Advancing Desulfurization in the Model Biocatalyst Rhodococcus qingshengii IGTS8 via an In Locus Combinatorial Approach.

Biodesulfurization poses as an ideal replacement to the high cost hydrodesulfurization of the recalcitrant heterocyclic sulfur compounds, such as dibenzothiophene (DBT) and its derivatives. The increasingly stringent limits on fuel sulfur content intensify the need for improved desulfurization biocatalysts, without sacrificing the calorific value of the fuel. Selective sulfur removal in a wide range of biodesulfurization strains, as well as in the model biocatalyst Rhodococcus qingshengii IGTS8, occurs via the 4S metabolic pathway that involves the dszABC operon, which encodes enzymes that catalyze the generation of 2-hydroxybiphenyl and sulfite from DBT. Here, using a homologous recombination process, we generate two recombinant IGTS8 biocatalysts, harboring native or rearranged, nonrepressible desulfurization operons, within the native dsz locus. The alleviation of sulfate-, methionine-, and cysteine-mediated dsz repression is achieved through the exchange of the native promoter Pdsz, with the nonrepressible Pkap1 promoter. The Dsz-mediated desulfurization from DBT was monitored at three growth phases, through HPLC analysis of end product levels. Notably, an 86-fold enhancement of desulfurization activity was documented in the presence of selected repressive sulfur sources for the recombinant biocatalyst harboring a combination of three targeted genetic modifications, namely, a dsz operon rearrangement, a native promoter exchange, and a dszA-dszB overlap removal. In addition, transcript level comparison highlighted the diverse effects of our genetic engineering approaches on dsz mRNA ratios and revealed a gene-specific differential increase in mRNA levels. IMPORTANCE Rhodococcus is perhaps the most promising biodesulfurization genus and is able to withstand the harsh process conditions of a biphasic biodesulfurization process. In the present work, we constructed an advanced biocatalyst harboring a combination of three genetic modifications, namely, an operon rearrangement, a promoter exchange, and a gene overlap removal. Our homologous recombination approach generated stable biocatalysts that do not require antibiotic addition, while harboring nonrepressible desulfurization operons that present very high biodesulfurization activities and are produced in simple and low-cost media. In addition, transcript level quantification validated the effects of our genetic engineering approaches on recombinant strains' dsz mRNA ratios and revealed a gene-specific differential increase in mRNA levels. Based on these findings, the present work can pave the way for further strain and process optimization studies that could eventually lead to an economically viable biodesulfurization process.

Incorporation of Magnetic Nanoparticles into Protoplasts of Microalgae Haematococcus pluvialis: A Tool for Biotechnological Applications.

Intensive research on the use of magnetic nanoparticles for biotechnological applications of microalgae biomass guided the development of proper treatment to successfully incorporate them into these single-cell microorganisms. Protoplasts, as cells lacking a cell wall, are extensively used in plant/microalgae genetic manipulation as well as various biotechnological applications. In this work, a detailed study on the formation of protoplasts from Haematococcus pluvialis with the use of enzymatic and mechanical procedures was performed. The optimization of several parameters affecting the formation of protoplasmic cells and cell recovery was investigated. In the enzymatic treatment, a solution of cellulase was studied at different time points of incubation, whereas in the mechanical treatment, glass beads vortexing was used. Mechanical treatment gave better results in comparison to the enzymatic one. Concerning the cell recovery, after the protoplast formation, it was found to be similar in both methods used; cell viability was not investigated. To enhance the protoplast cell wall reconstruction, different "recovery media" with an organic source of carbon or nitrogen were used. Cell morphology during all treatments was evaluated by electron microscopy. The optimal conditions found for protoplast formation and cell reconstruction were successfully used to produce Haematococcus pluvialis cells with magnetic properties.

3-Chloro-1,2-propanediol biodegradation by Ca-alginate immobilized Pseudomonas putida DSM 437 cells applying different processes: mass transfer effects.

