Statistical optimization of hydrolysis process for banana peels using cellulolytic and pectinolytic enzymes

Dried and ground banana peels (BP) were pretreated and hydrolyzed using a combination of cellulolytic and pectinolytic enzymes. Central composite design (CCD) was used to optimize cellulase, β-glucosidase and pectinase concentrations and hydrolysis time for production of glucose and reducing sugars. Design expert software was used to analyze and evaluate the data. The interactions between filter paper cellulase and β-glucosidase concentrations were statistically significant at a 95% confidence level in production of glucose and reducing sugars from BP. The validation experiment was carried out with cellulase, β-glucosidase and pectinase at 8 FPU/g cellulose, 15 IU/g cellulose and 66 IU/g pectin, respectively, for 15 h in a laboratory fermenter. The glucose and reducing sugars concentrations of 28.2 and 48 g/l, respectively, obtained through the validation experiment were higher than the theoretical values for glucose and reducing sugars predicted by the software. Glucose, galactose, arabinose, xylose and galacturonic acid concentrations increased with time, but a significant increase in fructose concentration was not observed. Process optimization also led to about 40% savings in hydrolysis time, indicating scale-up potential for the process.     

Optimization of the fermentation conditions and partial characterization for acido-thermophilic <Emphasis Type="Italic">α</Emphasis>-amylase from <Emphasis Type="Italic">Aspergillus niger</Emphasis> NCIM 548

A high performance thermostable α-amylase at low pH values has been synthesized. Sugarcane bagasse was hydrolyzed in a dilute acid solution and utilized as carbon source for the growth of Aspergillus niger strain NCIM 548. Glucose, xylose and arabinose with the ratio of 1.0: 0.9: 0.3 (w/w/w) were detected in the hydrolyzate by HPLC analysis. Optimization of the fermentation conditions for α-amylase production was performed by varying four influential parameters such as Sugarcane bagasse hydrolyzate (SBH), NH₄Cl, pH and incubation time using a central composite design (CCD) under response surface methodology (RSM). The optimum values of SBH, NH₄Cl, pH and incubation time were 20.49, 2.34 g/l, 5.65 and 76.67 h, respectively. The acido-thermophilic enzyme showed maximum stability at 70°C and pH value of 4. The rate constant, K _m and maximum reaction rate, V _max were 18.79 g/l and 15.85 g/l·min, respectively.     

Screening of <Emphasis Type="Italic">Candida utilis</Emphasis> and medium optimization for co-production of <Emphasis Type="Italic">S</Emphasis>-adenosylmethionine and glutathione

An effective S-adenosylmethionine and glutathione enriching yeast mutant of Candida utilis CCTCC M 209298 was first screened from plates containing 0.5 g/L of DL-ethionine by complex mutagenesis with UV and γ-ray in this study. Medium components optimization for enhanced co-production of S-adenosylmethionine and glutathione by C. utilis CCTCC M 209298 was further carried out using response surface methodology. The significant factors influencing S-adenosylmethionine and glutathione co-production were selected by Plackett-Burman design as sucrose, KH₂PO₄ and L-methionine, and Box-Behnken design was applied for further optimization studies. Based on these approaches, the optimized concentrations on medium components for higher co-production of S-adenosylmethionine and glutathione were sucrose 35.4 g/L, (NH₄)₂SO₄ 10 g/L, KH₂PO₄ 12.3 g/L, MgSO₄·7H₂O, 0.05 g/L, CaCl₂ 0.05 g/L and L-methionine 4.6 g/L. The medium optimization by response surface methodology led to a total production of 589.3 mg/L on S-adenosylmethionine and glutathione, which was 2.4-fold increased compared with the medium without optimization.     

Fractionation of barley straw with dilute sulfuric acid for improving hemicellulose recovery

Dilute acid fractionation of barley straw improves dissolving hemicellulose fraction of the straw, while leaving the cellulose more reactive and accessible to enzyme as a strategy of pretreatment. To characterize the fractionation process, the effects of the acid concentration, temperature and reaction time on the hemicellulose removal as well as on the formation of by-products (furfural, 5-hydroxymethylfurfural and acetic acid) were investigated. The optimum fractionation conditions of barley straw were 1% (w/v) concentration of sulfuric acid, 158 °C of reaction temperature and 15 min of reaction time. Under the optimum conditions, 87% of xylan was hydrolyzed and recovered in liquid hydrolyzate, which was 7% higher than that of the predicted yield. The hydrolyzate contained glucose 2.44 g/L, arabinose 1.70 g/L, xylose 13.41 g/L, acetic acid 1.55 g/L, levulinic acid 0.03 g/L, 5-HMF 0.03 g/L and furfural 0.75 g/L.     

