work, acetone–butanol–ethanol (ABE) fermentation characteristics of cassava starch and cassava chips when using Clostridium saccharoperbutylacetonicum N1-4 was presented. The obtained results in batch mode using a 1-L fermenter showed that C. saccharoperbutylacetonicum N1-4 was a hyperamylolytic strain and capable of producing solvents efficiently from cassava starch and cassava chips, which was comparable to when glucose was used. Batch fermentation of cassava starch and cassava chips resulted in 21.0 and 19.4 g/L of total solvent as compared with 24.2 g/L of total solvent when using glucose. Solvent productivity in fermentation of cassava starch was from 42% to 63% higher than that obtained in fermentation using corn and sago starches in the same condition. In fermentation of cassava starch and cassava chips, maximum butanol concentration was 16.9 and 15.5 g/L, respectively. Solvent yield and butanol yield (based on potential glucose) was 0.33 and 0.41, respectively, for fermentation of cassava starch and 0.30 and 0.38, respectively for fermentation using cassava chips.     

Ultrasound assisted pseudo-digestion for determination of iron and manganese in citric acid fermentation mediums by electrothermal atomic absorption spectroscopy

A sensitive, simple and rapid method for ultra-trace determination of iron and manganese based on ultrasound assisted pseudodigestion in citric acid fermentation medium samples (beet and cane molasses and raw sugar based mediums) is described. Parameters influencing pseudo-digestion, such as sonication time, sample mass and solvent system were fully optimized. Final solutions obtained upon sonication were analyzed by electrothermal atomic absorption spectrometry (ETAAS). The best conditions for metal pseudo-digestion were as follows: a 25, 30 and 20 min sonication time for beet molasses, cane molasses and raw sugar based medium samples, respectively, 0.7 g sample mass of raw sugar based samples, 0.5 g sample mass of molasses based samples and an extraction mixture of concentrated HNO₃-H₂O₂, in 25 mL of solvent. Analytical results obtained for the two metals by ultrasound assisted pseudo-digestion and conventional wet digestion methods showed a good agreement. This method reduces the time required for all treatments (heating to dryness, cooling and separation) in comparison with conventional wet digestion method. The accuracy of the method was tested by comparing the obtained results with that of conventional wet digestion method.      

High-productivity continuous biofilm reactor for butanol production

Corn steep liquor (CSL), a byproduct of the corn wet-milling process, was used in an immobilized cell continuous biofilm reactor to replace the expensive P2 medium ingredients. The use of CSL resulted in the production of 6.29 g/L of total acetone-butanol-ethanol (ABE) as compared with 6.86 g/L in a control experiment. These studies were performed at a dilution rate of 0.32 h⁻¹. The productivities in the control and CSL experiment were 2.19 and 2.01 g/(L·h), respectively. Although the use of CSL resulted in a 10% decrease in productivity, it is viewed that its application would be economical compared to P2 medium. Hence, CSL may be used to replace the P2 medium. It was also demonstrated that inclusion of butyrate into the feed was beneficial to the butanol fermentation. A control experiment produced 4.77 g/L of total ABE, and the experiment with supplemented sodium butyrate produced 5.70 g/L of total ABE. The butanol concentration increased from 3.14 to 4.04 g/L. Inclusion of acetate in the feed medium of the immobilized cell biofilm reactor was not found to be beneficial for the ABE fermentation, as reported for the batch ABE fermentation. Names are necessary to report factually on available data. However, the USDA neither guarantees nor warrants the standard of the product, and the use of the names by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Effect of media composition and growth conditions on production of β-glucosidase by Aspergillus niger C-6

The hydrolytic activity of fungal originated β-glucosidase is exploited in several biotechnological processes to increase the rate and extent of saccharification of several cellulosic materials by hydrolyzing the cellobiose which inhibits cellulases. In a previous presentation, we reported the screening and liquid fermentation with Aspergillus niger, strain C-6 for β-glucosidase production at shake flask cultures in a basal culture medium with mineral salts, corn syrup liquor, and different waste lignocellulosic materials as the sole carbon source obtaining the maximum enzymatic activity after 5–6 d of 8.5 IU/mL using native sugar cane bagasse. In this work we describe the evaluation of fermentation conditions: growth temperature, medium composition, and pH, also the agitation and aeration effects for β-glucosidase production under submerged culture using a culture media with corn syrup liquor (CSL) and native sugar cane bagasse pith as the sole carbon source in a laboratory fermenter. The maximum enzyme titer of 7.2 IU/mL was obtained within 3 d of fermentation. This indicates that β-glucosidase productivity by Aspergillus niger C-6 is function of culture conditions, principally temperature, pH, culture medium conditions, and the oxygen supply given in the bioreactor. Results obtained suggest that this strain is a potential microorganism that can reach a major level of enzyme production and also for enzyme characterization.     

