Efficacy of acidic pretreatment for the saccharification and fermentation of alginate from brown macroalgae

This study was performed to evaluate the effectiveness of acidic pretreatment in increasing the enzymatic digestibility of alginate from brown macroalgae. Pretreatment with 1 % (w/v) sulfuric acid at 120 °C for 30 min produced oligosaccharides, mannuronic acid, and guluronic acid. Enzymatic saccharification of pretreated alginate by alginate lyases produced 52.2 % of the theoretical maximal sugar yield, which was only 7.5 % higher than the sugar yield obtained with unpretreated alginate. Mass spectrometric analyses of products of the two reactions revealed that acidic pretreatment and enzymatic saccharification produced saturated monomers (i.e., mannuronic and guluronic acid) with saturated oligosaccharides and unsaturated monomers (i.e., 4-deoxy-l-erythro-5-hexoseulose uronic acid; DEH), respectively. While DEH is further metabolized by microorganisms, mannuronic acid and guluronic acid are not metabolizable. Because of the poor efficacy in increasing enzymatic digestibility and owing to the formation of non-fermentable saturated monomers, acidic pretreatment cannot be recommended for enzymatic saccharification and fermentation of alginate.     

Simultaneous saccharification of cassava starch and fermentation of algae for biodiesel production

A simpler approach to produce biodiesel from cassava starch was established, which successfully integrates the simultaneous saccharification and heterotrophic algal fermentation in an identical system. Batch experiments were investigated to verify the feasibility of raw starchy substrates fermentation for microalgal oil. The highest cell density (49.34 g L⁻¹) and oil content (54.60%) were obtained in 5-L fed-batch cultivation via simultaneous saccharification and fermentation (SSF). It is demonstrated that the previous multistep hydrolysis and fermentation for feedstock oil could be replaced by SSF with higher energy efficiency and lower facility costs.     

Detection of alginate lyases by isoelectric focusing and activity staining

A sensitive method has been developed for the rapid analysis of mutliple forms of alginate lyases in crude bacterial extracts. The technique is based on isoelectric focusing with a substrate-overlay technique for the direct measurement of enzyme activity. Isoelectric point values have been determined for the alginate lyases present in five strains of bacteria using, typically, 5.7 × 10⁻ units of activity. Multiple forms of these enzymes have been observed in three of the five bacterial strains studied. The method has also been used to compare the pI value of the -guluronate lyase from Klebsiella pneumoniae with those for the cloned gene products in strains of Escherichia coli.     

马铃薯淀粉同步糖化发酵制备L-乳酸条件的统计学优化

以马铃薯淀粉为原料,采用同步糖化发酵方法制备乳酸。通过Plackett-Burman实验设计对影响乳酸产量的7个因子进行筛选,结果表明淀粉质量浓度、糖化酶用量和发酵温度3个因素对乳酸产量影响显著。利用最陡爬坡试验逼近最大响应区,采用中心复合实验设计及响应面分析法进行回归分析,建立影响乳酸产量的二次模型。模型求解得出最优淀粉质量浓度为271.89g/L,糖化酶用量为265.09U/g,发酵温度为39.05℃,最大理论乳酸产量为196.99g/L。3批验证实验结果平均值与预测值接近,表明该模型与实际情况拟合良好,实际最大乳酸产量为193.6g/L,较优化前提高了13.9%,L-乳酸的平均纯度达到95.2%。     

Simultaneous saccharification and fermentation of cellulose for lactic acid production

Lactic acid production from α-cellulose by simultaneous saccharification and fermentation (SSF) was studied. The cellulose was converted in a batch SSF using cellulase enzyme Cytolase CL to produce glucose sugar andLactobacillus delbrueckii to ferment the glucose to lactic acid. The effects of temperature, pH, yeast extract loading, and lactic acid inhibition were studied to determine the optimum conditions for the batch processing. Cellulose was converted efficiently to lactic acid, and enzymatic hydrolysis was the rate controlling step in the SSF. The highest conversion rate was obtained at 46°C and pH 5.0. The observed yield of lactic acid from α-cellulose was 0.90 at 72 hours. The optimum pH of the SSF was coincident with that of enzymatic hydrolysis. The optimum temperature of the SSF was chosen as the highest temperature the microorganism could withstand. The optimum yeast extract loading was found to be 2.5 g/L. Lactic acid was observed to be inhibitory to the microorganisms’ activity.     

Characterization of two new aromatic amino acid lyases from actinomycetes for highly efficient production of p-coumaric acid

Bioprocess and Biosystems Engineering - p-Coumaric acid (p-CA) is a bioactive natural product and an important industrial material for pharmaceuticals and nutraceuticals. It can be synthesized from...     