Chemical characterization and liberation of pentose sugars from brewer's spent grain

The chemical composition of brewer's spent grain (BSG), generated from a process using 100% malted barley, was investigated. BSG is mainly composed of (g kg^?1) hemicellulose (284), lignin (278), cellulose (168) and protein (152.5), but it also contains extractives and ashes in smaller proportions. Minerals in BSG include calcium, sodium, potassium, magnesium, aluminum, iron, barium, strontium, manganese, copper, zinc, phosphorus, sulfur, chromium and silicon. In order to find alternative uses for BSG, this by‐product was subjected to acid hydrolysis process to recover the hemicellulosic sugars, which have a large number of industrial applications. The process was carried out under different conditions of liquid‐to‐solid ratio and acid concentration to evaluate the influence of these two variables on BSG hemicellulose hydrolysis. Under all the hydrolysis conditions evaluated, arabinose was recovered with higher efficiency than xylose. Under the best evaluated reaction conditions (liquid‐to‐solid ratio of 10 g g^?1 and 120 mg H₂SO₄ g^?1 dry matter) 76.2% of the hemicellulose was hydrolyzed and the xylose and arabinose sugars were recovered with 67 and 97.8% efficiency, respectively. Copyright © 2005 Society of Chemical Industry     

Medium optimization for lipid production through co‐fermentation of glucose and xylose by the oleaginous yeast Lipomyces starkeyi

Co‐fermentation of lignocellulose‐based carbohydrates is a potential solution to improve the economics of microbial lipid production. In the present paper, experiments were performed to optimize the media composition for lipid production by the oleaginous yeast Lipomyces starkeyi AS 2.1560 through co‐fermentation of glucose and xylose (2 : 1 wt/wt). Statistical screening of nine media variables was performed by a Plackett–Burman design. Three factors, namely mixed sugar, yeast extract and FeSO₄, were found as significant components influencing cellular lipid accumulation. Further optimization was carried out using a Box–Behnken factorial design to study the effects of these three variables on lipid production. A mathematical model with the R² value at 96.66% was developed to show the effect of each medium composition and their interactions on the lipid production. The model estimated that a maximal lipid content of 61.0 wt‐% could be obtained when the concentrations of mixed sugar, yeast extract and FeSO₄ were at 73.3 g/L (glucose 48.9 g/L, xylose 24.4 g/L), 7.9 g/L and 4.0 mg/L, respectively. The predicted value was in good accordance with the experimental data of 61.5%. Compared with the initial media, the optimized media gave 1.59‐fold and 2.03‐fold increases for lipid content and lipid productivity, respectively.     

Enhanced microbial oil production by activated sludge microorganisms via co‐fermentation of glucose and xylose

The co‐fermentation of glucose and xylose by activated sludge microorganisms for the production of microbial oils for use as biodiesel feedstock was investigated. Various carbon sources at initial concentration of 60 g/L and C:N ratio 70:1 were investigated: xylose, glucose, and 2:1 and 1:2 (by mass) glucose/xylose mixtures. Oil accumulation ranged between 12 to 22% CDW, the highest of which was obtained when xylose was the sole substrate used. Kinetic modeling of the fermentation data showed that specific growth and oil accumulation rates were similar in all substrate types and the lipid coefficient ranged from 0.02 to 0.06 g/g of sugar consumed. The fatty acid methyl ester yield and composition of the lipids showed their suitability for conversion to biodiesel. Based on the results, lignocellulose sugars could be used as fermentation substrates by activated sludge microorganisms for enhancing the oil content of sewage sludge for its use as a sustainable biofuel feedstock source. © 2013 American Institute of Chemical Engineers AIChE J, 59: 4036–4044, 2013     

Statistical methodology for optimizing the dilute acid hydrolysis of sugarcane bagasse

