Stirred Tank Fermentor Research Articles

Scaled-up production and recovery of lipopeptide biosurfactant and its application for washing petroleum-contaminated drill cuttings

A simple and efficient fermentation strategy using chitosan-immobilized Bacillus subtilis GY19 was developed for large-scale lipopeptide production from waste glycerol and palm oil. When a fed-batch fermentation approach was employed, excessive foaming was minimized, while lipopeptide production was greater than that of conventional batch fermentation. When a 700L stirred tank fermenter with a 400L working volume was used, a lipopeptide concentration and yield of 7.55g/L and 0.190g/g of substrate, respectively, were achieved. The pilot-scale recovery process involved sequential bacterial cell removal via centrifugation, elimination of impurities through acidification of the cell-free broth, and partial purification and concentration of the lipopeptides via ultrafiltration. A 5kDa stabilized cellulose-based membrane maintained a constant permeate flux and resulted in 67.2% lipopeptide recovery. The concentrated lipopeptide retentate contained 16.8g/L lipopeptides and a surface tension of 28.82 mN/m. A biobased washing agent for the treatment of petroleum-contaminated drill cuttings was later formulated with the retentate and various vegetable oil-based surfactants via the simplex−lattice mixture design methodology. The mixture of 1% retentate and 4% fatty alcohol ethoxylate 5EO presented the greatest removal efficiency of 83.2% for drill cuttings containing 15% (w/w) total petroleum hydrocarbons. The formulation also mobilized petroleum hydrocarbons into a distinct free oil layer, which would be easy to separate and subsequently recycle. These findings demonstrate the scalability of the lipopeptide production and recovery process, while the biobased washing agent offers an environmentally friendly approach for petroleum remediation.

Novel Batch and Repeated-Batch Butanol Fermentation from Sweet Sorghum Stem Juice by Co-Culture of Arthrobacter and Immobilized Clostridium in Scaled-Up Bioreactors

This research aims to study butanol fermentation from sweet sorghum stem juice (SSJ) by immobilized Clostridium beijerinckii TISTR 1461 cells on bamboo chopsticks using Arthrobacter sp. as an efficient bacterium for creating anaerobic conditions in scaled-up bioreactors. For batch culture in a 1-L screw-capped bottle, a butanol concentration (PB), butanol productivity (QB), and butanol yield (YB/S) were 12.09 g/L, 0.26 g/L·h and 0.28 g/g, respectively. These values were ~8 to 14% higher than those of a single culture using oxygen-free nitrogen (OFN) gas to generate anaerobic conditions. When butanol fermentation by the co-culture was scaled-up to 5-L and 30-L stirred-tank fermenters, the butanol production efficiency was not different from that using the 1-L bottles. Additionally, repeated-batch butanol fermentation in the 1-L bottles by the co-culture was successfully operated for four successive cycles with high butanol production. All results clearly indicate that Arthrobacter sp. is promising for creation of anaerobic conditions for butanol production by immobilized Clostridium in large scale bioreactors.

Investigation into struvite precipitation: A commonly encountered problem during fermentations on chemically defined media.

Chemically defined mineral media are widely used in bioprocesses, as these show less batch to batch variation compared with complex media. Nonetheless, the recommended media formulations often lead to the formation of precipitants at elevated pH values. These precipitates are insoluble and reduce the availability of macronutrients to the cells, which can result in limiting growth rates and lower productivity. They can also damage equipment by clogging pipes, hoses, and spargers in stirred tank fermenters. In this study, the observed precipitate was analyzed via X-ray fluorescence spectroscopy and identified as the magnesium ammonium phosphate salt struvite (MgNH4 PO4 × 6H2 O). The solubility of struvite crystals is known to be extremely low, causing the macronutrients magnesium, phosphate, and ammonium to be bound in the struvite crystals. Here, it was shown that struvite precipitates can be redissolved under common fermentation conditions. Furthermore, it was found that the struvite particle size distribution has a significant effect on the dissolution kinetics, which directly affects macronutrient availability. At a certain particle size, struvite crystals rapidly dissolved and provided unlimiting growth conditions. Therefore, struvite formation should be considered during media and bioprocess development, to ensure that the dissolution kinetics of struvite are faster than the growth kinetics.

