Simultaneous saccharification and fermentation of kraft pulp by recombinant Escherichia coli for phenyllactic acid production

Simultaneous saccharification and fermentation (SSF) of renewable cellulose for the production of 3-phenyllactic acid (PhLA) by recombinant Escherichia coli was investigated. Kraft pulp recovered from biomass fractionation processes was used as a model cellulosic feedstock and was hydrolyzed using 10–50 filter paper unit (FPU) g⁻¹ kraft pulp of a commercial cellulase mixture, which increased the glucose yield from 21% to 72% in an enzyme dose-dependent manner. PhLA fermentation of the hydrolyzed kraft pulp by a recombinant E. coli strain expressing phenylpyruvate reductase from Wickerhamia fluorescens TK1 produced 1.9 mM PhLA. The PhLA yield obtained using separate hydrolysis and fermentation was enhanced from 5.8% to 42% by process integration into SSF of kraft pulp (20 g L⁻¹) in a complex medium (pH 7.0) at 37 °C. The PhLA yield was negatively correlated with the initial glucose concentration, with a five-fold higher PhLA yield observed in culture medium containing 10 g L⁻¹ glucose compared to 100 g L⁻¹. Taken together, these results suggest that the PhLA yield from cellulose in kraft pulp can be improved by SSF under glucose-limited conditions.     

Optimization of fermentation conditions for the production of ethanol from sago starch by co-immobilized amyloglucosidase and cells of Zymomonas mobilis using response surface methodology

Statistical experimental design was used to optimize the conditions of simultaneous saccharification and fermentation (SSF), viz. temperature, pH and time of fermentation of ethanol from sago starch with co-immobilized amyloglucosidase (AMG) and Zymomonas mobilis MTCC 92 by submerged fermentation. Maximum ethanol concentration of 55.3 g/l was obtained using a starch concentration of 150 g/l. The optimum conditions were found to be a temperature of 32.4 °C, pH of 4.93 and time of fermentation of 17.24 h. Thus, by using SSF process with co-immobilized AMG and Z. mobilis cells MTCC 92, the central composite design (CCD) was found to be the most favourable strategy investigated with respect to ethanol production and enzyme recovery.     

Characteristics of enzymes from Acremonium cellulolyticus strains and their utilization in the saccharification of potato pulp

In this study, the abilities to produce enzymes by four Acremonium cellulolyticus strains were analyzed. Saccharification of potato pulp was performed to investigate the effects of the enzymes produced by A. cellulolyticus and to confirm the possibility of using A. cellulolyticus in the saccharification of potato pulp. Amylase, pectinase, galactosidase, and cellulase were produced by A. cellulolyticus from several carbon sources. Potato pulp was found to be a suitable substrate for A. cellulolyticus growth. The addition of cellulose not only improved the activity of cellulase but also improved the activity of α-galactosidase. Lactose and galactose induced the production of β-galactosidase and pectinase. Four strains of A. cellulolyticus were cultured in potato pulp to evaluate their abilities to produce cellulase, amylase, pectinase and galactosidase. Among them, A. cellulolyticus strain CF-2612 exhibited the highest production of all the enzymes. By using the crude enzymes from A. cellulolyticus strain CF-2612, 86% yield for glucose and 94% yield for galactose were achieved after 80 h of saccharification of potato pulp.     

Glucoamylase production by the marine yeast Aureobasidium pullulans N13d and hydrolysis of potato starch granules by the enzyme

Under the optimal conditions, 10 U/ml of glucoamylase was produced by the marine yeast Aureobasidium pullulans N13d. It was noticed that the crude glucoamylase actively hydrolyzed potato starch granules, but poorly digested raw corn starch and sweet potato starch, resulting in conversion of 68.5, 19 and 22% of them into glucose within 6 h of incubation in the presence of 40 g/l of potato starch granules and 20 U/ml of the crude enzyme. When potato starch granules concentration was increased from 10 to 80 g/l, hydrolysis extent was decreased from 85.6 to 60%, while potato starch granules concentration was increased from 80 to 360 g/l, hydrolysis extent was decreased from 60 to 56%. Ratio of hydrolysis extent of potato starch granules to hydrolysis extent of gelatinized potato starch was 86.0% and the hydrolysis extent of potato starch granules by action of the crude glucoamylase (1.0 U/ml) was 18.5% within 30 min at 60 °C. Only glucose was detected during the hydrolysis, indicating that the crude enzyme could hydrolyze both α-1,4 and α-1,6 linkages of starch molecule in the potato starch.     