In converting advanced biomass to fuel, one pretreatment that has been extensively explored is a high temperature, dilute-sulfuric acid (H₂SO₄) process. This effectively hydrolyzes the hemicellulosic portion of the lignocellulosic biomass to fermentable sugars. Our aim was to optimize the concentration of sulfuric acid and residence time to release xylose from the hemicellulose of sugarcane bagasse. According to response surface methodology (RSM), the optimum concentrations and residence time were determined. The experimental maximum yield for xylose production was found to be 78.9% at 170 °C, 0.24% acid, for 15 min, and 76.4% at 200 °C, 0.22% acid, for 6 min. The predicted maximum yield obtained for the fitted model was found to be 80.3% and 78.1% for the conditions stated above, respectively. The experimental yield was around 1.5% lower than that of the predicted yield. It was confirmed in this study that pentose sugars (xylose and arabinose) derived from hemicellulose fraction were further degraded. The statistical optimization method, which incorporates reaction time, temperature and acid concentration, did prove to provide a useful means of trading off the combined effects of these three variables on total xylose recovery yields.     

Production of xylose,furfural, fermentable sugars and ethanol from agricultural residues

With the developing shortage of petroleum, reliance on biomass as a source of chemicals and fuels will increase. In the present work, bagasse and rice husk were subjected to dilute acid (H₂SO₄) hydrolysis using pressurised water to obtain furfural and fermentable sugars. Various process conditions such as particle size, solid-liquid ratio, acid concentration, reaction time and temperature have been studied to optimise yields of furfural, xylose and other fermentable sugars. The use of particle sizes smaller than 495 μm did not further increase the yield of reducing sugars. A solid-liquid ratio of 1:15 was found to be the most suitable for production of reducing sugars. Hydrolysis using 0.4% H₂SO₄ at 453 K resulted in selective yields (g per 100 g of dried agricultural residues) of xylose from bagasse (22.5%) and rice husk (21.5%). A maximum yield of furfural was obtained using 0.4% H₂SO₄ at 473 K from bagasse (11.5%) and rice husk (10.9%). It was also found that hydrolysis using 1% H₂SO₄ at 493 K resulted in maximum yields of total reducing sugar from bagasse (53.5%) and rice husk (50%). The reducing sugars obtained were fermented to ethanol after removal of furfural. The effect of furfural on the fermentation of sugars to ethanol was also studied. Based on these studies, an integrated two-step process for the production of furfural and fermentable sugars could be envisaged. In the first step, using 0.4% H₂SO₄ at 473 K, furfural could be obtained, while in the second step, the use of 1% H₂SO₄ at 493 K should result in the production of fermentable sugars.     

虫草菌中多糖的气相色谱分析

采用气相色谱法研究自虫草菌中分离、纯化的多糖的单糖组成,结果表明其单糖的摩尔组成分别为阿拉伯糖：1,木糖：2,甘露糖：1,葡萄糖：3,半乳糖：3。     

Dynamic control of arabinose and xylose utilization in E. coli

The common bacterium Escherichia coli (E. coli) can utilize the pentose sugars arabinose and xylose for growth and energy. When fed both these sugars, the bacterium preferentially utilizes arabinose and only when all the arabinose is exhausted from the media does it start to use xylose. This hierarchical utilization of the two sugars is dictated by two proteins: AraC and XylR. These proteins act as controllers of sugar utilization and dictate the timing and rate of utilization of these sugars. While the biochemical interactions defining individual arabinose and xylose utilization systems are well understood, it is not completely understood how the hierarchical utilization is maintained by the bacterium, and how the regulatory crosstalk between the two systems facilitates this hierarchy. To help answer these questions, in this work, we systematically experimentally characterize the regulatory crosstalk between the two sugar utilization systems. Our work demonstrates extensive interaction between the two sugar systems. Specifically, data from our experiments suggest that the xylose system can regulate arabinose gene expression and consequently, cellular physiology dynamically via promiscuous transport and maybe through cross interactions between regulator and non‐cognate sugar. Put together, we demonstrate that arabinose and xylose utilization networks exhibit an example of distributed control in a biological system. This design likely ensures that the system does not fail under perturbations (mutations). Our results help understand multi‐process control in biological systems and bring to light design criteria for synthetic biology applications.