Lipase Production by Limtongozyma siamensis, a Novel Lipase Producer and Lipid Accumulating Yeast.

Lipase is a well-known and highly in-demand enzyme. During the last decade, several lipase optimization studies have been reported. However, production costs have always been a bottleneck for commercial-scale microbial enzyme production. This research aimed to optimize the conditions for lipase production by Limtongozyma siamensis DMKU-WBL1-3 via a One-Factor-At-a-Time (OFAT) approach combined with statistical methods while using a low-cost substrate. Results suggest that low-cost substrates can be substituted for all media components. An optimal medium was found, using response surface methodology (RSM) and central composite design (CCD), to consist of 0.50% (w/v) sweet whey, 0.40% (w/v) yeast extract (food grade), and 2.50% (v/v) palm oil with the medium pH adjusted to 4 under shaking flask cultivation. From an economic point of view, this work was successful in reducing production costs while increasing lipase productivity. The medium costs were reduced by 87.5% of the original cost while lipase activity was increased by nearly 6-fold. Moreover, lipase production was further studied in a 2-L stirred-tank fermentor. Its activity was 1,055.6 ± 0.0 U/ml when aeration and agitation rates were adjusted to 1 vvm and 170 rpm, respectively. Interestingly, under this optimal lipase production, the yeast showed accumulated lipids inside the cells. The primary fatty acid is a monounsaturated fatty acid (MUFA) that is typically linked to health benefits. This study hence reveals promising lipase production and lipid accumulation by L. siamensis DMKU-WBL1-3 that are worthy of further study.

Continuous lactate-driven dark fermentation of restaurant food waste: Process characterization and new insights on transient feast/famine perturbations

The effect of hydraulic retention time (HRT) on the continuous lactate-driven dark fermentation (LD-DF) of food waste (FW) was investigated. The robustness of the bioprocess against feast/famine perturbations was also explored. The stepwise HRT decrease from 24 to 16 and 12 h in a continuously stirred tank fermenter fed with simulated restaurant FW impacted on hydrogen production rate (HPR). The optimal HRT of 16 h supported a HPR of 4.2 L H2/L-d. Feast/famine perturbations caused by 12-h feeding interruptions led to a remarkable peak in HPR up to 19.2 L H2/L-d, albeit the process became stable at 4.3 L H2/L-d following perturbation. The occurrence of LD-DF throughout the operation was endorsed by metabolites analysis. Particularly, hydrogen production correlated positively with lactate consumption and butyrate production. Overall, the FW LD-DF process was highly sensitive but resilient against transient feast/famine perturbations, supporting high-rate HPRs under optimal HRTs.

New two-stage pH combined with dissolved oxygen control strategy for cyclic β-1,2 glucans synthesis.

On the basis of a novel two-stage pH combined with dissolved oxygen (DO) control strategy in fed-batch fermentation, this research addresses the influence of pH on cyclic β-1,2-glucans (CβGs) biosynthesis and melanin accumulation during the production of CβGs by Rhizobium radiobacter ATCC 13,333. Under these optimal fermentation conditions, the maximum cell concentration and CβGs concentration in a 7-L stirred-tank fermenter were 7.94gL-1 and 3.12gL-1, which were the maximum production reported for R. radiobacter. The melanin concentration of the fermentation broth was maintained at a low level, which was beneficial to the subsequent separation and purification of the CβGs. In addition, a neutral extracellular oligosaccharide (COGs-1) purified by the two-stage pH combined with DO control strategy fermentation medium was structurally characterized. Structural analyses indicated that COGs-1 was a family of unbranched cyclic oligosaccharides composed of only β-1,2-linked D-glucopyranose residues with degree of polymerization between 17 and 23, namely CβGs. This research provides a reliable source of CβGs and structural basis for further studies of biological activity and function. KEY POINTS: • A two-stage pH combined with DO control strategy was proposed for CβGs production and melanin biosynthesis by Rhizobium radiobacter. • The final extracellular CβGs production reached 3.12 gL-1, which was the highest achieved by Rhizobium radiobacter. • The existence of CβGs could be detected by TLC quickly and accurately.