3-Chloro-1,2-propanediol (3-CPD) biodegradation by Ca-alginate immobilized Pseudomonas putida cells was performed in batch system, continuous stirred tank reactor (CSTR), and packed-bed reactor (PBR). Batch system exhibited higher biodegradation rates and 3-CPD uptakes compared to CSTR and PBR. The two continuous systems (CSTR and PBR) when compared at 200 mg/L 3-CPD in the inlet exhibited the same removal of 3-CPD at steady state. External mass-transfer limitations are found negligible at all systems examined, since the observable modulus for external mass transfer Ω â‰ª 1 and the Biot number Bi > 1. Intra-particle diffusion resistance had a significant effect on 3-CPD biodegradation in all systems studied, but to a different extent. Thiele modulus was in the range of 2.5 in batch system, but it was increased at 11 when increasing cell loading in the beads, thus lowering significantly the respective effectiveness factor. Comparing the systems at the same cell loading in the beads PBR was less affected by internal diffusional limitations compared to CSTR and batch system, and, as a result, exhibited the highest overall effectiveness factor.

Biotechnological production of ethanol from renewable resources by Neurospora crassa: an alternative to conventional yeast fermentations?

Microbial production of ethanol might be a potential route to replace oil and chemical feedstocks. Bioethanol is by far the most common biofuel in use worldwide. Lignocellulosic biomass is the most promising renewable resource for fuel bioethanol production. Bioconversion of lignocellulosics to ethanol consists of four major unit operations: pretreatment, hydrolysis, fermentation, and product separation/distillation. Conventional bioethanol processes for lignocellulosics apply commercial fungal cellulase enzymes for biomass hydrolysis, followed by yeast fermentation of resulting glucose to ethanol. The fungus Neurospora crassa has been used extensively for genetic, biochemical, and molecular studies as a model organism. However, the strain's potential in biotechnological applications has not been widely investigated and discussed. The fungus N. crassa has the ability to synthesize and secrete all three enzyme types involved in cellulose hydrolysis as well as various enzymes for hemicellulose degradation. In addition, N. crassa has been reported to convert to ethanol hexose and pentose sugars, cellulose polymers, and agro-industrial residues. The combination of these characteristics makes N. crassa a promising alternative candidate for biotechnological production of ethanol from renewable resources. This review consists of an overview of the ethanol process from lignocellulosic biomass, followed by cellulases and hemicellulases production, ethanol fermentations of sugars and lignocellulosics, and industrial application potential of N. crassa.

Bioconversion of dilute-acid pretreated sorghum bagasse to ethanol by Neurospora crassa.

Bioethanol production from sweet sorghum bagasse (SB), the lignocellulosic solid residue obtained after extraction of sugars from sorghum stalks, can further improve the energy yield of the crop. The aim of the present work was to evaluate a cost-efficient bioconversion of SB to ethanol at high solids loadings (16 % at pretreatment and 8 % at fermentation), low cellulase activities (1-7 FPU/g SB) and co-fermentation of hexoses and pentoses. The fungus Neurospora crassa DSM 1129 was used, which exhibits both depolymerase and co-fermentative ability, as well as mixed cultures with Saccharomyces cerevisiae 2541. A dilute-acid pretreatment (sulfuric acid 2 g/100 g SB; 210 °C; 10 min) was implemented, with high hemicellulose decomposition and low inhibitor formation. The bioconversion efficiency of N. crassa was superior to S. cerevisiae, while their mixed cultures had negative effect on ethanol production. Supplementing the in situ produced N. crassa cellulolytic system (1.0 FPU/g SB) with commercial cellulase and ß-glucosidase mixture at low activity (6.0 FPU/g SB) increased ethanol production to 27.6 g/l or 84.7 % of theoretical yield (based on SB cellulose and hemicellulose sugar content). The combined dilute-acid pretreatment and bioconversion led to maximum cellulose and hemicellulose hydrolysis 73.3 % and 89.6 %, respectively.