     

不同C源对丙酮丁醇梭菌产丁醇的影响

对丙酮丁醇梭菌在以葡萄糖、木糖、蔗糖、混合糖、玉米芯酸解糖液分别作C源的P2培养基中的产丁醇状况进行研究。结果表明:不同C源对丙酮丁醇梭菌发酵产丁醇有显著的影响;葡萄糖为底物时,丁醇产量最高达到13.50 g/L,总溶剂为19.66 g/L;蔗糖为底物时,丁醇所占比例都在70%以上,丁醇产量可达12 g/L;木糖、混合糖为底物时,丁醇产量在10 g/L左右;只有丙酮丁醇梭菌I4-28能利用玉米芯酸解糖液发酵产丁醇,丁醇产量为7 g/L。     

CHEMICAL CHARACTERIZATION OF BEECH CONDENSATE

ABSTRACT

Beech (Fagus sylvatica L.) condensate from a steaming operation was collected and analyzed. The condensate exhibited a low pH, and contained carbohydrates and phenolic compounds. A total of five phenolic acids and aldehydes (syringic acid, vanillic acid, p-hydroxybenzoic acid, syringaldehyde, vanillin) were identified. TLC of the carbohydrates showed the presence of glucose, galactose, xylose, mannose and/or arabinose in the condensate. Fructose, saccharose, raffinose and stachyose were also identified. The COD, BOD5, pH, conductivity and suspended solids (TSS) were determined. The chemical oxygen demand (COD) in the beech condensate varied from 2016 to 1393O mg/L. This value is two to three fold higher than the corresponding biological oxygen demand (BOD₅).     

Optimization of medium for phenylalanine ammonia lyase production in <Emphasis Type="Italic">E. coli</Emphasis> using response surface methodology

A culture medium for phenylalanine ammonia lyase (PAL) production in E. coli was developed following preliminary studies by means of response surface methodology (RSM). The medium components having significant effect on the production were first identified by using a fractional factorial design. Then, central composite design (CCD) was used to optimize the medium constituents and explain the combined effects of four medium constituents: glucose, yeast extract, (NH₄)₂HPO₄ and MgSO₄. A quadratic model was found to fit the PAL production. CCD revealed that the optimum values of the test variables for PAL production were glucose 28.2 g/L, yeast extract 5.01 g/L, (NH₄)₂HPO₄ 7.02 g/L and MgSO₄ 1.5 g/L. PAL production of 62.85 U/g, which was in agreement with the prediction, was observed in the verification experiment. In comparison to the production of basal medium, 1.8-fold increase was obtained.     

Modeling and optimization of dilute nitric acid hydrolysis on corn stover

BACKGROUND: Because of its high cost, nitric acid has not been widely employed as the catalyst for hydrolysis of lignocellulosic biomass to obtain fermentable sugars. However, recently more and more research results have reported that nitric acid was more effective than other acids for the hydrolysis of lignocellulose. Therefore, it is necessary to find an optimum condition for nitric acid pretreatment and a means of reducing the cost. RESULTS: In this work, low concentrations of nitric acid and short reaction times were considered to optimize the pretreatment process. The kinetic parameters of models to predict the concentrations of xylose, glucose, arabinose, acetic acid and furfural in the hydrolysates were obtained. Applying the kinetic models, the optimum conditions were: 150 °C, 0.6% HNO₃ and 1 min, which yielded a solution containing up to 22.01 g L^?1 xylose, 1.91 g L^?1 glucose, 2.90 g L^?1 arabinose, 2.42 g L^?1 acetic acid and 0.21 g L^?1 furfural, which were consistent with the predicted values. The influence of temperature was also studied using the Arrhenius equation. CONCLUSIONS: A combination of experimental data and model analysis suggested that 96% xylose yield can be achieved by using low concentration nitric acid for a short reaction time, which could greatly reduce the pretreatment cost. Therefore, dilute nitric acid could be considered a good choice for the hydrolysis of corn stover. Copyright © 2010 Society of Chemical Industry     

Determination of optimal conditions for ribonucleic acid production by <Emphasis Type="Italic">Candida tropicalis</Emphasis> no. 121

The experiments were based on multivariate statistical concepts, and response surface methodology (RSM) was applied to optimize the fermentation medium for the production of ribonucleic acid (RNA) by Candida tropicalis no. 121. The process involved the individual adjustment and optimization of various medium components at shake flask level. The two-level Plackett-Burman (PB) design was used to screen the medium components, which significantly influenced RNA production. Among seven variables, the concentrations of molasses, ZnSO₄, and H₃PO₄ were found to be the important factors that significantly affected RNA production (confidence levels above 95%). These factors were further optimized using a central composite design (CCD) and RSM. The optimum values for the critical components were as follows: molasses 47.21 g/L: ZnSO₄ 0.048 g/L; H₃PO₄ 1.19 g/L. Under optimal conditions, RNA production was 2.56 g/L, which was in excellent agreement with the predicted value (2.561 g/L), and led to a 2.1-fold increase compare with that using the original medium in RNA production.     