Effects of Fe2+ addition to sugarcane molasses on poly-γ-glutamic acid production in Bacillus licheniformis CGMCC NO. 23967

BackgroundPoly-γ-glutamic acid (γ-PGA) is biodegradable, water-soluble, environment-friendly, and edible. Consequently, it has a variety of industrial applications. It is crucial to control production cost and increase output for industrial production γ-PGA.ResultsHere γ-PGA production from sugarcane molasses by Bacillus licheniformis CGMCC NO. 23967 was studied in shake-flasks and bioreactors, the results indicate that the yield of γ-PGA could reach 40.668 g/L in a 5L stirred tank fermenter. Further study found that γ-PGA production reached 70.436 g/L, γ-PGA production and cell growth increased by 73.20% and 55.44%, respectively, after FeSO4·7H2O was added. Therefore, we investigated the metabolomic and transcriptomic changes following FeSO4·7H2O addition. This addition resulted in increased abundance of intracellular metabolites, including amino acids, organic acids, and key TCA cycle intermediates, as well as upregulation of the glycolysis pathway and TCA cycle.ConclusionsThese results compare favorably with those obtained from glucose and other forms of biomass feedstock, confirming that sugarcane molasses can be used as an economical substrate without any pretreatment. The addition of FeSO4·7H2O to sugarcane molasses may increase the efficiency of γ-PGA production in intracellular.

Enhanced iturin a production in a two-compartment biofilm reactor by Bacillus velezensis ND.

In this study, a two-compartment biofilm reactor was designed for iturin A production. The biofilm reactor consists of a stirred-tank fermentor containing exclusively suspended cells and a packing column where the biofilm is attached. Polyester fiber with sphere shape and rough surfaces was chosen as the carrier of biofilm in packing column. Batch, fed-batch, and repeated-batch fermentation using Bacillus velezensis ND in the biofilm reactor were studied. Compared to conventional suspended cell fermentations, the productivity of iturin A in batch and fed-batch biofilm fermentation were increased by 66.7% and 63.3%, respectively. Maximum itutin A concentration of 6.8 ± 0.1g/L and productivity of 46.9 ± 0.2mg/L/h were obtained in fed-batch biofilm fermentation. Repeated-batch fermentation showed high stability, with almost same profile as batch fermentation. After a step-wise temperature control strategy was introduced in the biofilm reactor, productivity of iturin A was increased by 131.9% compared to suspended cell reactor. This superior performance of biofilm reactor confirms that it has great potential in industrial production of iturin A.

Novel multiphase loop reactor with improved aeration prevents excessive foaming in Rhamnolipid production by Pseudomonas putida

Rhamnolipids are biosurfactants that tend to cause strong foaming, making microbial production in an aerated stirred tank fermenter challenging. The continuous removal of rhamnolipids from the cultivation broth via in situ liquid-liquid extraction can remedy this foam challenge, and thereby supports long-term cultivation and production. However, for efficient processing and stable phase separation, a specialized apparatus is required. In this study, the novel multiphase loop reactor, which is a modified airlift reactor with an internal loop enabling continuous in situ liquid-liquid extraction, was designed and adapted to produce rhamnolipids with a recombinant bacterium, Pseudomonas putida KT2440. The initially designed multiphase loop reactor showed a low oxygen transfer rate, unable to meet the oxygen demand of the whole-cell biocatalyst, resulting in inefficient growth and production. A re-design of the sparger via 3D printing enabled a high oxygen supply allowing rhamnolipid production at key performance indicators that matched stirred-tank reactor cultivations. Advantageously, the multiphase loop reactor allowed stable and constant phase separation and solvent removal enabling continuous cultivation in the future. Concluding, the successful use of the multiphase loop reactor for rhamnolipid synthesis is presented, highlighting its potential to become a new platform technology for intensified bioprocessing.

