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.

     

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.     

Analysis of exposed cellulose surfaces in pretreated wood biomass using carbohydrate‐binding module (CBM)–cyan fluorescent protein (CFP)

In enzymatic saccharification of lignocellulosics, the access of the enzymes to exposed cellulose surfaces is a key initial step in triggering hydrolysis. However, knowledge of the structure–hydrolyzability relationship of the pretreated biomass is still limited. Here we used fluorescent‐labeled recombinant carbohydrate‐binding modules (CBMs) from Clostridium josui as specific markers for crystalline cellulose (CjCBM3) and non‐crystalline cellulose (CjCBM28) to analyze the complex surfaces of wood tissues pretreated with NaOH, NaOH–Na₂S (kraft pulping), hydrothermolysis, ball‐milling, and organosolvolysis. Japanese cedar wood, one of the most recalcitrant softwood species was selected for the analysis. The binding analysis clarified the linear dependency of the exposure of crystalline and non‐crystalline cellulose surfaces for enzymatic saccharification yield by the organosolv and kraft delignification processes. Ball‐milling for 5–30 min increased saccharification yield up to 77%, but adsorption by the CjCBM–cyan fluorescent proteins (CFPs) was below 5%. Adsorption of CjCBM–CFPs on the hydrothermolysis pulp were less than half of those for organosolvolysis pulp, in coincidence with low saccharification yields. For all the pretreated wood, crystallinity index was not directly correlated with the overall saccharification yield. Fluorescent microscopy revealed that CjCBM3–CFP and CjCBM28–CFP were site‐specifically adsorbed on external fibrous structures and ruptured or distorted fiber surfaces. The assay system with CBM–CFPs is a powerful measure to estimate the initiation sites of hydrolysis and saccharification yields from chemically delignified wood pulps. Biotechnol. Bioeng. 2010; 105: 499–508. © 2009 Wiley Periodicals, Inc.     

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.     

Production of thermostable pectinase and xylanase for their potential application in bleaching of kraft pulp

A very high level of alkalophilic and thermostable pectinase and xylanase has been produced from newly isolated strains of Bacillus subtilis and Bacillus pumilus respectively. Enzyme production for pectinase was carried out under SSF using combinations of cheap agricultural residues while xylanase was produced under submerged fermentation using wheat bran as substrate to minimize the cost of production of these enzymes Among the various substrates tested, the highest yield of pectinase production was observed by using combination of WB + CW (6592 U/g of dry substrate) supplemented with 4% yeast extract when incubated at 37 °C for 72 h using deionized water of pH 7.0 as moistening agent. The biobleaching effect of these cellulase free enzymes on kraft pulp was determined. Both xylanase and pectinase showed stability over a broad range of pH from 6 to 10 and temperature from 55 to 70 °C. The bleaching efficiency of the pectinase and xylanase on kraft pulp was maximum after 150 min at 60 °C using enzyme dosage of 5 IU/ml of each enzyme at 10% pulp consistency with about 16% reduction in kappa number and 84% reduction in permanganate number. Enzyme treated pulp when subjected to CDED₁D₂ steps, 25% reduction in chlorine consumption and upto 19% reduction in consumption of chlorine dioxide was observed for obtaining the same %ISO brightness. Also an increase of 22 and 84% in whiteness and fluorescence respectively and a decrease of approximately 19% in the yellowness of the biotreated pulp were observed by pretreatment of the pulp with our enzymatic mixture.     

A thermophilic ionic liquid-tolerant cellulase cocktail for the production of cellulosic biofuels