Effects of intermittent addition of cellulase for production of L-lactic acid from wastewater sludge by simultaneous saccharification and fermentation

An attempt was made to create L-lactic acid, a precursor of poly-lactic acid, which is a biodegradable plastic, from wastewater sludge from the paper-manufacturing industry. The sludge contained a high percentage of cellulose and needed to be hydrolyzed to glucose by the action of the cellulase before being treating with lactic acid bacteria. Therefore, a method involving simultaneous saccharification and fermentation (SSF) was carried out. The optimum pH of the SSF for production of the lactic acid by the newly isolated lactic acid bacterium with a high selectively of L-lactic acid was found out to be around pH = 5.0, and the optimum temperature to be approximately 40 degrees C. On the basis of the measurement of the cell density changes in the lactic acid bacteria, it was ascertained that the bacterial activity could continue at a high level for a relatively long period of time, and that the L-lactic acid productivity was diminished by the rapid deactivation of the cellulase. With the intermittent addition of cellulase once daily for the sake of compensating for the cellulase deactivation, the L-lactic acid attained a maximum concentration of 16.9 g/L, i.e., a 72.2% yield based on the potential glucose contained in the sludge under optimum pH and temperature conditions.     

Multi-step feeding systems for lactic acid production by simultaneous saccharification and fermentation of processed wood

Eucalyptus globulus wood samples were delignified in acetic acid media and swelled with NaOH solutions in a further stage. Solid residues from treatments were used as substrates for lactic acid production by Simultaneous Saccharification and Fermentation (SSF) in media containing Trichoderma reesei cellulases and Lactobacillus delbrueckii cells. The improvements in the overall process derived from adding fresh enzymes and/or substrate during the SSF process were assessed. In order to obtain comparative data on the efficiency of substrate utilization, enzymatic hydrolysis runs (in absence of microorganisms) were also carried out. Lactic acid concentrations in the range 48-62 g/l were obtained in SSF experiments. The solid residues after SSF (made up of microbial biomass and the non-hydrolyzed fraction of substrate) were characterized for measuring their potential as feed additives.     

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.     

Purification and characterization of a novel alginate lyase from the marine bacterium Cobetia sp. NAP1 isolated from brown algae

The application of marine resources, instead of fossil fuels, for biomass production is important for building a sustainable society. Seaweed is valuable as a source of marine biomass for producing biofuels such as ethanol, and can be used in various fields. Alginate is an anionic polysaccharide that forms the main component of brown algae. Various alginate lyases (e.g. exo- and endo-types and oligoalginate lyase) are generally used to degrade alginate. We herein describe a novel alginate lyase, AlgC-PL7, which belongs to the polysaccharide lyase 7 family. AlgC-PL7 was isolated from the halophilic Gram-negative bacterium Cobetia sp. NAP1 collected from the brown algae Padina arborescens Holmes. The optimal temperature and pH for AlgC-PL7 activity were 45 °C and 8, respectively. Additionally, AlgC-PL7 was thermostable and salt-tolerant, exhibited broad substrate specificity, and degraded alginate into monosaccharides. Therefore, AlgC-PL7 is a promising enzyme for the production of biofuels.     

Dilute acid pretreatment and enzymatic saccharification of sugarcane tops for bioethanol production

The aim of this work was to study the feasibility of using sugarcane tops as feedstock for the production of bioethanol. The process involved the pretreatment using acid followed by enzymatic saccharification using cellulases and the process was optimized for various parameters such as biomass loading, enzyme loading, surfactant concentration and incubation time using Box–Behnken design. Under optimum hydrolysis conditions, 0.685 g/g of reducing sugar was produced per gram of pretreated biomass. The fermentation of the hydrolyzate using Saccharomyces cerevisae produced 11.365 g/L of bioethanol with an efficiency of about 50%. This is the first report on utilization of sugarcane tops for bioethanol production.     

稀酸水解玉米芯制备丁二酸

利用正交设计得到稀H2SO4水解玉米芯制备混合糖液的优化工艺:玉米芯料液比1∶5(质量体积比),物料粒径250~380μm、H2SO4用量3%(体积分数)、水解温度126℃、反应时间2.5 h。此工艺条件下的总糖收率达90%,总糖质量浓度为60 g/L,发酵抑制物糠醛含量为0.87 g/L,5-羟甲基糠醛含量为0.68 g/L。在此基础上利用活性炭吸附和Ca(OH)2中和对玉米芯混合糖液进行脱毒及脱盐处理,SO42-脱除率达96%,色素脱除率为96%,糠醛、5-羟甲基糠醛及多酚类物质脱除率均高于50%。处理后的玉米芯多组分糖液作为产琥珀酸放线杆菌(Actinobacillus succino-genes)NJ113的发酵C源,当培养基中初始总糖质量浓度为50 g/L时,丁二酸收率为61.68%,丁二酸质量浓度为30.8 g/L;初始总糖质量浓度为70 g/L时,丁二酸收率仍可达50%以上,丁二酸质量浓度为35.2 g/L。发酵实验表明,将经过脱毒脱盐处理的玉米芯多组分糖液替代葡萄糖作为C源发酵制备丁二酸具有可行性。     

