INVOLVEMENT OF INDOLE‐3‐ACETIC ACID PRODUCED BY THE GROWTH‐PROMOTING BACTERIUM AZOSPIRILLUM SPP. IN PROMOTING GROWTH OF CHLORELLA VULGARIS1

Involvement of indole‐3‐acetic acid (IAA), produced by the microalgae‐growth‐promoting bacteria Azospirillum brasilens and A. lipoferum, in promoting growth of the microalga Chlorella vulgaris Beij. was studied. Four wildtype strains of Azospirillum and their IAA‐deficient mutants were co‐immobilized with C. vulgaris in alginate beads. Cultures were grown in synthetic growth medium supplemented with tryptophan. Growth promotion of microalgae and production of exogenous IAA by Azospirillum spp. were monitored. All wildtype Azospirillum spp. produced significant but varying amounts of IAA, while their mutant forms produced significantly less. The results demonstrated a significant growth promotion in Chlorella cultures when immobilized with the four wildtype strains of Azospirillum, while very low or no enhanced growth was induced by the four IAA‐deficient mutants, compared to when C. vulgaris is immobilized alone. A complementation experiment, where an IAA‐attenuated mutant (A. brasilense SpM7918) was supplemented with IAA produced by its parental wildtype strain (A. brasilense Sp6), restored growth promotion in the microalgae‐mutant culture.     

Improving carbohydrate production of Chlorella sorokiniana NIES‐2168 through semi‐continuous process coupled with mixotrophic cultivation

Biofuels from microalgae is now a hot issue of great potential. However, achieving high starch productivity with photoautotrophic microalgae is still challenging. A feasible approach to enhance the growth and target product of microalgae is to conduct mixotrophic cultivation. The appropriate acetate addition combined with CO₂ supply as dual carbon sources (i.e., mixotrophic cultivation) could enhance the cell growth of some microalgae species, but the effect of acetate‐mediated mixotrophic culture mode on carbohydrate accumulation in microalgae remains unclear. Moreover, there is still lack of the information concerning how to increase the productivity of carbohydrates from microalgae under acetate‐amended mixotrophic cultivation and how to optimize the engineering strategies to achieve the goal. This study was undertaken to develop an optimal acetate‐contained mixotrophic cultivation system coupled with effective operation strategies to markedly improve the carbohydrate productivity of Chlorella sorokiniana NIES‐2168. The optimal carbohydrate productivity of 695 mg/L/d was obtained, which is the highest value ever reported. The monosaccharide in the accumulated carbohydrates is mainly glucose (i.e., 85–90%), which is very suitable for bio‐alcohols fermentation. Hence, by applying the optimal process developed in this study, C. sorokiniana NIES‐2168 has a high potential to serve as a feedstock for subsequent biofuels conversion.     

Factors Affecting the Mixotrophic Flagellate Poterioochromonas malhamensis Grazing on Chlorella Cells

Grazing behaviour between protozoa and phytoplankton exists widely in planktonic ecosystems. Poterioochromonas malhamensis is a well‐known and widespread mixotrophic flagellate, which is recognized to play an important role within marine and freshwater planktonic ecosystems and regarded as the greatest contamination threat for mass algal cultures of Chlorella. In this study, a comprehensive range of factors, including morphological characters, biochemical compositions, and specific growth rate of ten species or strains of Chlorella, were evaluated for their effect on the feeding ability of P. malhamensis, which was assessed by two parameters: the clearance rate of P. malhamensis on Chlorella spp. and the specific growth rate of P. malhamensis. The results showed that the clearance rate of P. malhamensis was negatively correlated with cell wall thickness and specific growth rate of Chlorella spp., while the specific growth rate of P. malhamensis was positively correlated with carbohydrate percentage and C/N ratio and negatively correlated with protein, lipid percentage, and nitrogen mass. In conclusion, the factors influencing feeding selectivity include not only the morphological character and chemical composition of Chlorella, but also its population dynamics. Our study provides useful insights into the key factors that affect the feeding selectivity of P. malhamensis and provides basic and constructive data to help in screening for grazing‐resistant microalgae.     