Formulation of a glycolipid:lipopeptide mixture as biosurfactant-based dispersant and development of a low-cost glycolipid production process

Biosurfactant-based dispersants were formulated by mixing glycolipids from Weissella cibaria PN3 and lipopeptides from Bacillus subtilis GY19 to enhance the synergistic effect and thereby achieve hydrophilic-lipophilic balance. The proportions of each biosurfactant and dispersant-to-oil ratios (DORs) were varied to obtain the appropriated formulations. The most efficient glycolipid:lipopeptide mixtures (F1 and F2) had oil displacement activities of 81–88% for fuel and crude oils. The baffled flask test of these formulations showed 77–79% dispersion effectiveness at a DOR of 1:25. To reduce the cost of the dispersant, this study optimized the glycolipid production process by using immobilized cells in a stirred tank fermenter. Semicontinuous glycolipid production was carried out conveniently for 3 cycles. Moreover, food wastes, including waste coconut water and waste frying oil, were found to promote glycolipid production. Glycolipids from the optimized process and substrates had similar characteristics but 20–50% lower cost than those produced from basal medium with soybean oil in shaking flasks. The lowest cost dispersant formulation (F2*) contained 10 g/L waste-derived cell-bound glycolipid and 10 g/L lipopeptide and showed high dispersion efficiency with various oils. Therefore, this biosurfactant-based dispersant could be produced on a larger scale for further application.

INVESTIGATION OF FERMENTATION CONDITION FOR PRODUCTION ENHANCEMENT OF POLYHYDROXYALKANOATE FROM CHEESE WHEY BY PSEUDOMONAS SP.

A newly acquired polyhydroxyalkanoate (PHA) producing Pseudomonas sp. was characterized based on 16S rRNA gene sequences (1,423 bp) and identified as P. Putida SS9. This strain P. putida SS9 was considered in respect of its potential for achieving maximum production of PHA under controlled volumetric oxygen transfer coefficient (kLa) using inexpensive substrate, cheese whey. The significant parameters such as pH, temperature, nitrogen source concentration were studied in shake flask. The influence of aeration and agitation on kLa and production of PHA was studied in stirred tank fermenter. Maximum PHA concentration of 5.98 and 4.33 g/l was obtained using cheese whey medium (CWM) and mineral salts medium (MSM) respectively. Further, the properties of PHA were analyzed by Differential scanning calorimetry, Thermal gravimetry analysis. The structure of the compound was elucidated by Fourier transform infrared and Nuclear magnetic resonance spectroscopy. The findings suggested that the cheese whey could be a most excellent alternative carbon source for PHA production using P. putida SS9 and thus it could effectively replace the synthetic mineral salts medium under optimum kLa. The nucleotide sequence proclaimed in this work will emerge in the GenBank nucleotide sequence database under accession number MF403057.

Intensive Multiple Sequential Batch Simultaneous Saccharification and Cultivation of Kluyveromyces marxianus SS106 Thermotolerant Yeast Strain for Single-Step Ethanol Fermentation from Raw Cassava Starch

We developed the intensive multiple sequential batch simultaneous saccharification and cultivation of the selected thermotolerant yeast strain for single-step ethanol production. The selection and high-cell-density inoculum production of thermotolerant yeast able to produce ethanol under the optimal conditions for single-step ethanol fermentation has become a necessity. In this study, the newly isolated Kluyveromyces marxianus SS106 could tolerate high temperatures (35–45 °C) and grow under a wide range of pH values (3.0–5.5), which are the optimum conditions of raw cassava starch hydrolyzing enzyme used in single-step ethanol fermentation. The high-cell-density concentration of K. marxianus SS106 was produced by a single batch and an intensive multiple sequential batch process in a 5-L stirred tank bioreactor using the simultaneous saccharification and cultivation (SSC) method. The single SSC process yielded the yeast cell biomass at a concentration of 39.30 g/L with a productivity of 3.28 g/L/h and a specific growth rate of 0.49 h−1. However, the yeast cell density concentration was higher in the intensive multiple sequential batch SSC than in the single batch process. This process yielded yeast cell biomass at concentrations of 36.09–45.82 g/L with productivities of 3.01–3.82 g/L/h and specific growth rates of 0.29–0.44 h−1 in the first six batch cycle. The results suggested that the intensive multiple sequential batch simultaneous saccharification and cultivation of K. marxianus SS106 would be a promising process for high-cell-density yeast production for use as the inoculum in single-step ethanol fermentation. Furthermore, we also experimented with single-step ethanol production from raw cassava starch by K. marxianus SS106 in a 5-L stirred tank fermenter. This produced ethanol at a concentration of 61.72 g/L with a productivity of 0.86 g/L/h.