Generation of biofuels from sugars in lignocellulosic biomass is a promising alternative to liquid fossil fuels, but efficient and inexpensive bioprocessing configurations must be developed to make this technology commercially viable. One of the major barriers to commercialization is the recalcitrance of plant cell wall polysaccharides to enzymatic hydrolysis. Biomass pretreatment with ionic liquids (ILs) enables efficient saccharification of biomass, but residual ILs inhibit both saccharification and microbial fuel production, requiring extensive washing after IL pretreatment. Pretreatment itself can also produce biomass-derived inhibitory compounds that reduce microbial fuel production. Therefore, there are multiple points in the process from biomass to biofuel production that must be interrogated and optimized to maximize fuel production. Here, we report the development of an IL-tolerant cellulase cocktail by combining thermophilic bacterial glycoside hydrolases produced by a mixed consortia with recombinant glycoside hydrolases. This enzymatic cocktail saccharifies IL-pretreated biomass at higher temperatures and in the presence of much higher IL concentrations than commercial fungal cocktails. Sugars obtained from saccharification of IL-pretreated switchgrass using this cocktail can be converted into biodiesel (fatty acid ethyl-esters or FAEEs) by a metabolically engineered strain of E. coli. During these studies, we found that this biodiesel-producing E. coli strain was sensitive to ILs and inhibitors released by saccharification. This cocktail will enable the development of novel biomass to biofuel bioprocessing configurations that may overcome some of the barriers to production of inexpensive cellulosic biofuels.     

Hydrolytic potential of five fungal supernatants to enhance a commercial enzyme cocktail

Objectives

To evaluate the potential of enzyme cocktails produced by five filamentous fungi to supplement the industrial cellulase cocktail, Celluclast 1.5L, in order to improve the efficiency of saccharification.

Results

The fungi were cultivated on wheat bran and the resulting supernatants were combined with Celluclast in enzymatic hydrolysis experiments to test their ability to hydrolyze wheat bran and five cellulose-rich substrates. The supernatant showing the best performance was that from an Aspergillus niger cellulase mutant. The addition of β-glucosidase only to the Celluclast cocktail was not as beneficial.

Conclusion

Supplementing commercial cocktails with enzymes from carefully selected fungi may result in significantly more efficient saccharification of lignocellulosic materials. Furthermore, such an approach could lead to the identification of novel enzyme activities crucial for saccharification.

     

Production of ethanol from potato pulp: Investigation of the role of the enzyme from Acremonium cellulolyticus in conversion of potato pulp into ethanol

In this study, the saccharification and fermentation of the by-product of starch manufacture, potato pulp, were investigated. Analytic results of the components show that the potato pulp contains large amounts of starch, cellulose, and pectin. A commercial enzyme from Acremonium cellulolyticus was found to be highly efficient in the saccharification of potato pulp, since it exhibited high pectinase, α-amylase, and cellulase activities. Hydrothermal treatment of the potato pulp increased the saccharification rate, with a corresponding glucose concentration of 114 g/L and yield of 68% compared to the glucose concentration of 47 g/L and yield of 28% in the untreated case. The hydrolyzate could be used as both nitrogen and carbon sources for ethanol fermentation, showing that bioconversion of potato pulp to ethanol is feasible.     

Laccase-initiated cross-linking of lignocellulose fibres using a ultra-filtered lignin isolated from kraft black liquor

In this work, the effect of Trametes pubescens laccase (TpL) used in combination with a low-molecular-weight ultra-filtered lignin (UFL) to improve mechanical properties of kraft liner pulp and chemi-thermo-mechanical pulp was studied. UFL was isolated by ultra-filtration from the kraft cooking black liquor obtained from softwood pulping. This by-product from the pulp industry contains an oligomeric lignin with almost twice the amount of free phenolic moieties than residual kraft pulp lignin. The reactivity of TpL on UFL and kraft pulp was studied by nuclear magnetic resonance spectroscopy and size exclusion chromatography. Laccase was shown to polymerise UFL and residual kraft pulp lignin in the fibres, seen by the increase in their average molecular weight and in the case of UFL as a decrease in the amount of phenolic hydroxyls. The laccase initiated cross-linking of lignin, mediated by UFL, which gives rise to more than a twofold increase in wet strength of kraft liner pulp handsheets without loosing other critical mechanical properties. Hence, this could be an interesting path to decrease mechano-sorptive creep that has been reported to lessen in extent as wet strength is given to papers. The laccase/2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) mediator system showed a greater increase in wet tensile strength of the resulting pulp sheets than the laccase/UFL system. However, other mechanical properties such as dry tensile strength, compression strength and Scott Bond internal strength were negatively affected by the laccase/ABTS system.     