Enzymatic solubilization of a pectinaceous dietary fiber fraction from potato pulp: Optimization of the fiber extraction process

Upgrading of potato pulp, a byproduct stream from industrial manufacture of potato starch, is important for the continued economic competitiveness of the potato starch industry. The major part of potato pulp consists of the tuber plant cell wall material which is particularly rich in galactan branched rhamnogalacturonan I type pectin. In the work reported here, the release of high-molecular weight pectinaceous dietary fiber polysaccharides from starch free potato pulp was accomplished by use of a multicomponent pectinase preparation from Aspergillus aculeatus (Viscozyme^® L). The enzyme reaction conditions for the solubilization were optimized via a surface response design to be addition of 0.27% Viscozyme^® L by weight of potato pulp substrate dry matter, 1 h treatment at pH 3.5, 62.5 °C. Analysis of the molecular size and monomer composition of the enzymatically released fibers showed that they were rich in galactose and uronic acid indicating that the solubilized fibers were mainly made up of galactan branched rhamnogalacturonan I type pectin polymers.     

Statistically designed optimisation of enzyme catalysed starch removal from potato pulp

Potato pulp is a high-volume, low-value byproduct stream resulting from the industrial manufacture of potato starch. The pulp is a rich source of biologically functional dietary fibers, but the targeted valorisation of the fibers requires removal of the residual starch from the pulp. The objective of this study was to release the residual starch, making up 21–22% by weight of the dry matter, from the potato pulp in a rational way employing as few steps, as few enzyme activities, as low enzyme dosages, as low energy input (temperature and time), and as high pulp dry matter as possible. Starch removal to obtain dietary fibers is usually accomplished via a three step, sequential enzymatic treatment procedure using a heat stable α-amylase, protease, and amyloglucosidase. Statistically designed experiments were performed to investigate the influence of enzyme dose, amount of dry matter, incubation time and temperature on the amount of starch released from the potato pulp. The data demonstrated that all the starch could be released from potato pulp in one step when 8% (w/w) dry potato pulp was treated with 0.2% (v/w) (enzyme/substrate (E/S)) of a thermostable Bacillus licheniformis α-amylase (Termamyl^® SC) at 70 °C for at least 65 min. The study also indicated that the amount of other carbohydrates released from the pulp during the release of starch was less than using the AOAC Official Method 985.29 and another recently published starch release method employed as a pretreatment for enzymatic upgrading of a pectinaceous potato pulp fiber.     

Continuous 2-keto-gluconic acid (2KGA) production from corn starch hydrolysate by Pseudomonas fluorescens AR4

A continuous fermentation process for 2-keto-gluconic acid (2KGA) production from cheap raw material corn starch hydrolysate was developed using the strain Pseudomonas fluorescens AR4. The dilution rate and feeding glucose concentration had a significant effect on the cell concentrations, glucose utilization and 2KGA production performance. The optimal operating factors were obtained as: 0.065 h⁻¹ of dilution rate, 180 g/L of feeding glucose concentration, and 16 h of batch fermentation time as the starting point. Under these conditions, the steady state had the 135.92 g/L of produced 2KGA concentration, 8.83 g/L.h of average volumetric productivity, and 0.9510 g/g of yield. In conclusion, the proposed efficient and stable continuous fermentation process for 2KGA production by the strain P. fluorescens AR4 is potentially competitive for industrial production from corn starch hydrolysate in terms of 2KGA productivity and yield.     

An industrial level system with nonisothermal simultaneous solid state saccharification,fermentation and separation for ethanol production

To alleviate the problems of low substrate loading, nonisothermal, end-product inhibition of ethanol during the simultaneous saccharification and fermentation, a nonisothermal simultaneous solid state saccharification, fermentation, and separation (NSSSFS) process was investigated; one novel pilot scale nonisothermal simultaneous solid state enzymatic saccharification and fermentation coupled with CO₂ gas stripping loop system was invented and tested. The optimal pretreatment condition of steam-explosion was 1.5 MPa for 5 min in industrial level. In the NSSSFS, enzymatic saccharification and fermentation proceeded at around 50 °C and 37 °C, respectively, and were coupled together by the hydrolyzate loop; glucose from enzymatic saccharification was timely consumed by yeast, and the formed ethanol was separated online by CO₂ gas stripping coupled with adsorption of activated carbon; the solids substrate loading reached 25%; ethanol yields from 18.96% to 30.29% were obtained in fermentation depending on the materials tested. Based on the pilot level of 300 L fermenter, a novel industrial-level of 110 m³ solid state enzymatic saccharification, fermentation and ethanol separation plant had been successfully established and operated. The NSSSFS was a novel and feasible engineering solution to the inherent problems of simultaneous saccharification and fermentation, which would be used in large scale and in industrial production of ethanol.     