Co-production of gallic acid and a novel cell-associated tannase by a pigment-producing yeast, Sporidiobolus ruineniae A45.2

BackgroundGallic acid has received a significant amount of interest for its biological properties. Thus, there have been recent attempts to apply this substance in various industries and in particular the feed industry. As opposed to yeasts, fungi and bacteria and their tannases have been well documented for their potential bioconversion and specifically for the biotransformation of tannic acid to gallic acid. In this research, Sporidiobolus ruineniae A45.2 is introduced as a newly pigment-producing and tannase-producing yeast that has gained great interest for its use as an additive in animal feed. However, there is a lack of information on the efficacy of gallic acid production from tannic acid and the relevant tannase properties. The objective of this research study is to optimize the medium composition and conditions for the co-production of gallic acid from tannic acid and tannase with a focus on developing an integrated production strategy for its application as a feed additive.ResultsTannase produced by S. ruineniae A45.2 has been classified as a cell-associated tannase (CAT). Co-production of gallic acid obtained from tannic acid and CAT by S. ruineniae A45.2 was optimized using response surface methodology and then validated with the synthesis of 11.2 g/L gallic acid from 12.3 g/L tannic acid and the production of 31.1 mU/mL CAT after 48 h of cultivation in a 1-L stirred tank fermenter. Tannase was isolated from the cell wall, purified and characterized in comparison with its native form (CAT). The purified enzyme (PT) revealed the same range of pH and temperature optima (pH 7) as CAT but was distinctively less stable. Specifically, CAT was stable at up to 70 °C for 60 min, and active under its optimal conditions (40 °C) at up to 8 runs.ConclusionCo-production of gallic acid and CAT is considered an integrated and green production strategy. S. ruineniae biomass could be promoted as an alternative source of carotenoids and tannase. Thus, the biomass, in combination with gallic acid that was formed in the fermentation medium, could be directly used as a feed additive. On the other hand, gallic acid could be isolated and purified for food and pharmaceutical applications. This paper is the first of its kind to report that the CAT obtained from yeast can be resistant to high temperatures of up to 70 °C.

Overexpression of an endogenous raw starch digesting mesophilic α-amylase gene in Bacillus amyloliquefaciens Z3 by in vitro methylation protocol.

Mesophilic α-amylases function effectively at low temperatures with high rates of catalysis and require less energy for starch hydrolysis. Bacillus amyloliquefaciens is an essential producer of mesophilic α-amylases. However, because of the existence of the restriction-modification system, introducing exogenous DNAs into wild-type B. amyloliquefaciens is especially tricky. α-Amylase producer B. amyloliquefaciens strain Z3 was screened and used as host for endogenous α-amylase gene expression. In vitro methylation was performed in recombinant plasmid pWB980-amyZ3. With the in vitro methylation, the transformation efficiency was increased to 0.96 × 102 colony-forming units μg-1 plasmid DNA. A positive transformant BAZ3-16 with the highest α-amylase secreting capacity was chosen for further experiments. The α-amylase activity of strain BAZ3-16 reached 288.70 ± 16.15 U mL-1 in the flask and 386.03 ± 16.25 U mL-1 in the 5-L stirred-tank fermenter, respectively. The Bacillus amyloliquefaciens Z3 expression system shows excellent genetic stability and high-level extracellular production of the target protein. Moreover, the synergistic interaction of AmyZ3 with amyloglucosidase was determined during the hydrolysis of raw starch. The hydrolysis degree reached 92.34 ± 3.41% for 100 g L-1 raw corn starch and 81.30 ± 2.92% for 100 g L-1 raw cassava starch after 24 h, respectively. Methylation of the plasmid DNA removes a substantial barrier for transformation of B. amyloliquefaciens strain Z3. Furthermore, the exceptional ability to hydrolyze starch makes α-amylase AmyZ3 and strain BAZ3-16 valuable in the starch industry. © 2020 Society of Chemical Industry.