Ethanol production from starch by recombinantEscherichia coli containing integrated genes for ethanol production and plasmid genes for saccharification

Ethanologenic strains ofEscherichia coli have been developed which can express thermostable enzymes for starch saccharification as intracellular products. These enzymes can be harvested within cells at the end of fermentation and liberated by heating to the temperature at which they exhibit maximal activity (60°C to 70°C). Organisms such as these could be used to supply enzymes for yeast-based fermentations while producing ethanol as a co-product.     

Optimization of saccharification and ethanol production by simultaneous saccharification and fermentation (SSF) from seaweed, Saccharina japonica

Ethanol was produced using the simultaneous saccharification and fermentation (SSF) method with macroalgae polysaccharide from the seaweed Saccharina japonica (Sea tangle, Dasima) as biomass. The seaweed was dried by hot air, ground with a hammer mill and filtered with a 200-mesh sieve prior to pretreatment. Saccharification was carried out by thermal acid hydrolysis with H(2)SO(4) and the industrial enzyme, Termamyl 120 L. To increase the yield of saccharification, isolated marine bacteria were used; the optimal saccharification conditions were 10% (w/v) seaweed slurry, 40 mM H(2)SO(4) and 1 g dcw/L isolated Bacillus sp. JS-1. Using this saccharification procedure, the reducing sugar concentration and viscosity were 45.6 ± 5.0 g/L and 24.9 cp, respectively, and the total yield of the saccharification with optimal conditions and S. japonica was 69.1%. Simultaneous saccharification and fermentation was carried out for ethanol production. The highest ethanol concentration, 7.7 g/L (9.8 ml/L) with a theoretical yield of 33.3%, was obtained by SSF with 0.39 g dcw/L Bacillus sp. JS-1 and 0.45 g dcw/L of the yeast, Pichia angophorae KCTC 17574.     

Caffeic acid production by simultaneous saccharification and fermentation of kraft pulp using recombinant Escherichia coli

Caffeic acid (3,4-dihydroxycinnamic acid) serves as a building block for thermoplastics and a precursor for biologically active compounds and was recently produced from glucose by microbial fermentation. To produce caffeic acid from inedible cellulose, separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) reactions were compared using kraft pulp as lignocellulosic feedstock. Here, a tyrosine-overproducing Escherichia coli strain was metabolically engineered to produce caffeic acid from glucose by introducing the genes encoding a 4-hydroxyphenyllactate 3-hydroxylase (hpaBC) from Pseudomonas aeruginosa and tyrosine ammonia lyase (fevV) from Streptomyces sp. WK-5344. Using the resulting recombinant strain, the maximum yield of caffeic acid in SSF (233 mg/L) far exceeded that by SHF (37.9 mg/L). In the SSF with low cellulase loads (≤2.5 filter paper unit/g glucan), caffeic acid production was markedly increased, while almost no glucose accumulation was detected, indicating that the E. coli cells experienced glucose limitation in this culture condition. Caffeic acid yield was also negatively correlated with the glucose concentration in the fermentation medium. In SHF, the formation of by-product acetate and the accumulation of potential fermentation inhibitors increased significantly with kraft pulp hydrolysate than filter paper hydrolysate. The combination of these inhibitors had synergistic effects on caffeic acid fermentation at low concentrations. With lower loads of cellulase in SSF, less potential fermentation inhibitors (furfural, 5-hydroxymethyfurfural, and 4-hydroxylbenzoic acid) accumulated in the medium. These observations suggest that glucose limitation in SSF is crucial for improving caffeic acid yield, owing to reduced by-product formation and fermentation inhibitor accumulation.

