Optimization of furfural production from hemicellulose extracted from delignified palm pressed fiber using a two-stage process

This study aims to optimize the conditions for furfural production from hemicellulose extracted from delignified palm pressed fiber (dPPF) via two-stage process: acid hydrolysis followed by dehydration, using response surface methodology (RSM). The extracted hemicellulose contained 80.8% xylose. In order to convert hemicellulose to xylose in the acid hydrolysis step, there were four important parameters consisting of reaction temperature (100–150 °C), sulfuric acid concentration (1–10% v/v), ratio of sulfuric acid to hemicellulose (L/S ratio) (10, 9, and 8 v/w), and reaction time (30–120 min). The maximum xylose production (12.58 g/L) was achieved at 125 °C, 5.5% sulfuric acid, L/S ratio of 9 mL/g for 30 min with the determination coefficient (R²) value of 0.90. For the dehydration process, two parameters; reaction temperature (120–160 °C) and reaction time (30–150 min), were optimized. The maximum furfural production (8.67 g/L) was achieved at a reaction temperature of 140 °C for 90 min with the determination coefficient (R²) value of 0.93.     

Improved xylanase production from a haloalkalophilic Staphylococcus sp. SG-13 using inexpensive agricultural residues

The production of an alkali-stable xylanase, with dual pH optima, from haloalkalophilic Staphylococcus sp. SG-13 has been enhanced using agro-residues in submerged fermentation and a biphasic growth system. The agro-residues such as wheat bran, sugarcane bagasse, corncobs and poplar wood when used as sole carbon source, improved the xylanase yield by five-fold as compared to xylose and xylan. Staphylococcus sp. SG-13 also produced equally good amounts of xylanase when grown simply in deionized water (pH 8.0) supplemented with agro-residues as sole carbon source. In the biphasic growth system (lower layer containing agricultural residue set in agar medium with liquid medium above it), the prime substrate, wheat bran (1% w/v), resulted in maximum xylanase production of 4525 U l^–1 (pH 7.5) and 4540 U l^–1 (pH 9.2) at an agar: broth ratio of 4.0 after 48 h of incubation at 37 °C under static conditions. In general, the cost-effective agro-residues were found to be more suitable inducers for xylanase production over expensive substrates like xylan.     

Production of cellulase-free thermostable xylanases by an isolated strain of Aspergillus niger PPI, utilizing various lignocellulosic wastes

A strain of Aspergillus niger PPI having prolific xylanolytic potential was isolated and the optimum conditions for maximum xylanase production was studied, resulting in the following: 4% substrate concentration, 10% v/v inoculum size, 72 h of incubation and pH 3.5–4.5 at 28 °C. The production profile of xylanase was examined with various lignocellulosics and maximum yield was achieved with oat. The hemicellulose content of wastes was also determined and oatmeal was found to have maximum hemicellulose content followed by wheat straw, sugarcane bagasse, rice husk and gram residue respectively. The enzyme showed maximum activity at pH 4 and temperature 60 °C. However, maximum stability was achieved at pH 3.5 and temperature 55 °C. Cellulase activity was found altogether absent in the enzyme broth.     

Fermentation of xylose and rice straw hydrolysate to ethanol byCandida shehatae NCL-3501

Candida shehatae NCL-3501 utilized glucose and xylose efficiently in batch cultures. The specific rate of ethanol production was higher with mixtures of glucose and xylose (0.64–0.83 g g^–1 cells d^–1) compared to that with individual sugars (0.38–0.58 g g^–1 cells d^–1). Although the optimum temperature for growth was 30°C, this strain grew and produced appreciable levels of ethanol at 45°C. A stable ethanol yield (0.40–0.43 g g^–1 substrate utilized) was obtained between 10 g L^–1 and 80 g L^–1 of initial xylose concentration. Conversion efficiency was further improved by immobilization of the cells in calcium alginate beads. Free or immobilized cells ofC. shehatae NCL-3501 efficiently utilized sugars present in rice straw hemicellulose hydrolysate, prepared by two different methods, within 48 h. Ethanol yields of 0.45 g g^–1 and 0.5 g g^–1 from autohydrolysate, and 0.37 g g^–1 from acid hydrolysate were produced by free and immobilized cells, respectively.     