Glucose and Nitrogen Amendments Can Mitigate Wastewater‐Borne Bacteria Competition Effect Against Algal Growth in Wastewater‐Based Systems

The reuse of wastewater is important for reducing costs involved with algal lipid production. However, nutrient limitations, wastewater‐borne microbes, and mixotrophic growth can significantly affect biomass yields and lipid/biomass ratios. This research compared the growth performances of both Chlorella vulgaris and Pseudokirchneriella subcapitata on domestic wastewater effluent. The experiments were conducted in the presence and absence of wastewater‐borne bacteria, while additionally assessing the impact of distinct nitrate and glucose supplementations. When compared to the sterilized controls, the presence of wastewater‐borne bacteria in the effluent reduced C. vulgaris and P. subcapitata total biomass production by 37% and 46%, respectively. In the corresponding treatments supplemented with glucose and nitrate, total biomass production increased by 12% and 61%, respectively. The highest biomass production of 1.11 and 0.72 g · L^?1 was, however, observed in the sterilized treatments with both glucose and nitrate supplementations for C. vulgaris and P. subcapitata, respectively. Lipid to biomass ratios were, on average, threefold higher when only nitrate was introduced in the sterilized treatments for both species (0.4 and 0.5, respectively). Therefore, the combination of nitrate and glucose supplementation is shown to be an important strategy for enhancing algal lipid and biomass production when those algae are grown in the presence of wastewater‐borne bacteria. On the other hand, in the absence of wastewater‐borne bacteria, only nitrate supplementation can significantly improve lipid/biomass ratios.     

Raw starch–degrading α‐amylase from Bacillus aquimaris MKSC 6.2: isolation and expression of the gene,bioinformatics and biochemical characterization of the recombinant enzyme

Aims

The aims were to isolate a raw starch–degrading α‐amylase gene baqA from Bacillus aquimaris MKSC 6.2, and to characterize the gene product through in silico study and its expression in Escherichia coli.

Methods and Results

A 1539 complete open reading frame of a starch–degrading α‐amylase gene baqA from B. aquimaris MKSC 6·2 has been determined by employing PCR and inverse PCR techniques. Bioinformatics analysis revealed that B. aquimaris MKSC 6.2 α‐amylase (BaqA) has no starch‐binding domain, and together with a few putative α‐amylases from bacilli may establish a novel GH13 subfamily most closely related to GH13_1. Two consecutive tryptophans (Trp201 and Trp202, BaqA numbering) were identified as a sequence fingerprint of this novel GH13 subfamily. Escherichia coli cells produced the recombinant BaqA protein as inclusion bodies. The refolded recombinant BaqA protein degraded raw cassava and corn starches, but exhibited no activity with soluble starch.

Conclusions

A novel raw starch–degrading B. aquimaris MKSC 6.2 α‐amylase BaqA is proposed to be a member of new GH13 subfamily.

Significance and Impact of the Study

This study has contributed to the overall knowledge and understanding of amylolytic enzymes that are able to bind and digest raw starch directly.     

Biomass and lipid productivities of <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> under autotrophic,heterotrophic and mixotrophic growth conditions

Biomass and lipid productivities of Chlorella vulgaris under different growth conditions were investigated. While autotrophic growth did provide higher cellular lipid content (38%), the lipid productivity was much lower compared with those from heterotrophic growth with acetate, glucose, or glycerol. Optimal cell growth (2 g l⁻¹) and lipid productivity (54 mg l⁻¹ day⁻¹) were attained using glucose at 1% (w/v) whereas higher concentrations were inhibitory. Growth of C. vulgaris on glycerol had a similar dose effects as those from glucose. Overall, C. vulgaris is mixotrophic.     

Inhibitory effects of sodium silicate on the fungal growth and secretion of cell wall‐degrading enzymes by Trichothecium roseum

Trichothecium roseum causes decay in muskmelons, apples, tomatoes and mangoes, which leads to economic losses. In this study, we investigated the effect of sodium silicate on the growth of T. roseum and the cell wall‐degrading enzymes (CWDEs) secreted by the hyphae. The results indicated that sodium silicate significantly inhibited mycelial growth and spore germination of T. roseum. The sodium silicate treatment also retarded the secretion of several CWDEs, including pectate lyase (PL), polygalacturonic acid transeliminase (PGTE), pectin methyltranseliminase (PMTE), pectin methylgalacturonase (PMG), polygalacturonase (PG), cellulase (Cx) and β‐glucosidase. These results suggest that sodium silicate exerts its effects on T. roseum through direct inhibition of its growth and secretion of CWDEs.     