The hydrothermal degradation of cellulosic matter to sugars and their fermentative conversion to protein

For the hydrothermal degradation of cellulosic matter, an apparatus was developed in which water is used as extraction medium. Samples, 0.15 g each, of pure cellulose (filter paper), natural straw, and ¹⁴C-labeled straw were treated at temperatures of between 200° and 275°C. Of the inserted cellulose, 65.7% was recovered at the optimum temperature as sugars and hydroxymethylfurfural. It was possible to degrade the straw selectively: at lower temperatures, the hemicellulose part of the plant matter was converted to xylose and arabinose; and then at higher temperatures, the cellulose was converted to glucose and cellobiose. At the same time, a certain amount of the sugars was transformed to furfural compounds. The growth behavior of the yeast Candida utilis (strain Weissenbach) was analyzed, using cellobiose, xylose, and glucose (standard) as carbon sources. The growth curves applying cellobiose were nearly identical to those of glucose. Xylose showed lower productivity than the hexoses. The main products of the hydrothermal degradation can, therefore, be used favorably as nutritive substances for this proteinproducing yeast.     

A study of mycelial growth and exopolysaccharide production from a submerged culture of <Emphasis Type="Italic">Mycoleptodonoides aitchisonii</Emphasis> in an air-lift bioreactor

We determined the optimal culture and medium conditions for effective production of mycelial mass and exopolysaccharide from a liquid culture of Mycoleptodonoides aitchisonii in an air-lift bioreactor. The mycelial growth and exopolysaccharide production were found to be optimal at a temperature of 25 °C and pH of 6.5. When 60 g/L of lactose was used as a carbon source, the maximum mycelial growth and exopolysaccharide production were obtained. The polypeptone and yeast extract were the most appropriate nitrogen sources for mycelial growth and exopolysaccharide production. In addition, when a mixture of 20 g/L of polypeptone and 5 g/L of yeast extract was used, the exopolysaccharide production increased 50% compared to that of the sole nitrogen source. CaCl₂·2H₂O (1.0 g/L) was the most effective mineral source. Using the optimal culture and medium conditions, batch cultures with basal and designed medium on mycelial growth and exopolysaccharide production in a 5 L air-lift bioreactor were carried out for 16 days. The mycelial growth and exopolysaccharide production increased with an increase of culture time at 14 days, and the maximum mycelial growth and exopolysaccharide production were 20.3 and 6.2 g/L, respectively, after 14 days of culture. The developed model in an air-lift bioreactor showed good agreement with experimental data. These results indicate that exopolysaccharide production is associated with the mycelial growth of M. aitchisonii in an air-lift bioreactor.     

Relationships between Hemicellulose Composition and Lignin Structure in Woods

The composition and absolute amount of neutral sugars were determined for 48 hardwood species (including 17 hardwoods of genera Acacia, 14 hardwoods of genera Eucalyptus, and 17 hardwoods of other genera) and 14 softwood species by alditol-acetate method, and their relationships to the syringyl ratio (syringyl/(syringyl+guaiacyl)) of lignin, which was determined by nitrobenzene oxidation, was investigated. In the hardwood species, an increase in the syringy ratio of lignin correlated with a tendency toward increased xylose/glucose, rhamnose/glucose, and arabinose/glucose ratios. However, the absolute amount of glucose in hardwood was maintained in a small range (0.4–0.5 g in 1 g sample), independent of changes in the syringyl ratio. In the softwood species, with increasing lignin content, the mannose/glucose ratio decreased, but the absolute amount of glucose remained almost constant. In both hardwood and softwood species, a strong correlation was suggested between lignin, indicated by higher syringy ratio, and hemicellulose, indicated by higher xylan/mannan ratio.