Liquefaction of dulse (Palmaria palmata (L.) Kuntze) by a commercial enzyme preparation and a purified endo ,β-1,4-D-xylanase

Liquefaction of dry and freshPalmaria palmata by food grade enzyme preparations and a purified endo--1,4-D-xylanase was studied.The endo--1,4-D-xylanase (EC 3.2.1.8) was purified to homogeneity from a commercial food grade enzyme prepared fromAspergillus niger. It has a molecular weight of 22 500, a pI of 3.5, is inactive toward corn arabinoxylan,p-nitrophenyl--D-xylose, carboxymethyl cellulose but shows a weak activity toward microcrystalline cellulose. It hydrolyzes oat and dulse xylan equally well in seawater and deionized water essentially into xylose and xylobiose. It is stable between pH 5.5 to 9.0 and 0 to 30 °C and its activity is optimal at pH 4.5–5.5 and 40–60 °C. It has a K_m of 2.2 and 2.8 mg ml^-1 and V_max of 3600 and 3900 nkat mg^-1 of protein on oat and dulse xylan, respectively.Acetate buffer, deionized water and seawater alone extracted 62.6 to 64.5 % of the dry weight of dry dulse, but the use of commercial food grade enzyme preparations or the purified xylanase improved liquefaction to 81.2–87.1 %. Xylose and galactose were the only sugars present in the soluble extracts. Deionized and seawater extracted 58.8–52.7 and 39.1–42.2% of the dry weight of the fresh algae collected in fall and summer, respectively. Only galactose was found in the seawater extract, while some xylose with galactose were measured in the deionized water extract of the fresh autumn algal sample. Purified and crude xylanase improved liquefaction of fresh algae to 79.8–81.4 and 71.9–77.9% of the fresh dry weight (fall and summer, respectively) in deionized and seawater, respectively, and increased the xylose content of the soluble fractions. Polysaccharides in the soluble residues were composed of 1,3/1,4-linked xylose, 1-linked galactose (floridoside) and 1,4-linked glucose (cellulose) and contained essentially 1,4-linked xylose and 1,4-linked glucose in insoluble fractions obtained after enzymatic treatment.The use of xylanase-containing food grade enzyme preparations improves liquefaction ofPalmaria palmata, particularly from fresh alga. This study indicates that processing such as drying may modify markedly the solubility ofP. palmata cell wall polysaccharides, which would imply the existence of some organization and/or other components in the fresh cell wall that lower xylan solubility in seawater.     

Methane fermentation of xylan by mesophilic and thermophilic methane sludges

The degradation of xylan during methane fermentation proceeded as a first-order reaction. The rate constants were calculated to be 0.40–0.09 day^–1 at 37° C and 0.341 day^–1 at 55° C. From calculations based on the experimental data, K _A and C _A values in the expression of the velocity of xylose consumption changed as the fermentation progressed. In the mesophilic fermentation, the degradation of xylan slowed down after 2 days of incubation, but the rate of consumption of xylose increased between days 3 and 4 of incubation and slow again at the 5th day of incubation. In the thermophilic fermentation, the degradation of xylan proceeded at a constant rate and the rate of consumption of xylose increased slightly on the 3rd day of incubation. When the velocity of gas evolution was determined, the C _G value for acetate at 55° C was about 1.8 times larger than the value at 37° C.     

Effects of acetic acid on the temperature range of ethanol tolerance inCandida shehate growing on D-xylose

Summary The yeastCandida shehatæ, growing on a mineral medium with vitamins and D-xylose as sole carbon and energy source, tolerated acetic acid up to 0.4% (v/v), at pH 4.5, the temperature range of growth narrowing from 5–34°C to 21–27°C, and the growth yield on D-xylose decreasing to 64%; tolerance to added ethanol dropped from 5% (v/v) to 2% (v/v). Acetic acid concertedly shifted the Arrhenius plots of thermal death and growth to lower temperatures, but left unchanged the former dissociative relations in the temperature profile. The entropy coefficient of acetic acid-enhanced thermal death was 183.3 entropy units. M^–1.     