One‐step bioconversion of hemicellulose polymers to rhamnolipids with Cellvibrio japonicus: A proof‐of‐concept for a potential host strain in future bioeconomy

The purpose of this study was to evaluate Cellvibrio japonicus as a potential host strain for one‐step bioconversion of hemicellulose polymers to value‐added products. C. japonicus could be cultivated on all main lignocellulose monosaccharides as well as xylan polymers as a sole carbon source. This is particularly interesting as most industrially relevant bacteria are neither able to depolymerize wood polymers nor metabolize most hemicellulose monosaccharides. As a result, lignocellulose raw materials typically have to be degraded employing additional processes while the complete conversion of all lignocellulose sugars remains a challenge. Exemplary for a value‐added product, a one‐step conversion of xylan polymers to mono‐rhamnolipid biosurfactants with C. japonicus after transformation with the plasmid pSynPro8oT carrying the genes rhlAB was demonstrated. As achieved product yields in this one‐step bioconversion process are comparably low, many challenges remain to be overcome for application on an industrial scale. Nonetheless, this study provides a first step in the search for establishing a future host strain for bioeconomy, which will ideally be used for bioconversion of lignocellulose polymers with as little exhaustive pretreatment as possible.     

Oxalyltransferase,a plant cell‐wall acyltransferase activity,transfers oxalate groups from ascorbate metabolites to carbohydrates

In the plant apoplast, ascorbate is oxidised, via dehydroascorbic acid, to O‐oxalyl esters [oxalyl‐l ‐threonate (OxT) and cyclic oxalyl‐l ‐threonate (cOxT)]. We tested whether OxT and cOxT can donate the oxalyl group in transacylation reactions to form oxalyl‐polysaccharides, potentially modifying the cell wall. [oxalyl‐¹⁴C]OxT was incubated with living spinach (Spinacia oleracea) and Arabidopsis cell‐suspension cultures in the presence or absence of proposed acceptor substrates (carbohydrates). In addition, [¹⁴C]OxT and [¹⁴C]cOxT were incubated in vitro with cell‐wall enzyme preparations plus proposed acceptor substrates. Radioactive products were monitored electrophoretically. Oxalyltransferase activity was detected. Living cells incorporated oxalate groups from OxT into cell‐wall polymers via ester bonds. When sugars were added, [¹⁴C]oxalyl‐sugars were formed, in competition with OxT hydrolysis. Preferred acceptor substrates were carbohydrates possessing primary alcohols e.g. glucose. A model transacylation product, [¹⁴C]oxalyl‐glucose, was relatively stable in vivo (half‐life >24 h), whereas [¹⁴C]OxT underwent rapid turnover (half‐life ~6 h). Ionically wall‐bound enzymes catalysed similar transacylation reactions in vitro with OxT or cOxT as oxalyl donor substrates and any of a range of sugars or hemicelluloses as acceptor substrates. Glucosamine was O‐oxalylated, not N‐oxalylated. We conclude that plants possess apoplastic acyltransferase (oxalyltransferase) activity that transfers oxalyl groups from ascorbate catabolites to carbohydrates, forming relatively long‐lived O‐oxalyl‐carbohydrates. The findings increase the range of known metabolites whose accumulation in vivo indicates vitamin C catabolism. Possible signalling roles of the resulting oxalyl‐sugars can now be investigated, as can the potential ability of polysaccharide oxalylation to modify the wall's physical properties.     

Potential of Chlorella vulgaris culture for waste treatment from anaerobic biomass biodigestion at the Piaszczyna (Poland) integrated facility

Many strains of microalgae are potentially useful for industrial purposes. Microalgal biomass and microalgae‐derived substances are becoming valuable products with a widening range of applications including biofuels and human food. In this study, the possibility of using the methane waste from biomass biodigestion in the cultivation of Chlorella vulgaris biomass with simultaneous waste treatment was investigated. The methane waste from biomass biodigestion was obtained from a multifunctional facility (Piaszczyna, Poland) producing bioethanol from plant biomass with several steps to reuse the wastes, heat, and carbon dioxide. The growth and biomass yield, as well as photosynthetic performance of C. vulgaris on diluted waste, were similar to the results obtained on the standard mineral medium. The cultivation of C. vulgaris was the waste, treatment step that significantly reduced chemical oxygen demand. The results indicated that the waste contained micro‐ and macronutrients sufficient to sustain the growth of C. vulgaris cell culture up to 2 g of dry biomass per liter of culture. The results contributed to the development of the waste treatment step in the Piaszczyna facility that allowed for a further decrease in emissions and may lead to development of microalgae biomass‐based products in the facility portfolio.     