Production of Butyric Acid from Glucose and Xylose with Immobilized Cells of <Emphasis Type="Italic">Clostridium tyrobutyricum</Emphasis> in a Fibrous-bed Bioreactor

Butyric acid has many applications in chemical, food, and pharmaceutical industries. In the present study, Clostridium tyrobutyricum ATCC 25755 was immobilized in a fibrous-bed bioreactor to evaluate the performance of butyrate production from glucose and xylose. The results showed that the final concentration and yield of butyric acid were 13.70 and 0.46 g g⁻¹, respectively, in batch fermentation when 30 g L⁻¹ glucose was introduced into the bioreactor. Furthermore, high concentration 10.10 g L⁻¹ and yield 0.40 g g⁻¹ of butyric acid were obtained with 25 g L⁻¹ xylose as the carbon source. The immobilized cells of C. tyrobutyricum ensured similar productivity and yield from repeated batch fermentation. In the fed-batch fermentation, the final concentration of butyric acid was further improved to 24.88 g L⁻¹ with one suitable glucose feeding in the fibrous-bed bioreactor. C. tyrobutyricum immobilized in the fibrous-bed bioreactor would provide an economically viable fermentation process to convert the reducing sugars derived from plant biomass into the final bulk chemical (butyric acid).     

Enhancing Clostridial Acetone-Butanol-Ethanol (ABE) Production and Improving Fuel Properties of ABE-enriched Biodiesel by Extractive Fermentation with Biodiesel

The extractive acetone–butanol–ethanol (ABE) fermentations of Clostridium acetobutylicum were evaluated using biodiesel as the in situ extractant. The biodiesel preferentially extracted butanol, minimized product inhibition, and increased production of butanol (from 11.6 to 16.5 gL⁻¹) and total solvents (from 20.0 to 29.9 gL⁻¹) by 42% and 50%, respectively. The fuel properties of the ABE-enriched biodiesel obtained from the extractive fermentations were analyzed. The key quality indicators of diesel fuel, such as the cetane number (increased from 48 to 54) and the cold filter plugging point (decreased from 5.8 to 0.2 °C), were significantly improved for the ABE-enriched biodiesel. Thus, the application of biodiesel as the extractant for ABE fermentation would increase ABE production, bypass the energy intensive butanol recovery process, and result in an ABE-enriched biodiesel with improved fuel properties.     

Continuous production of butanol by clostridium acetobutylicum immobilized in a fibrous bed bioreactor

We explored the influence of dilution rate and pH in continuous cultures of Clostridium acetobutylicum. A 200-mL fibrous bed bioreactor was used to produce high cell density and butyrate concentrations at pH 5.4 and 35°C. By feeding glucose and butyrate as a cosubstrate, the fermentation was maintained in the solventogenesis phase, and the optimal butanol productivity of 4.6g/(L h) and a yield of 0.42 g/g were obtained at a dilution rate of 0.9h⁻¹ and pH 4.3. Compared to the conventional acetone-butanol-ethanol fermentation, the new fermentation process greatly improved butanol yield, making butanol production from corn an attractive alternative to ethanol fermentation.     

Biosynthesis of poly-β-hydroxybutyrate and exopolysaccharides on azotobacter chroococcum strain 6B utilizing simple and complex carbon sources

Coproduction of poly-β-hydroxybutyrate (PHB) and exopolysaccharides (EPS) was investigated with Azotobacter chroococcum strain 6B isolated from soil samples. The bacterium was cultured using various carbon sources solely or with 0.1 g/L of ammonium sulfate. Ammonium addition resulted in reduced PHB and EPS production with glucose, fructose, and sucrose media, but cellular mass remained constant except for sucrose. Protein was nearly twofold higher in ammonium-grown cultures. Glucose and fructose alone biosynthesized high amounts of EPS (maximum 2.1 and 1.1 g/L, respectively, at 72 h), whereas PHB was accumulated only in glucose-grown cells. Sucrose almost did not produce EPS. Conversely, PHB content was the highest obtained from all experimented conditions (1.1 g/L at 48 h, 40% cell dry wt). When a complex carbon source such as sugar cane molasses was utilized, PHB was accumulated concomitant with EPS production from the initial time to 48 h (0.75 g/L, 37% cell dry wt and 0.6 g/L, respectively), and then PHB decayed at 72 h (0.2 g/L). On the other hand, EPS continued to be biosynthesized (1.1 g/L, 72 h). PHB fractions of total intra- and extracellular biopolymers were calculated. Sucrose-modified Burk’s medium without ammonium addition is suggested as a medium capable of diverting the carbon source for the production of intracellular PHB rather than EPS with A. chroococcum 6B.