Production of acetate by mutant strains of Clostridium thermoaceticum

Summary Mutant strains were derived from Clostridium thermoaceticum ATCC 39 289 by treatment with chemical mutagenic agents and selective enrichment procedures. Some mutant strains exhibited growth when cultured in media containing 20 mabetm (1.75 g l^–1) pyruvate of high-magnesium lime (dolime) above pH 6.0. One strain (G-20) grew and produced acetate when 80 mabetm (7 gl^–1) pyruvate or 50 mabetm (2.3 g l^–1) formate at pH 5.6 was the sole energy source. In a fed-batch process controlled at pH 6.2, this mutant produced 52.5 g l^–1 acetate (equivalent to 72.5 g l^–1 Na acetate) and 67 g l^–1 calcium-magnesium acetate (CMA) in 140 h when dolime was the neutralizing agent, with 93% substrate utilization. This mutant strain holds promise for CMA production due to its better tolerance of dolime and its ability to synthesize high levels of acetic acid. Offprint requests to: S. R. Parekh     

Effect of temperature and pH onCandida blankii in chemostat culture

The growth characteristics ofCandida blankii as a function of temperature and pH in a simulated bagasse hemicellulose hydrolysate were determined in chemostat culture. The highest maximum specific growth rate of 0.44h^–1 was reached at 38°C and at pH 5.5, with a sharp decrease in growth rate on either side of this temperature. Growth occurred at 46°C but not at 48°C. The protein and cell yields varied little below 40°C and the respective values were 0.22 and 0.5 g/g at 38°C. At the lower pH values, a severe linear decrease in cell and protein yields occurred, whereas a small increase in these yields at decreasing pH values was found when acetic acid was omitted from the medium. In the presence of acetic acid, a very sharp decrease in the growth rate at pH values below pH 4.5 was noted, despite the very low residual acetic acid concentrations, of less than 50 mg/l, in the culture.     

Cultivation of Candida langeronii in sugar cane bagasse hemicellulosic hydrolyzate for the production of single cell protein

Sugar cane bagasse hemicellulosic fraction was hydrolysed by treatment with 70 mg of sulphuric acid per gram of dry mass at 125 °C for 2 h. The hydrolysate was used as the substrate to grow Candida langeronii RLJ Y-019 at 42 °C; initial pH 6.0; stirring at 700 rev/min and aeration at 1.0 and 2.0 v/v/min. The utilization of D-xylose, L-arabinose, and acetic acid were delayed due to the presence of D-glucose, but after D-glucose depletion the other carbon sources were utilized. The kinetic parameters calculated for both cultivations at 1.0 and 2.0 v/v/min included: maximum specific growth rate (_max) of 0.29 ± 0.01 h^–1 and 0.43 ± 0.016 h^–1, yields (Y _x/s) of 0.36 ± 0.012 and 0.40 ± 0.012 g_x/g_s and productivity (Q _x) of 0.81 ± 0.016 and 0.97 ± 0.012 g_x/l/h, respectively, and compared favourably with published results obtained with Candida utilis and Geotrichum candidum. Candida langeronii appeared superior to C. utilis for biomass production from hemicellulose hydrolysate, in that it utilized L-arabinose and was capable of growth at higher temperatures. The biomass contained 48.2, 1.4, 5.8 and 23.4% of total protein, DNA, RNA and carbohydrate, respectively and contained essential amino acids for animal feed.     

Acetate production byClostridium thermoaceticum in corn steep liquor media

A low-cost nutrient medium based on corn steep liquor (CSL) was developed for the production of acetates byClostridium thermoaceticum. Pre-treatment of CSL with dolime and vitamin supplementation increased the rate of acetate production. Adding excess nutrients in a fed-batch mode minimized by-product formation and increased final acetate concentration from 19 g L^–1 to 40 g L^–1 acetic acid. High yields of acetic acid (0.95 g g^–1 glucose in fed-batch mode) was probably due to the conversion of the lactic acid in CSL into acetic acid by the organism.     

Effects of acetic acid on the temperature profile of ethanol tolerance inSaccharomyces cerevisiae

Summary In a strain ofSaccharomyces cerevisiae, acetic acid at concentrations up to 1% (v/v) depressed the tolerance to added ethanol, from 11% (v/v) down to zero, and simultaneously narrowed the temperature range of growth from 3–42°C to 19–26°C. In addition, acetic acid shifted the associative temperature profile of growthand death to lower temperatures, and depressed the growth yield on glucose.     