Interplay between Sulfur Assimilation and Biodesulfurization Activity in Rhodococcus qingshengii IGTS8: Insights into a Regulatory Role of the Reverse Transsulfuration Pathway.

Biodesulfurization is a process that selectively removes sulfur from dibenzothiophene and its derivatives. Several natural biocatalysts harboring the highly conserved desulfurization operon dszABC, which is significantly repressed by methionine, cysteine, and inorganic sulfate, have been isolated. However, the available information on the metabolic regulation of gene expression is still limited. In this study, scarless knockouts of the reverse transsulfuration pathway enzyme genes cbs and metB were constructed in the desulfurizing strain Rhodococcus sp. strain IGTS8. We provide sequence analyses and report the enzymes' involvement in the sulfate- and methionine-dependent repression of biodesulfurization activity. Sulfate addition in the bacterial culture did not repress the desulfurization activity of the Δcbs strain, whereas deletion of metB promoted a significant biodesulfurization activity for sulfate-based growth and an even higher desulfurization activity for methionine-grown cells. In contrast, growth on cysteine completely repressed the desulfurization activity of all strains. Transcript level comparison uncovered a positive effect of cbs and metB gene deletions on dsz gene expression in the presence of sulfate and methionine, but not cysteine, offering insights into a critical role of cystathionine ß-synthase (CßS) and MetB in desulfurization activity regulation. IMPORTANCE Precise genome editing of the model biocatalyst Rhodococcus qingshengii IGTS8 was performed for the first time, more than 3 decades after its initial discovery. We thus gained insight into the regulation of dsz gene expression and biocatalyst activity, depending on the presence of two reverse transsulfuration enzymes, CßS and MetB. Moreover, we observed an enhancement of biodesulfurization capability in the presence of otherwise repressive sulfur sources, such as sulfate and l-methionine. The interconnection of cellular sulfur assimilation strategies was revealed and validated.

Deciphering the biodesulfurization potential of two novel Rhodococcus isolates from a unique Greek environment.

Sustainable biodesulfurization (BDS) processes require the use of microbial biocatalysts that display high activity against the recalcitrant heterocyclic sulfur compounds and can simultaneously withstand the harsh conditions of contact with petroleum products, inherent to any industrial biphasic BDS system. In this framework, the functional microbial BDS-related diversity in a naturally oil-exposed ecosystem, was examined through a 4,6-dimethyl-dibenzothiophene based enrichment process. Two new Rhodococcus sp. strains were isolated, which during a medium optimization process revealed a significantly enhanced BDS activity profile when compared to the model strain R. qingshengii IGTS8. In biocatalyst stability studies conducted in biphasic mode using partially hydrodesulfurized diesel under various process conditions, the new strains also presented an enhanced stability phenotype. In these studies, it was also demonstrated for all strains, that the BDS activity losses were decoupled from the overall cells' viability, in addition to the fact that the use of whole-broth biocatalyst positively affected BDS performance.

Magnetic Immobilization and Growth of Nannochloropsis oceanica and Scenedasmus almeriensis.

Microalgae are used in industrial and pharmaceutical applications. Their performance on biological applications may be improved by their immobilization. This study presents a way of cell immobilization using microalgae carrying magnetic properties. Nannochloropsis oceanica and Scenedasmus almeriensis cells were treated enzymatically (cellulase) and mechanically (glass beads), generating protoplasts as a means of incorporation of magnetic nanoparticles. Scanning electron microscopy images verified the successful cell wall destruction for both of the examined microalgae cells. Subsequently, protoplasts were transformed with magnetic nanoparticles by a continuous electroporation method and then cultured on a magnetic surface. Regeneration of transformed protoplasts was optimized using various organic carbon and amino acid supplements. Both protoplast preparation methods demonstrated similar efficiency. Casamino acids, as source of amino acids, were the most efficient compound for N. oceanica protoplasts regeneration in enzymatic and mechanical treatment, while for S. almeriensis protoplasts regeneration, fructose, as source of organic carbon, was the most effective. Protoplasts transformation efficiency values with magnetic nanoparticles after enzymatic or mechanical treatments for N. oceanica and S. almeriensis were 17.8% and 10.7%, and 18.6% and 15.7%, respectively. Finally, selected magnetic cells were immobilized and grown on a vertical magnetic surface exposed to light and without any supplement.