Isolation and characterization of lignin‐degrading bacteria from rainforest soils

The deconstruction of lignin to enhance the release of fermentable sugars from plant cell walls presents a challenge for biofuels production from lignocellulosic biomass. The discovery of novel lignin‐degrading enzymes from bacteria could provide advantages over fungal enzymes in terms of their production and relative ease of protein engineering. In this study, 140 bacterial strains isolated from soils of a biodiversity‐rich rainforest in Peru were screened based on their oxidative activity on ABTS, a laccase substrate. Strain C6 (Bacillus pumilus) and strain B7 (Bacillus atrophaeus) were selected for their high laccase activity and identified by 16S rDNA analysis. Strains B7 and C6 degraded fragments of Kraft lignin and the lignin model dimer guaiacylglycerol‐β‐guaiacyl ether, the most abundant linkage in lignin. Finally, LC–MS analysis of incubations of strains B7 and C6 with poplar biomass in rich and minimal media revealed that a higher number of compounds were released in the minimal medium than in the rich one. These findings provide important evidence that bacterial enzymes can degrade and/or modify lignin and contribute to the release of fermentable sugars from lignocellulose. Biotechnol. Bioeng. 2013; 110: 1616–1626. © 2013 Wiley Periodicals, Inc.     

EFFICIENCY OF GROWTH AND NUTRIENT UPTAKE FROM WASTEWATER BY HETEROTROPHIC,AUTOTROPHIC, AND MIXOTROPHIC CULTIVATION OF CHLORELLA VULGARIS IMMOBILIZED WITH AZOSPIRILLUM BRASILENSE1

Heterotrophic growth of microalgae presents significant economic advantages over the more common autotrophic cultivation. The efficiency of growth and nitrogen, phosphorus, and glucose uptake from synthetic wastewater was compared under heterotrophic, autotrophic, and mixotrophic regimes of Chlorella vulgaris Beij. immobilized in alginate beads, either alone or with the bacterium Azospirillum brasilense. Heterotrophic cultivation of C. vulgaris growing alone was superior to autotrophic cultivation. The added bacteria enhanced growth only under autotrophic and mixotrophic cultivations. Uptake of ammonium by the culture, yield of cells per ammonium unit, and total volumetric productivity of the culture were the highest under heterotrophic conditions when the microalga grew without the bacterium. Uptake of phosphate was higher under autotrophic conditions and similar under the other two regimes. Positive influence of the addition of A. brasilense was found only when light was supplied (autotrophic and mixotrophic), where affinity to phosphate and yield per phosphate unit were the highest under heterotrophic conditions. The pH of the culture was significantly reduced in all regimes where glucose was consumed, similarly in heterotrophic and mixotrophic cultures. It was concluded that the heterotrophic regime, using glucose, is superior to autotrophic and mixotrophic regimes for the uptake of ammonium and phosphate. Addition of A. brasilense positively affects the nutrient uptake only in the two regimes supplied with light.     

No trade‐offs in interspecific interference ability and predation susceptibility in newt larvae

Coexistence of species with similar requirements is allowed, among others, through trade‐offs between competitive ability and other ecological traits. Although interspecific competition is based on two mechanisms, exploitation of resources and physical interference, trade‐off studies largely consider only species’ ability to exploit resources. Using a mesocosm experiment, we examined the trade‐off between interference competition ability and susceptibility to predation in larvae of two newt species, Ichthyosaura alpestris and Lissotriton vulgaris. In the presence of heterospecifics, L. vulgaris larvae slowed somatic growth and developmental rates, and experienced a higher frequency of injuries than in conspecific environments which suggests asymmetrical interspecific interference. During short‐term predation trials, L. vulgaris larvae suffered higher mortality than I. alpestris. Larvae of the smaller species, L. vulgaris, had both lower interference and antipredator performance than the larger I. alpestris, which suggests a lack of trade‐off between interference competition ability and predator susceptibility. We conclude that interference competition may produce a positive rather than negative relationship with predation susceptibility, which may contribute to the elimination of subordinate species from common habitats.     