鸡尾酒δ整合策略构建表达三类纤维素酶的酿酒酵母工程菌株及初步评价

木质纤维素乙醇具有替代化石燃料的潜力,其生产过程包括生物质预处理、纤维素酶生产、水解和发酵等多个步骤。将纤维素酶生产、水解和发酵组合在一起的统合生物加工过程（consolidated bioprocessing,CBP）由于能降低水解和发酵成本而具有应用于纤维素乙醇生产的潜力,该技术的关键是构建能有效降解纤维素的工程菌株,而构建表达纤维素酶的酿酒酵母即是其中一种选择。采用鸡尾酒多拷贝δ整合的策略将7种纤维素酶基因（Trichoderma reesei cbh1、cbh2和egl2,Aspergillus aculeatus cbh1、egl1和bgl1）表达盒整合至酿酒酵母W303-1A染色体上,经4轮整合筛选得到菌株LA1、LA2、LA3和LA4。对这4个菌株进行纤维素酶活性测定,结果表明从LA1到LA3各种纤维素酶活性呈递增趋势,而LA4的酶活性与LA3的酶活水平相当。对菌株LA3进行酸碱预处理玉米芯料的发酵评价,结果表明：①在外加商品化纤维素酶的情况下,与对照菌株W303-1A和AADY相比,LA3能有效利用纤维素料发酵产醇;②与分步整合的菌株W3相比,发酵性能更优;③培养基中的营养成分影响菌株发酵性能。这些结果表明,鸡尾酒δ整合是一种有效的构建酿酒酵母CBP菌株的方法。     

Saccharification of cellulosic substrates byAspergillus niger cellulase

Chemical pre-treatment enhanced saccharification of agricultural lignocellulosic residues usingAspergillus niger cellulase by between 16 and 38%. Maximum saccharification (76%) of alkali-treated bagasse was with 0.5% substrate over 48 h using 0.3 U enzyme/ml.

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Résumé Le pré-traitement chimique augmente de 16 à 38% la saccharification de résidus agricoles ligno-cellulosiques lorsque l'on utilise la cellulase d'Aspergillus niger. On a obtenu la saccharification maximum (76%) de la bagasse traitée à l'alcali avec 0.5% de substrat en 48 h de traitement avec 0.3 unités d'enzyme par ml.

     

Nitrification, denitrification, and dechlorination in bleached kraft pulp mill wastewater

This study deals with combining the biologi cal removal of organic halogens with the removal of nitrogen from bleached kraft pulp mill wastewater in fluidized-bed reactors under nitrifying and denitrifying conditions. Untreated and biotreated bleached kraft pulp mill wastewaters had no detrimental effect on nitrification or denitrification. The nitrifying biofilm reactor, pregrown on synthetic inorganic feed with ammonia, removed without a lag phase adsorbable organic halogens [7.2 mg Cl (g biomass volatile solids)⁻¹day⁻¹] from bleached kraft pulp mill wastewater and selected chlorophenols from synthetic wastewater. Electron microscopical examination of the biofilm showed that bacteria, morphologically similar to the nitrifying species Nitrosomonas or Nitrobacter, and Nitrosospira were dominant. The denitrifying fluidized-bed reactor, pregrown on nitrate and methanol, denitrified without a lag phase bleached kraft pulp mill wastewater. Under denitrifying conditions, 35% of the total organic carbon content of untreated bleached kraft pulp mill waste water was removed. The reducing power delivered by untreated bleached kraft pulp mill wastewater for denitrification was 2 mmol electrons/mmol carbon mineralized. Dechlorination under denitrifying conditions was negligible. Received: 21 November 1996 / Received revision: 27 January 1997 / Accepted: 1 February 1997     

Saccharification of cellulosics by Microbispora bispora

Summary The saccharification efficiency of cellulase from the thermophilic actinomycete Microbispora bispora was evaluated using commercially available feedstocks. The enzyme preparation was effective against refuse derived cellulose with 30% being converted to glucose in a 24 hour period. Pretreatment of the refuse with cadoxen resulted in an increase in saccharification efficiency to 70%.     