Optimization of Microalga Chlorella vulgaris Magnetic Harvesting.

Harvesting of microalgae is a crucial step in microalgae-based mass production of different high value-added products. In the present work, magnetic harvesting of Chlorella vulgaris was investigated using microwave-synthesized naked magnetite (Fe3O4) particles with an average crystallite diameter of 20 nm. Optimization of the most important parameters of the magnetic harvesting process, namely pH, mass ratio (mr) of magnetite particles to biomass (g/g), and agitation speed (rpm) of the C. vulgaris biomass-Fe3O4 particles mixture, was performed using the response surface methodology (RSM) statistical tool. Harvesting efficiencies higher than 99% were obtained for pH 3.0 and mixing speed greater or equal to 350 rpm. Recovery of magnetic particles via detachment was shown to be feasible and the recovery particles could be reused at least five times with high harvesting efficiency. Consequently, the described harvesting approach of C. vulgaris cells leads to an efficient, simple, and quick process, that does not impair the quality of the harvested biomass.

Catalytic and thermodynamic properties of an acidic α-amylase produced by the fungus Paecilomyces variotii ATHUM 8891.

An extracellular acid stable α-amylase from Paecilomyces variotii ATHUM 8891 (PV8891 α-amylase) was purified to homogeneity applying ammonium sulfate fractionation, ion exchange and gel filtration chromatography and exhibited a reduced molecular weight of 75 kDa. The purified enzyme was optimally active at pH 5.0 and 60 °C and stable in acidic pH (3.0-6.0). K m, v max and k cat for starch hydrolysis were found 1.1 g L-1, 58.5 µmole min-1 (mg protein)-1, and 73.1 s-1, respectively. Amylase activity was marginally enhanced by Ca2+ and Fe2+ ions while Cu2+ ions strongly inhibited it. Thermodynamic parameters determined for starch hydrolysis (Ε α, ΔH*, ΔG*, ΔS*, Δ G E - S ∗ and Δ G E - T ∗ ) suggests an effective capacity of PV8891 α-amylase towards starch hydrolysis. Thermal stability of PV8891 α-amylase was assessed at different temperatures (30-80 οC). Thermodynamic parameters ( E a d , ΔH*, ΔG*, ΔS*) as well as the integral activity of a continuous system for starch hydrolysis by the PV8891 α-amylase revealed satisfactory thermostability up to 60 °C. The acidic nature and its satisfactory performance at temperatures lower than the industrially used amylases may represent potential applications of PV8891 α-amylase in starch processing industry.

A Study on the Effect of Macro- and Micro- Nutrients on Nannochloropsis oceanica Growth, Fatty Acid Composition and Magnetic Harvesting Efficiency.

The effect of iron, manganese, phosphorus and nitrogen on growth and lipid synthesis of the microalgae Nannochloropsis oceanica CCMP1779, as well as their impact on the magnetic harvesting efficiency, are examined under their depriving cell culture conditions. Herein, it is demonstrated that nitrogen and manganese depletion primarily reduced cell growth while phosphorus and iron restriction led to higher dry biomass. Subsequently, the role of those nutrients on fatty acids profile was examined. Phosphorus and nitrogen restriction resulted in lower and higher lipid content, respectively. High amounts of polyunsaturated fatty acids like eicosapentaenoic acid are produced under iron and manganese depletion. Phosphorus deprivation favors monounsaturated fatty acids such as C18:1 and C16:1, while nitrogen restriction favors saturated fatty acid production like C14:0, C16:0 and C18:0. Since the presence/absence of macro- and micro-elements may affect the overall electrostatic charges on the outmost microalgae surface, it was also analyzed how these elements affect the magnetic harvesting efficiency. Results showed that phosphorus deprivation led to the best magnetic harvesting efficiency of N. oceanica cells (93%) as compared to other nutrient starvation as well as standard medium.