The effect of prehydrolysis and improved mixing on high-solids batch simultaneous saccharification and fermentation of spruce to ethanol

In the production of ethanol from lignocellulosic material, it is necessary to reach a high ethanol concentration after fermentation. Simply increasing the substrate concentration leads to stirring problems and inhibition of the enzymes and yeast in the process.Batch simultaneous saccharification and fermentation (SSF) of steam-pretreated spruce with 13.7% water-insoluble solids (WIS) (25% total solids (TS)) was run in a stirred-tank reactor as well as in two reactors designed to handle solid or semi-solid material. In all reactors, the overall ethanol yields were only between 5 and 6%. Fermentation of the liquid fraction of the steam-pretreated spruce slurry resulted in an overall ethanol yield of 85%.22 h of prehydrolysis at 48 °C prior to SSF at 32 °C significantly increased the overall ethanol yield to 72% (final ethanol concentration of 47.8 g/L), using the whole slurry of steam-pretreated spruce at a dry matter content of 13.7% WIS (25% TS).     

Enzymatic hydrolysis of corncob and ethanol production from cellulosic hydrolysate

 Enzymatic hydrolysis of corncob and ethanol fermentation from cellulosic hydrolysate were investigated. After corncob was pretreated by 1% H₂SO₄ at 108 °C for 3 h, the cellulosic residue was hydrolyzed by cellulase from Trichoderma reesei ZU-02 and the hydrolysis yield was 67.5%. Poor cellobiase activity in T. reesei cellulase restricted the conversion of cellobiose to glucose, and the accumulation of cellobiose caused severe feedback inhibition to the activities of β-1,4-endoglucanase and β-1,4-exoglucanase in cellulase system. Supplementing cellobiase from Aspergillus niger ZU-07 greatly reduced the inhibitory effect caused by cellobiose, and the hydrolysis yield was improved to 83.9% with enhanced cellobiase activity of 6.5 CBU g⁻¹ substrate. Fed-batch hydrolysis process was started with a batch hydrolysis containing 100 g l⁻¹ substrate, with cellulosic residue added at 6 and 12 h twice to get a final substrate concentration of 200 g l⁻¹. After 60 h of reaction, the reducing sugar concentration reached 116.3 g l⁻¹ with a hydrolysis yield of 79.5%. Further fermentation of cellulosic hydrolysate containing 95.3 g l⁻¹ glucose was performed using Saccharomyces cerevisiae 316, and 45.7 g l⁻¹ ethanol was obtained within 18 h. The research results are meaningful in fuel ethanol production from agricultural residue instead of grain starch.     

Cellulose ethanol production from waste newsprint by simultaneous saccharification and fermentation using Saccharomyces cerevisiae KNU5377

A thermotolerant ethanol-fermenting yeast, Saccharomyces cerevisiae KNU5377, isolated from a sludge of a local industrial complex stream in Korea, was evaluated for its capability for lignocellulosic ethanol production from waste newsprint in high temperature. In this fermentation, most of dry-defibrated waste newspaper was first saccharified at 50 °C for 108 h using a commercial cellulase and, then with the last addition of dry-defibrated newsprints to the pre-saccharified broth, simultaneous saccharification and fermentation (SSF) of 1.0 L of reaction mixture was carried out at 40 °C, slowly being dropped from 50 °C, for further 72 h in a 5 L fermentor by inoculating the overnight culture of KNU5377. The maximum production of 8.4% (v/v) ethanol was obtained when 250 g (w/v)/L of dry-defibrated waste newspaper was used for ethanol production by SSF. These results suggest that S. cerevisiae KNU5377 is very useful for cellulose ethanol production by the SSF system.     

Enhanced ethanol production via fermentation of rice straw with hydrolysate-adapted Candida tropicalis ATCC 13803

Neutralized hydrolysate and pretreated rice straw obtained from a 2% (w/v) sulfuric acid pretreatment were mixed at 10% (w/v) and subjected to simultaneous saccharification and co-fermentation (SSCF), with cellulase, β-glucosidase, and Candida tropicalis cells at 15 FPU/g-ds, 15 IU/g-ds and 1 × 10⁹ cells/ml, respectively. A 36-h SSCF with adapted cells resulted in YP/S and ethanol volumetric productivity of 0.36 g/g and 0.57 g/l/h, respectively. In addition to ethanol, insignificant amounts of glycerol and xylitol were also produced. Adapted C. tropicalis cells produced nearly 1.6 times more ethanol than non-adapted cells. Ethanol yield (Yp/s), ethanol volumetric productivity and a xylitol concentration of 0.48 g/g, 0.33 g/l/h and 0.89 g/l, respectively, were produced from fermentation of remaining hydrolysate with adapted C. tropicalis cells. The 0.20 g/g ethanol yield and 77% production efficiency from SSCF of pretreated rice straw indicate scale-up potential for the process. This study demonstrated that C. tropicalis produced ethanol and xylitol from a mixed-sugar stream, although cell adaptation affected ethanol and xylitol yields. Scanning electron microscopy indicated agglomeration of cellulose microfibrils and globular deposition of lignin in acid-pretreated rice straw.     