Xylanolytic anaerobic thermophiles from Icelandic hot-springs

Anaerobic enrichment cultures inoculated with neutral and alkaline (pH 7.0–9.0) sediment and biomat samples from hot-springs in Hveragerdi and Fluir, Iceland, were screened for growth on beech xylan from pH 8.0 to 10.0 at 68° C: no growth occured in cultures above pH 8.4. Five anaerobic xylanolytic bacteria were isolated from enrichment cultures at pH 8.4; all five microbes were Gram-positive rods with terminal spores, and produced CO₂, H₂, acetate, lactate and ethanol from xylan and xylose. One of the isolates, strain A2, grew from 50 to 75° C, with optimum growth near 68° C, and from pH 5.2 to 9.0 with an optimum between 6.8 and 7.4. Taxonomically, strain A2 was most similar to Clostridium thermohydrosulfuricum. At pH 7.0, the supernatant xylanases of strain A2 had a temperature range from 50 to 78° C with an optimum between 68 and 78° C. At 68° C, xylanase activity occurred from pH 4.9 to 9.1, with an optimum from pH 5.0 to 6.6. At pH 7.0 and 68° C, the K _m of the supernatant xylanases was 2.75 g xylan/l and the V _max was 2.65 × 10^–6 kat/l culture supernatant. When grown on xylose, xylanase production was as high as when grown on xylan. Correspondence to: B. K. Ahring     

Purification and characterization of a high-thermostable β-xylanase from newly isolated Thermomyces lanuginosus THKU-49

Highly thermostable β-xylanase produced by newly isolated Thermomyces lanuginosus THKU-49 strain was purified in a four-step procedure involving ammonium sulfate precipitation and subsequent separation on a DEAE-Sepharose fast flow column, hydroxylapatite column, and Sephadex G-100 column, respectively. The enzyme purified to homogeneity had a specific activity of 552 U/mg protein and a molecular weight of 24.9 kDa. The optimal temperature of the purified xylanase was 70°C, and it was stable at temperatures up to 60°C at pH 6.0; the optimal pH was 5.0–7.0, and it was stable in the pH range 3.5–8.0 at 4°C. Xylanase activity was inhibited by Mn²⁺, Sn²⁺, and ethylenediaminetetraacetic acid. The xylanase showed a high activity towards soluble oat spelt xylan, but it exhibited low activity towards insoluble oat spelt xylan; no activity was found to carboxymethylcellulose, avicel, filter paper, locust bean gum, cassava starch, and p-nitrophenyl β-d-xylopyranoside. The apparent K _m value of the xylanase on soluble oat spelt xylan and insoluble oat spelt xylan was 7.3 ± 0.236 and 60.2 ± 6.788 mg/ml, respectively. Thin-layer chromatography analysis showed that the xylanase hydrolyzed oat spelt xylan to yield mainly xylobiose and xylose as end products, but that it could not release xylose from the substrate xylobiose, suggesting that it is an endo-xylanase.     

Acid hydrolysis of sugarcane bagasse for lactic acid production

In order to use sugarcane bagasse as a substrate for lactic acid production, optimum conditions for acid hydrolysis of the bagasse were investigated. After lignin extraction, the conditions were varied in terms of hydrochloric (HCl) or sulfuric (H₂SO₄) concentration (0.5–5%, v/v), reaction time (1–5 h) and incubation temperature (90–120 °C). The maximum catalytic efficiency (E) was 10.85 under the conditions of 0.5% of HCl at 100 °C for 5 h, which the main components (in g l⁻¹) in the hydrolysate were glucose, 1.50; xylose, 22.59; arabinose, 1.29; acetic acid, 0.15 and furfural, 1.19. To increase yield of lactic acid production from the hydrolysate by Lactococcus lactis IO-1, the hydrolysate was detoxified through amberlite and supplemented with 7 g l⁻¹ of xylose and 7 g l⁻¹ of yeast extract. The main products (in g l⁻¹) of the fermentation were lactic acid, 10.85; acetic acid, 7.87; formic acid, 6.04 and ethanol, 5.24.     

Production of alkali-tolerant cellulase-free xylanase by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. WLUN024 with wheat bran as the main substrate

An alkali-tolerant cellulase-free xylanase producer, WLI-11, was screened from soil samples collected from a pulp and paper mill in China. It was subsequently identified as a Pseudomonas sp. A mutant, WLUN024, was selected by consecutive mutagenesis by u.v. irradiation and NTG treatment using Pseudomonas sp. WLI-11 as parent strain. Pseudomonas sp. WLUN024 produced xylanase when grown on xylosidic materials, such as hemicellulose, xylan, xylose, and wheat bran. Effects of various nutritional factors on xylanase production by Pseudomonas sp. WLUN024 with wheat bran as the main substrate were investigated. A batch culture of Pseudomonas sp. WLUN024 was conducted under suitable fermentation conditions, where the maximum activity of xylanase reached 1245 U ml⁻¹ after incubating at 37 °C for 24 h. Xylanase produced by Pseudomonas sp. WLUN024 was purified and the molecular weight was estimated as 25.4 kDa. Primary studies on the characteristics of the purified xylanase revealed that this xylanase was alkali-tolerant (optimum pH 7.2–8.0) and cellulase-free. In addition, the xylanase was also capable of producing high quality xylo-oligosaccharides, which indicated its application potential in not only pulp bio-bleaching processes but also in the nutraceutical industry.     