Efficient Recovery of Glucose and Fructose via Enzymatic Saccharification of Rice Straw with Soft Carbohydrates

Soft carbohydrates, defined as readily-recoverable carbohydrates via mere extraction from the biomass or brief enzymatic saccharification, were found in significant amounts in rice straw as forms of free glucose, free fructose, sucrose, starch, and β-1,3-1,4-glucan. In this study, we investigated their amounts in rice straw (defined as culm and leaf sheath), and developed an easy method for glucose and fructose recovery from them with heat-pretreatment and subsequent 4-h enzymatic saccharification with an enzyme cocktail of cellulase and amyloglucosidase. The recovery of glucose and fructose exhibited good correlation with the amounts of soft carbohydrates. The maximum yields of glucose and fructose in the rice straw per dry weight at the heading stage and the mature stage were 43.5% in cv. Habataki and 34.1% in cv. Leafstar. Thus, rice straw with soft carbohydrates can be regarded as a novel feedstock for economically feasible production of readily-fermentable glucose and fructose for bioethanol.     

Enzymatic hydrolysis and simultaneous saccharification and fermentation of alkali/peracetic acid-pretreated sugarcane bagasse for ethanol and 2,3-butanediol production

The enzymatic digestibility of alkali/peracetic acid (PAA)-pretreated bagasse was systematically investigated. The effects of initial solid consistency, cellulase loading and addition of supplemental β-glucosidase on the enzymatic conversion of glycan were studied. It was found the alkali-PAA pulp showed excellent enzymatic digestibility. The enzymatic glycan conversion could reach about 80% after 24 h incubation when enzyme loading was 10 FPU/g solid. Simultaneous saccharification and fermentation (SSF) results indicated that the pulp could be well converted to ethanol. Compared with dilute acid pretreated bagasse (DAPB), alkali-PAA pulp could obtain much higher ethanol and xylose concentrations. The fermentation broth still showed some cellulase activity so that the fed pulp could be further converted to sugars and ethanol. After the second batch SSF, the fermentation broth of alkali-PAA pulp still kept about 50% of initial cellulase activity. However, only 21% of initial cellulase activity was kept in the fermentation broth of DAPB. The xylose syrup obtained in SSF of alkali-PAA pulp could be well converted to 2,3-butanediol by Klebsiella pneumoniae CGMCC 1.9131.     

Pretreatment Of Hardwood (Eucalyptus Regnans) Sawdust By Autohydrolysis Explosion And Its Saccharification By Trichodermal Cellulases

Autohydrolysis explosion pretreatment of hardwood (Eucalyptus regnans) sawdust at 200°C and 6.9 MPa gas pressure (steam + nitrogen) for 5 min solubilized 85% of the total hemicellulose components and produced a pulp that was highly accessible to attack by cellulases from Trichoderma reesei C-30 and by a commercial preparation, Meicelase. The autohydrolysis liquor, representing 15% of the original weight of the sawdust on a solids basis, consisted mainly of xylose, xylose oligomers and minor amounts of galactose, mannose, arabinose, glucose and uronic acids. Enzymic hydrolysis of pretreated E. regnans pulps using Trichodermal cellulases resulted in saccharification yields of <50% within 24 h from 10% (w/v) substrate slurries and 20 cellulase (FPU) units per g of pretreated pulp. The cellulose-to-glucose conversions were lower and this was attributable to the production of a compound(s) during enzymic hydrolysis that was inhibitory to the β-glucosidase component, but not the cellulases, in the Trichodermal cellulase preparations. Enzymic digests supplemented with Novozym 188 β-glucosidase showed >70% cellulose-to-glucose conversion within 24 h under similar conditions of hydrolysis. The inhibitor compound was not inhibitory to the Novozym 188 β-glucosidases. Alkali-extracted autohydrolysis-exploded pulps were less susceptible to hydrolysis than unextracted pulps. Factors that influenced the extent of cellulose conversion into glucose such as enzyme-substrate and cellulase-to-β-glucosidase ratios are also discussed.     