A novel co-culture process with Zymomonas mobilis and Pichia stipitis for efficient ethanol production on glucose/xylose mixtures

An efficient conversion of glucose and xylose is a requisite for a profitable process of bioethanol production from lignocellulose. Considering the approaches available for this conversion, co-culture is a simple process, employing two different organisms for the fermentation of the two sugars. An innovative fermentation scheme was designed, co-culturing immobilized Zymomonas mobilis and free cells of Pichia stipitis in a modified fermentor for the glucose and xylose fermentation, respectively. A sugar mixture of 30 g/l glucose and 20 g/l of xylose was completely converted to ethanol within 19 h. This gave a volumetric ethanol productivity of 1.277 g/l/h and an ethanol yield of 0.49–0.50 g/g, which is more than 96% of the theoretical value. Extension of this fermentation scheme to sugarcane bagasse hydrolysate resulted in a complete sugar utilisation within 26 h; ethanol production peaked at 40 h with a yield of 0.49 g/g. These values are comparable to the best results reported. Cell interaction was observed between Z. mobilis and P. stipitis. Viable cells of Z. mobilis inhibited the cell activity of P. stipitis and the xylose fermentation. Z. mobilis showed evidence of utilising a source other than glucose for growth when co-cultured with P. stipitis.     

Production of 1,3-propanediol, 2,3-butanediol and ethanol by a newly isolated Klebsiella oxytoca strain growing on biodiesel-derived glycerol based media

The production of 1,3-propanediol, 2,3-butanediol and ethanol was studied, during cultivations of strain Klebsiella oxytoca FMCC-197 on biodiesel-derived glycerol based media. Different kinds of glycerol feedstocks and experimental conditions had an important impact upon the distribution of metabolic products; production of 1,3-propanediol was positively influenced by stable pH conditions and by the absence of N₂ gas infusions throughout the fermentation. Thus, during batch bioreactor fermentations conducted at increasing glycerol concentrations, 1,3-propanediol at 41.3 g/L and yield ～47% (w/w) was achieved at initial glycerol concentration ～120 g/L. At even higher initial glycerol media (150 and 170 g/L), growth was not ceased, but 1,3-propanediol production declined. During fed-batch fermentation under optimal experimental conditions, 126 g/L of glycerol were converted into 50.1 g/L of 1,3-propanediol. In this experiment, also 25.2 g/L of ethanol (conversion yield ～20%, w/w) were formed. A batch-bioreactor culture was performed under non-sterilized conditions and the 1,3-propanediol production was almost equivalent to the sterilized process. Concerning 2,3-butanediol formation, the most detrimental parameter was the absence of N₂ sparging and as a result, no 2,3-butanediol was produced. The presence of glucose as co-substrate seriously enhanced 2,3-butanediol production; when commercial glucose was employed as sole substrate, 32.1 g/L of 2,3-butanediol were formed.     

Effect of hydrothermolysis process conditions on pretreated switchgrass composition and ethanol yield by SSF with Kluyveromyces marxianus IMB4

Hot compressed liquid water was used to treat switchgrass in a method called hydrothermolysis to disrupt lignin, dissolve hemicellulose, and increase accessibility of cellulose to cellulase. Three temperatures (190, 200, and 210 °C) and hold times (10, 15, and 20 min) were tested. Switchgrass treated at 190 °C for 10 min had the greatest xylan recovery in the prehydrolyzate. Less than 0.65 g/L glucose were released into the prehydrolyzate for all pretreatment conditions, indicating most glucose was retained as cellulose in the solid substrate. 5-Hydroxymethylfurfural (HMF) and furfural formation in the prehydrolyzate were found to be less than 1 g/L for all treatments. The highest concentration of ethanol, 16.8 g/L (72% of theoretical), was produced from switchgrass pretreated at 210 °C and 15 min using simultaneous saccharification and fermentation (SSF) at 45 °C with the thermotolerant yeast Kluyveromyces marxianus IMB4 and 15 FPU cellulase/g glucan.     