     

Optimization of pretreatment and saccharification for the production of bioethanol from water hyacinth by Saccharomyces cerevisiae

Alkaline-oxidative (A/O) pretreatment and enzymatic saccharification were optimized for bioethanol fermentation from water hyacinth by Saccharomyces cerevisiae. Water hyacinth was subjected to A/O pretreatment at various NaOH and H(2)O(2) concentrations and reaction temperatures for the optimization of bioethanol fermentation by S. cerevisiae. The most effective condition for A/O pretreatment was 7% (w/v) NaOH at 100 °C and 2% (w/v) H(2)O(2). The carbohydrate content was analyzed after reaction at various enzyme concentrations and enzyme ratios using Celluclast 1.5 L and Viscozyme L to determine the effective conditions for enzymatic saccharification. After ethanol fermentation using S. cerevisiae KCTC 7928, the concentration of glucose, ethanol and glycerol was analyzed by HPLC using a RI detector. The yield of ethanol in batch fermentation was 0.35 g ethanol/g biomass. Continuous fermentation was carried out at a dilution rate of 0.11 (per h) and the ethanol productivity was 0.77 [g/(l h)].     

Seasonal variations in the production of antifungal substances by some dictyotales (brown algae) from the french mediterranean coast

Hexane extracts of some algae belonging to the Dictyotales collected over a 12 month period were tested for their antifungal activity using human pathogenic fungi (yeasts, moulds and dermatophytes) and phytopathogenic fungi responsible for diseases in Mediterranean plants and trees. The three algal species tested (Dictyota dichotoma, Dictyota dichotoma var. implexa, Dilophus spiralis) exhibited a wide spectrum of antifungal activity which varied during the seasons.     

d-Lactic acid production from dry biomass of Hydrodictyon reticulatum by simultaneous saccharification and co-fermentation using Lactobacillus coryniformis subsp. torquens

d-Lactic acid production from dry biomass of the microalga, Hydrodictyon reticulatum, was carried out in a 5-l jar fermentor (initial pH 6, 34?°C using CaCO₃ as a neutralizing agent) through simultaneous saccharification and co-fermentation using the Lactobacillus coryniformis subsp. torquens. After 36?h, 36.6?g lactic acid/l was produced from 80?g H. reticulatum/l in the medium containing 3?g yeast extract/l and 3?g peptone/l in the absence of mineral salts. The maximum productivity, average productivity and yield were 2.38?g/l?h, 1.02?g/l?h and 45.8?%, respectively. The optical purity of d-Lactic acid ranged from 95.8–99.6?%. H. reticulatum is thus a promising biomass material for the production of d-Lactic acid.     

Simultaneous saccharification and fermentation of acid-pretreated rapeseed meal for succinic acid production using Actinobacillus succinogenes

Rapeseed meal was evaluated for succinic acid production by simultaneous saccharification and fermentation using Actinobacillus succinogenes ATCC 55618. Diluted sulfuric acid pretreatment and subsequent hydrolysis with pectinase was used to release sugars from rapeseed meal. The effects of culture pH, pectinase loading and yeast extract concentration on succinic acid production were investigated. When simultaneous saccharification and fermentation of diluted acid pretreated rapeseed meal with a dry matter content of 12.5% (w/v) was performed at pH 6.4 and a pectinase loading of 2% (w/w, on dry matter) without supplementation of yeast extract, a succinic acid concentration of 15.5 g/L was obtained at a yield of 12.4 g/100g dry matter. Fed-batch simultaneous saccharification and fermentation was carried out with supplementation of concentrated pretreated rapeseed meal and pectinase at 18 and 28 h to yield a final dry matter content of 20.5% and pectinase loading of 2%, with the succinic acid concentration enhanced to 23.4 g/L at a yield of 11.5 g/100g dry matter and a productivity of 0.33 g/(Lh). This study suggests that rapeseed meal may be an alternative substrate for the efficient production of succinic acid by A. succinogenes without requiring nitrogen source supplementation.     

Production of D-lactic acid from defatted rice bran by simultaneous saccharification and fermentation

Production of d-lactic acid from rice bran, one of the most abundant agricultural by-products in Japan, is studied. Lactobacillus delbrueckii subsp. delbrueckii IFO 3202 and defatted rice bran powder after squeezing rice oil were used for the production. Since the rice bran contains polysaccharides as starch and cellulose, we coupled saccharification with amylase and cellulase to lactic acid fermentation. The indigenous bacteria in the rice bran produced racemic lactic acid in the saccharification at pH 6.0-6.8. Thus the pH was controlled at 5.0 to suppress the growth of the indigenous bacteria. L. delbrueckii IFO 3202 produced 28 kgm(-3) lactic acid from 100 kgm(-3) rice bran after 36 h at 37 degrees C. The yield based on the amount of sugars soluble after 36-h hydrolysis of the bran by amylase and cellulase (36 kgm(-3) from 100 kgm(-3) of the bran) was 78%. The optical purity of produced d-lactic acid was 95% e.e.