Biodiesel production from heterotrophic oleaginous microalga Thraustochytrium sp. BM2 with enhanced lipid accumulation using crude glycerol as alternative carbon source

Aiming to improve the economy and sustainability of biodiesel production, the scale-up of lipid production by heterotrophic Thraustochytrium sp. BM2 utilizing crude glycerol as a low cost carbon source was optimized in stirred tank fermenter. The issues of impurities such as excess ions, methanol, soap and other organic impurities as well as different pretreatment techniques were explored and tackled for industrial application of crude glycerol as carbon source. For process engineering strategies to enhance lipid production, semi-batch operation outperformed fed-batch cultivation and achieved higher lipid yield and overall lipid productivity primarily due to shorter fermentation time. The two-step esterification/transesterification method achieved high fatty acid methyl ester (FAME) conversion rate up to 91.8%, which was two to three folds higher compared with the one-step process.

Making sense of parameter estimation and model simulation in bioprocesses.

Most articles that report fitted parameters for kinetic models do not include meaningful statistical information. This study demonstrates the importance of reporting a complete statistical analysis and shows a methodology to perform it, using functionalities implemented in computational tools. As an example, alginate production is studied in a batch stirred-tank fermenter and modeled using the kinetic model proposed by Klimek and Ollis (1980). The model parameters and their 95% confidence intervals are estimated by nonlinear regression. The significance of the parameters value is checked using a hypothesis test. The uncertainty of the parameters is propagated to the output model variables through prediction intervals, showing that the kinetic model of Klimek and Ollis (1980) can simulate with high certainty the dynamic of the alginate production process. Finally, the results obtained in other studies are compared to show how the lack of statistical analysis can hold back a deeper understanding about bioprocesses.

Feeding Strategies of Two-Stage Fed-Batch Cultivation Processes for Microbial Lipid Production from Sugarcane Top Hydrolysate and Crude Glycerol by the Oleaginous Red Yeast Rhodosporidiobolus fluvialis.

Microbial lipids are able to produce from various raw materials including lignocellulosic biomass by the effective oleaginous microorganisms using different cultivation processes. This study aimed to enhance microbial lipid production from the low-cost substrates namely sugarcane top hydrolysate and crude glycerol by Rhodosporidiobolus fluvialis DMKU-SP314, using two-stage fed-batch cultivation with different feeding strategies in a 3 L stirred-tank fermenter. The effect of two feeding strategies of 147.5 g/L crude glycerol solution was evaluated including pulse feeding at different starting time points (48, 24, and 72 h after initiation of batch operation) and constant feeding at different dilution rates (0.012, 0.020, and 0.033 h−1). The maximum lipid concentration of 23.6 g/L and cell mass of 38.5 g/L were achieved when constant feeding was performed at the dilution rate of 0.012 h−1 after 48 h of batch operation, which represented 1.24-fold and 1.27-fold improvements in the lipid and cell mass concentration, respectively. Whereas, batch cultivation provided 19.1 g/L of lipids and 30.3 g/L of cell mass. The overall lipid productivity increased to 98.4 mg/L/d in the two-stage fed-batch cultivation. This demonstrated that the two-stage fed-batch cultivation with constant feeding strategy has the possibility to apply for large-scale production of lipids by yeast.

Scale-up of a Type I secretion system in E. coli using a defined mineral medium.