Enhanced ethanol and chitosan production from wheat straw by Mucor indicus with minimal nutrient consumption

Recently, Mucor indicus was introduced as a promising ethanol producing microorganism for fermentation of lignocellulosic hydrolysates, showing a number of advantages over Saccharomyces cerevisiae. However, high nutrient requirement is the main drawback of the fungus in efficient ethanol production from lignocelluloses. In this study, application of fungal extract as a potential nutrient source replacing all required nutrients in fermentation of wheat straw by M. indicus was investigated. Wheat straw was pretreated with N-methylmorpholine-N-oxide (NMMO) at 120 °C for 1–5 h prior to enzymatic hydrolysis. Hydrolysis yield was improved at least by 6-fold for 3 h pretreated straw compared with that of untreated one. A fungal extract was produced by autolysis of M. indicus biomass, an unavoidable byproduct of fermentation. Maximum free amino nitrogen (2.04 g/L), phosphorus (1.50 g/L), and total nitrogen (4.47 g/L) as well as potassium, magnesium, and calcium in the fungal extract were obtained by autolysis of the biomass at 50 °C and pH 5.0. The fungal extract as a nutrient-rich supplement substituted yeast extract and all other required minerals in fermentation and enhanced the ethanol yield up to 92.1% of the theoretical yield. Besides, appreciate amounts of chitosan were produced as another valuable product of the autolysis.     

Mixed culture of Saccharomyces cerevisiae and Acetobacter pasteurianus for acetic acid production

Mixed culture of Saccharomyces cerevisiae and Acetobacter pasteurianus was carried out for high yield of acetic acid. Acetic acid production process was divided into three stages. The first stage was the growth of S. cerevisiae and ethanol production, fermentation temperature and aeration rate were controlled at 32 °C and 0.2 vvm, respectively. The second stage was the co-culture of S. cerevisiae and A. pasteurianus, fermentation temperature and aeration rate were maintained at 34 °C and 0.4 vvm, respectively. The third stage was the growth of A. pasteurianus and production of acetic acid, fermentation temperature and aeration rate were controlled at 32 °C and 0.2 vvm, respectively. Inoculation volume of A. pasteurianus and S. cerevisiae was 16% and 0.06%, respectively. The average acetic acid concentration was 52.51 g/L under these optimum conditions. To enhance acetic acid production, a glucose feeding strategy was subsequently employed. When initial glucose concentration was 90 g/L and 120 g/L glucose was fed twice during fermentation, acetic acid concentration reached 66.0 g/L.     

Conversion of cassava flour to fuel ethanol by sequential solid state and submerged cultures

A process for conversion of cassava flour to ethanol was developed. This involved direct inoculation of Aspergillus awamori spores into a cassava flour paste and incubation for some period during which hydrolytic enzymes are produced (solid state culture or koji production) and subsequent addition of water and yeast cells, during which there is simultaneous hydrolysis and ethanol production (submerged culture). When cassava flour alone was used for the solid state phase, the paste was very sticky, making mixing and aeration difficult. However, addition of rice bran improved the texture and enzyme production. The optima rice bran concentration, spore inoculum concentration, and duration of solid state culture before submerged culture were 20%, 6.16 × 10⁶ spores/100 g, and 2 days, respectively. Under these optimum conditions, a high ethanol concentration of 120 g/L and ethanol yield of 0.309 g-ethanol/g-cassava flour were obtained. This ethanol yield corresponds to 0.44 g-ethanol/g-cassava starch.     

High efficiency bioethanol production from barley straw using a continuous pretreatment reactor

We developed a new pretreatment process for producing high-efficiency bioethanol from a lignocellulosic biomass. Barley straw was pretreated with sodium hydroxide in a twin-screw extruder for continuous pretreatment. The biomass to ethanol ratio (BTER) for optimal pretreatment conditions was evaluated by response surface methodology. Simultaneous saccharification and fermentation (SSF) was conducted to investigate the BTER with 30 FPU/g cellulose of enzyme and 7% (v/v) yeast (Saccharomyces cerevisiae CHY 1011) using 10% (w/v) pretreated biomass under various pretreatment conditions. The maximum BTER was 73.00% under optimal pretreatment conditions (86.61 °C, 0.58 M, and 84.79 mL/min for temperature, sodium hydroxide concentration, and solution flow rate, respectively) and the experimental BTER was 70.01 ± 0.59%. SSF was performed to investigate the optimal enzyme and biomass dosage. As a result, maximum ethanol concentration and ethanol yield were 46.00 g/L and 77.36% at a loading pretreated biomass of 20% with 30 FPU/g cellulose of the enzyme dosage for barley straw to bioethanol. These results are a significant contribution to the production of bioethanol from barley straw.