Statistical evaluation and modeling of cheap substrate-based cultivation medium of Chlorella vulgaris to enhance microalgae lipid as new potential feedstock for biolubricant

Chlorella vulgaris (C. vulgaris) microalga was investigated as a new potential feedstock for the production of biodegradable lubricant. In order to enhance microalgae lipid for biolubricant production, mixotrophic growth of C. vulgaris was optimized using statistical analysis of Plackett–Burman (P-B) and response surface methodology (RSM). A cheap substrate-based medium of molasses and corn steep liquor (CSL) was used instead of expensive mineral salts to reduce the total cost of microalgae production. The effects of molasses and CSL concentration (cheap substrates) and light intensity on the growth of microalgae and their lipid content were analyzed and modeled. Designed models by RSM showed good compatibility with a 95% confidence level when compared to the cultivation system. According to the models, optimal cultivation conditions were obtained with biomass productivity of 0.123 g L^?1 day^?1 and lipid dry weight of 0.64 g L^?1 as 35% of dry weight of C. vulgaris. The extracted microalgae lipid presented useful fatty acid for biolubricant production with viscosities of 42.00 cSt at 40°C and 8.500 cSt at 100°C, viscosity index of 185, flash point of 185°C, and pour point of ?6°C. These properties showed that microalgae lipid could be used as potential feedstock for biolubricant production.     

Investigation of mixotrophic,heterotrophic, and autotrophic growth of Chlorella vulgaris under agricultural waste medium

Growth of Chlorella vulgaris and its lipid production were investigated under autotrophic, heterotrophic, and mixotrophic conditions. Cheap agricultural waste molasses and corn steep liquor from industries were used as carbon and nitrogen sources, respectively. Chlorella vulgaris grew remarkably under this agricultural waste medium, which resulted in a reduction in the final cost of the biodiesel production. Maximum dry weight of 2.62 g L^?1 was obtained in mixotrophic growth with the highest lipid concentration of 0.86 g L^?1. These biomass and lipid concentrations were, respectively, 140% and 170% higher than autotrophic growth and 300% and 1200% higher than heterotrophic growth. In mixotrophic growth, independent or simultaneous occurrence of autotrophic and heterotrophic metabolisms was investigated. The growth of the microalgae was observed to take place first heterotrophically to a minimum substrate concentration with a little fraction in growth under autotrophic metabolism, and then the cells grew more autotrophically. It was found that mixotrophic growth was not a simple combination of heterotrophic and autotrophic growth.     

A novel kinase function of a nucleoside‐diphosphate‐kinase homologue in Porphyromonas gingivalis is critical in subversion of host cell apoptosis by targeting heat‐shock protein 27

We have previously shown that a homologue of a conserved nucleoside‐diphosphate‐kinase (Ndk) family of multifunctional enzymes and secreted molecule in Porphyromonas gingivalis can modulate select host molecular pathways including downregulation of reactive‐oxygen‐species generation to promote bacterial survival in human gingival epithelial cells (GECs). In this study, we describe a novel kinase function for bacterial effector, P. gingivalis‐Ndk, in abrogating epithelial cell death by phosphorylating heat‐shock protein 27 (HSP27) in GECs. Infection by P. gingivalis was recently suggested to increase phosphorylation of HSP27 in cancer‐epithelial cells; however, the mechanism and biological significance of antiapoptotic phospho‐HSP27 during infection has never been characterised. Interestingly, using glutathione S‐transferase‐rNdk pull‐down analysed by mass spectrometry, we identified HSP27 in GECs as a strong binder of P. gingivalis‐Ndk and further verified using confocal microscopy and ELISA. Therefore, we hypothesised P. gingivalis‐Ndk can phosphorylate HSP27 for inhibition of apoptosis in GECs. We further employed P. gingivalis‐Ndk protein constructs and an isogenic P. gingivalis‐ndk‐deficient‐mutant strain for functional examination. P. gingivalis‐infected GECs displayed significantly increased phospho‐HSP27 compared with ndk‐deficient‐strain during 24 hr infection. Phospho‐HSP27 was significantly increased by transfection of GFP‐tagged‐Ndk into uninfected‐GECs, and in vitro phosphorylation assays revealed direct phosphorylation of HSP27 at serines 78 and 82 by P. gingivalis‐Ndk. Depletion of HSP27 via siRNA significantly reversed resistance against staurosporine‐mediated‐apoptosis during infection. Transfection of recombinant P. gingivalis‐Ndk protein into GECs substantially decreased staurosporine‐induced‐apoptosis. Finally, ndk‐deficient‐mutant strain was unable to inhibit staurosporine‐induced Cytochrome C release/Caspase‐9 activation. Thus, we show for the first time the phosphorylation of HSP27 by a bacterial effector—P. gingivalis‐Ndk—and a novel function of Ndks that is directly involved in inhibition of host cell apoptosis and the subsequent bacterial survival.     