Cloning and expression in Escherichia coli of the Clostridium thermoaceticum gene encoding thermostable formyltetrahydrofolate synthetase

Formyltetrahydrofolate synthetase (FTHFS) (EC 6.3.4.3), a thermostable protein of four identical subunits from Clostridium thermoaceticum was cloned into Escherichia coli SK1592. The clone (CRL47) contained a 9.5 kb EcoRI fragment of C. thermoaceticum DNA ligated into pBR322. It produced catalytically active, thermostable FTHFS, that was not found in E. coli SK1592 containing native pBR322. The identity of the expressed enzyme was confirmed by specific binding of rabbit polyclonal anti-FTHFS serum produced against C. thermoaceticum FTHFS. The specific activities (mol·min^-1·mg^-1) of FTHFS in cell free extracts of CRL47 were 28–89 when assayed at 50°C and pH8. This was from 3–10-fold higher than in C. thermoaceticum extracts. FTHFS was purified to homogeneity from CRL47. The purified enzyme behaved during electrophoresis and gel chromatography and it had similar specific activity and thermostability as the enzyme purified from C. thermoaceticum.Abbreviations FTHFS formyltetrahydrofolate synthetase - kb kilobase - H₄-folate tetrahydrofolate - SDS sodium dodecyl sulfate A preliminary account of this work was presented at the annual meeting of the American Society for Microbiology, Atlanta, GA, 1987 (C. R. Lovell, A. Przybyla and L. G. Ljungdahl, Abstr. Annu. Meet. Am. Soc. Microbiol. 1987, K126, p. 223).     

A novel strain of Streptomyces malaysiensis isolated from Brazilian soil produces high endo-β-1,4-xylanase titres

One hundred and sixty two actinomycete strains isolated from Brazilian soils were screened for xylanase activity, according to the size of the hydrolysis zones observed in oat spelts xylan agar plates. The strain AMT-3, later identified as Streptomyces malaysiensis, was selected as the best producer. In subsequent shake flasks fermentations using growth media contanning 1% (w/v) of either birchwood, or oat spelts xylan, plus organic nitrogen and salts, high endo--1,4-xylanase titres (EC 3.2.1.8) (116 U ml^–1) were observed in the larchwood medium within 6 days. This is the first report concerning xylanase production by streptomyces malaysiensis, which has been recently described as a new species.     

Production of thermostable xylanases in batch and continuous culture by Thermomonospora fusca KW 3

Summary The actinomycete Thermomonospora fusca KW 3 produced novel thermostable xylanases in batch and continuous cultures in media containing insoluble xylan. The production of xylanases could be induced with oat spelt or beech xylan. Very low activities were detected when the strain was grown on glucose or xylose. In continuous cultivations, mycelial wall growth could be prevented using a stirrer speed controller. Homogeneous mixing of the insoluble substrate was obtained by vibrating the flexible tubes. T. fusca KW 3 could be grown on insoluble xylan at growth rates as high as 0.23 h^–1, equivalent to a doubling time of 3 h. Xylanase activity decreased from maximum levels of 2.5 units (U) ml^–1 with increasing dilution rate and was nearly constant at a level of 0.5 U ml^–1 with dilution rates greater than 0.1 h^–1. Correspondence to: P. Röthlisberger     

Effect of inoculum level on xylitol production from rice straw hemicellulose hydrolysate byCandida guilliermondii

The effect of inoculum level on xylitol production byCandida guilliermondii was evaluated in a rice straw hemicellulose hydrolysate. High initial cell density did not show a positive effect in this bioconversion since increasing the initial cell density from 0.67 g L^–1 to 2.41 g L^–1 decreased both the rate of xylose utilization and xylitol accumulation. The maximum xylitol yield (0.71 g g^–1) and volumetric productivity (0.56 g L^–1 h^–1) were reached with an inoculum level of 0.9 g L^–1. These results show that under appropriate inoculum conditions rice straw hemicellulose hydrolysate can be converted into xylitol by the yeastC. guilliermondii with efficiency values as high as 77% of the theoretical maximum.