Secretion of heterologous proteins into the culture supernatant in laboratory strains of Escherichia coli is possible by utilizing a Type I secretion system (T1SS). One prominent example for a T1SS is based on the hemolysin A toxin. With this system, heterologous protein secretion has already been achieved. However, no cultivations in a defined mineral medium and in stirred tank bioreactors have been described in literature up to now, hampering the broad applicability of the system. In this study, a mineral medium was developed for cultivation under defined conditions. With this medium, the full potential and advantage of a secretion system in E. coli (low secretion of host proteins, no contamination with proteins from complex media compounds) can now be exploited. Additionally, quantification of the protein amount in the supernatant was demonstrated by application of the Bradford assay. In this work, host cell behavior was described in small scale by online monitoring of the oxygen transfer rate. Scalability was demonstrated by stirred tank fermentation yielding 540 mg/L HlyA1 in the supernatant. This work enhances the applicability of a protein secretion system in E. coli and paves the way for an industrial application.

Optimization and characterization of pullulan production by a newly isolated high-yielding strain Aureobasidium melanogenum

Pullulan is an extracellular water-soluble polysaccharide with wide applications. In this study, we screened strains that could selectively produce high molecular weight pullulan for application in industrial pullulan production. A new fungus strain A4 was isolated from soil and identified as Aureobasidium melanogenum based on colony characteristics, morphology, and internally transcribed spacer analysis. Thin-layer chromatography, Fourier-transform infrared spectroscopy, and nuclear magnetic resonance analysis suggested that the dominant exopolysaccharide produced by this strain, which presented a molecular weight of 1.384 × 106 Dalton in in-gel permeation chromatography, was pullulan. The culture conditions for A. melanogenum A4 were optimized at 30 °C and 180 rpm: carbon source, 50 g/L maltose; initial pH 7; and 8 g/L Tween 80. Subsequently, batch fermentation was performed under the optimized conditions in a 5-L stirred-tank fermentor with a working volume of 3 L. The fermentation broth contained 303 g/L maltose, which produced 122.34 g/L pullulan with an average productivity of 1.0195 g/L/h and 82.32 g/L dry biomass within 120 h. The conversion efficiency of maltose to pullulan (Y%) and specific production rate (g/h/g dry cells) (Qs) reached 40.3% and 0.0251 g/L/g dry cells, respectively. The results showed strain A4 could be a good candidate for industrial production.

Selection of thermotolerant Saccharomyces cerevisiae for high temperature ethanol production from molasses and increasing ethanol production by strain improvement.

A thermotolerant ethanol fermenting yeast strain is a key requirement for effective ethanol production at high temperature. This work aimed to select a thermotolerant yeast producing a high ethanol concentration from molasses and increasing its ethanol production by mutagenesis. Saccharomyces cerevisiae DMKU 3-S087 was selected from 168 ethanol producing strains because it produced the highest ethanol concentration from molasses at 40°C. Optimization of molasses broth composition was performed by the response surface method using Box-Behnken design. In molasses broth containing optimal total fermentable sugars (TFS) of 200g/L and optimal (NH4)2SO4 of 1g/L, with an initial pH of 5.5 by shaking flask cultivation at 40°C ethanol, productivity and yield were 58.4 ± 0.24g/L, 1.39g/L/h and 0.29g/g, respectively. Batch fermentation in a 5L stirred-tank fermenter with 3L optimized molasses broth adjusted to an initial pH of 5.5 and fermentation controlled at 40°C and 300rpm agitation resulted in 72.4g/L ethanol, 1.21g/L/h productivity and 0.36g/g yield at 60h. Strain DMKU 3-S087 improvement was performed by mutagenesis using ultraviolet radiation and ethyl methane sulfonate (EMS). Six EMS mutants produced higher ethanol (65.2 ± 0.48-73.0 ± 0.54g/L) in molasses broth containing 200g/L TFS and 1g/L (NH4)2SO4 by shake flask fermentation at 37°C than the wild type (59.8 ± 0.25g/L). Among these mutants, only mutant S087E100-265 produced higher ethanol (62.5 ± 0.26g/L) than the wild type (59.5 ± 0.02g/L) at 40°C. In addition, mutant S087E100-265 showed better tolerance to high sugar concentration, furfural, hydroxymethylfurfural and acetic acid than the wild type.