A thermostable feruloyl-esterase from the hemicellulolytic bacterium <Emphasis Type="Italic">Thermobacillus xylanilyticus</Emphasis> releases phenolic acids from non-pretreated plant cell walls

A gene (Tx-est1) encoding a thermostable feruloyl-esterase was isolated from the genome of the Gram-positive hemicellulolytic thermophilic bacterium Thermobacillus xylanilyticus. This gene contains an open reading frame of 1,020 bp encoding a protein with molecular mass of 37.4 kDa, similar to feruloyl-esterases from cellulolytic bacteria and fungi. The recombinant enzyme Tx-Est1 was expressed and produced in Escherichia coli. Tx-Est1 contains the conserved putative lipase residues Ser 202, Asp 287, and His 322 which act as catalytic triad in its C-terminus part. Purified Tx-Est1 was active against phenolic acid derivatives and stable at high temperatures. Optimal activity was observed at 65 °C and the optimal pH was around 8.5. The kinetic parameters of the esterase were determined on various substrates. The enzyme displayed activity against methyl esters of hydrocinnamic acids and feruloylated arabino-xylotetraose, exhibiting high specificity and affinity for the latter. Our results showed that Tx-Est1 is a thermostable feruloyl-esterase which could be useful to hydrolyze arabinoxylans from graminaceous plant cell walls as the enzyme is able to release phenolic acids from a lignocellulose biomass.     

<Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> produces a diverse array of extracellular enzymes when grown on sorghum

In an effort to understand how fungi degrade biomass, we grew Phanerochaete chrysosporium on sorghum stover and chronicled the growth of the fungus over the course of 14 days. The fungal mass grew steadily until the fifth day, reaching 0.06 mg of cells per milligram of dry mass, which fell by the seventh day and stayed at nearly the same level until day 14. After 1 day, hemicellulases, cellulases, and polygalacturonases were detected in the extracellular fluid at 1.06, 0.34, and 0.20 U/ml, respectively. Proteomic studies performed with the extracellular fluid using liquid chromatography–tandem mass spectrometry identified 57, 116, and 102 degradative enzymes targeting cellulose, hemicellulose, pectin, lignin, proteins, and lipids on days 1, 7, and 14, respectively. Significant concentrations of breakdown products of the sorghum polysaccharides were detected in the extracellular fluid indicating that the enzymes were breaking the polysaccharides, and after 14 days, almost 39% of the sorghum sugars had been used by the fungus. Our results suggest that P. chrysosporium produces a set of enzymes to degrade the components of lignocellulose from the beginning of its growth, but modifies the complement of enzymes it secretes over time to adapt to the particular substrate available.     

Saccharification of Lignocelluloses by Carbohydrate Active Enzymes of the White Rot Fungus Dichomitus squalens

White rot fungus Dichomitus squalens is an efficient lignocellulose degrading basidiomycete and a promising source for new plant cell wall polysaccharides depolymerizing enzymes. In this work, we focused on cellobiohydrolases (CBHs) of D. squalens. The native CBHI fraction of the fungus, consisting three isoenzymes, was purified and it maintained the activity for 60 min at 50°C, and was stable in acidic pH. Due to the lack of enzyme activity assay for detecting only CBHII activity, CBHII of D. squalens was produced recombinantly in an industrially important ascomycete host, Trichoderma reesei. CBH enzymes of D. squalens showed potential in hydrolysis of complex lignocellulose substrates sugar beet pulp and wheat bran, and microcrystalline cellulose, Avicel. Recombinant CBHII (rCel6A) of D. squalens hydrolysed all the studied plant biomasses. Compared to individual activities, synergistic effect between rCel6A and native CBHI fraction of D. squalens was significant in the hydrolysis of Avicel. Furthermore, the addition of laccase to the mixture of CBHI fraction and rCel6A significantly enhanced the amount of released reducing sugars from sugar beet pulp. Especially, synergy between individual enzymes is a crucial factor in the tailor-made enzyme mixtures needed for hydrolysis of different plant biomass feedstocks. Our data supports the importance of oxidoreductases in improved enzyme cocktails for lignocellulose saccharification.