Conversion of cellulose to glucose with cellulase of Trichoderma viride ITCC-1433

Trichoderma viride ITCC-1433 secretes a cellulase complex that is rich in β-glucosidase and therefore well suited for the saccharification of cellulosic materials. The cellulase was investigated with respect to optimum conditions of reaction and enzyme stability. Avicelase, CMCase, and β-glucosidase differed considerably in their physicochemical properties. At temperatures above 50°C, β-glucosidase is not very stable. Therefore, as a compromise the conditions of hydrolysis were chosen to be 50°C and pH 4.5. With the crude culture filtrate of T. viride ITCC-1433 a nearly pure glucose solution of 4% is reached from a 10% cellulose suspension. Wood pulp and newsprint are hydrolyzed to a much smaller extent. With an enzyme concentrate up to 8% glucose accumulated in the reaction fluid within 48 hr. At this time the glucose-cellobiose ratio was 75:1. Glucose was demonstrated to be the most potent inhibitor of total hydrolysis. The addition of glucose to the enzyme-substrate solution at zero time completely stopped its own formation and cellobiose and reducing groups (oligosaccharides) accumulated. By removing glucose through an ultrafilter device about 90% saccharification of cellulose to glucose was achieved in 48 hr without any accumulation of cellobiose.     

Enhanced production of cellulase from a novel strain Trichoderma gamsii M501 through response surface methodology and its application in biomass saccharification

Cellulolytic enzymes produced by Trichoderma sp. have attracted interest in converting the biomass to simple sugars in the production of cellulosic ethanol. In this work, a novel cellulolytic strain M501 was isolated and identified as T. gamsii by sequencing the ITS rDNA region. The production of cellulase (CMCase) by T. gamsii M501 was enhanced by employing statistical methods. The strain grown in the optimized production medium composed of mineral salts, microcrystalline cellulose (13.7 g/l), tryptone (4.8 g/l) and trace elements (2 mL/l) at pH 5.5 and 28 °C for 72 h produced a maximum CMCase of 61.3 U/mL. The optimized production medium also showed the other enzyme activity of FPU (2.6 U/mL), β-glucosidase (2.1 U/mL), xylanase (681 U/mL) and β- xylosidase (0.6 U/mL). The crude cellulase cocktail produced by T. gamsii M501 efficiently hydrolyzed alkali pretreated sugarcane bagasse with glucose and xylose yield of 78 % and 74 % respectively at 10 % solid loading. This study is the first of its kind research on biomass saccharification using T. gamsii cellulase cocktail. Therefore, the novel strain T. gamsii M501 would be useful for further development of an enzyme cocktail for cellulosic ethanol production.     

Saccharification of used paper with different cellulases

Various used paper materials have been exposed to the action of cellulases from Penicillium funiculosum, Trichoderma reesei, Trichoderma viride and Aspergillus niger. A 2 h incubation period showed cellulase from T. viride the most active except for office paper that was maximally degraded by A. niger cellulase. Cellulase mixtures increased saccharification while sequential treatment with cellulases from T. reesei and P. funiculosum increased biodegradation at values between 15% and 190%. The maximum increase of saccharification (190%) was obtained when T. reesei cellulase initiated the sequential treatment of newspaper relative to the sole action of P. funiculosum cellulase on this non-pretreated and pretreated material.     

Microscopy studies reveal delignification and sterol removal from eucalypt kraft pulps by laccase–HBT

Fungal laccases in the presence of mediators are powerful biocatalysts to degrade lignin. Pycnoporus cinnabarinus laccase and 1-hydroxybenzotriazole (HBT) have been successfully used to delignify eucalypt kraft pulp once integrated in a totally chlorine-free bleaching sequence. Real time delignification of kraft pulp by laccase–HBT was verified in situ by monitoring the loss of lignin autofluorescence during the enzymatic treatment using confocal laser scanning microscopy. The highest delignification of pulp fibers occurred over a very short time-span (5 min). Moreover, we demonstrate the removal of sterols, responsible for pitch deposits in hardwood kraft pulps, as an additional effect of laccase-HBT. Spherical structures between pulp fibers localized by low temperature scanning electron microscopy were removed by laccase–HBT. The use of filipin, a specific stain, revealed the sterol nature of many of these structures. At the end of the enzyme-aided bleaching sequence, the fluorescent sterols–filipin signals were almost completely absent.