Detoxification of 2,4-dichlorophenol by the marine microalga Tetraselmis marina.

Xenobiotic chlorinated phenols have been found in fresh and marine waters and are toxic to many aquatic organisms. Metabolism of 2,4-dichlorophenol (2,4-DCP) in the marine microalga Tetraselmis marina was studied. The microalga removed more than 1mM of 2,4-DCP in a 2l photobioreactor over a 6 day period. Two metabolites, more polar than 2,4-DCP, were detected in the growth medium by reverse phase HPLC and their concentrations increased at the expense of 2,4-DCP. The metabolites were isolated by a C8 HPLC column and identified as 2,4-dichlorophenyl-beta-d-glucopyranoside (DCPG) and 2,4-dichlorophenyl-beta-d-(6-O-malonyl)-glucopyranoside (DCPGM) by electrospray ionization-mass spectrometric analysis in a negative ion mode. The molecular structures of 2,4-DCPG and 2,4-CPGM were further confirmed by enzymatic and alkaline hydrolyses. Thus, it was concluded that the major pathway of 2,4-DCP metabolism in T. marina involves an initial conjugation of 2,4-DCP to glucose to form 2,4-dichlorophenyl-beta-d-glucopyranoside, followed by acylation of the glucoconjugate to form 2,4-dichlorophenyl-beta-d-(6-O-malonyl)-glucopyranoside. The microalga ability to detoxify dichlorophenol congeners other than 2,4-DCP was also investigated. This work provides the first evidence that microalgae can use a combined glucosyl and malonyl transfer to detoxify xenobiotics such as dichlorophenols.

Effect of pectate lyase bioscouring on physical, chemical and low-stress mechanical properties of cotton fabrics.

The main objective of the present study was to meticulously investigate an inclusive set of physicochemical and handle properties (determined through Kawabata evaluation system) of bioscoured cotton fabrics. The application of a commercial pectinase preparation, Bioprep 3000L, for a range of concentrations and treatment times, could create a pectin-free textile with low wax content. Multiple regression analysis was used to describe the effect of enzymatic process variables on pectin and waxes removal. Comparison of fabrics' properties such as wettability, whiteness, crystallinity index, and dyeing behaviour, confirmed that bioscouring could be as much effective as the conventional alkaline process. Uncovering the relationship between the composition of materials and their physicochemical properties was attempted. The application of higher enzyme concentrations generated fabrics with improved low-stress mechanical properties. Bending and shear rigidity, compressional resilience, as well as, extensibility of enzymatically treated cotton fabrics could be efficiently predicted by means of a single independent variable, the crystallinity index.

Microbial Production of Violacein and Process Optimization for Dyeing Polyamide Fabrics With Acquired Antimicrobial Properties.

In the present study, crude bacterial extract containing violacein is investigated for the preparation of antimicrobial polyamide fabrics. The optimal culture conditions of Janthinobacterium lividum (JL) for maximum biomass and violacein production were found to be 25°C, pH 7.0, while the addition of ampicillin of 0.2 mg mL-1 in the small scale increased violacein production 1.3-fold. In scale-up trials, the addition of 1% (v/v) glycerol in a fed-batch bioreactor, resulted in fivefold extracted crude violacein increase with final concentration of 1.828 g L-1. Polyamide 6.6 fabrics were dyed following three different processes; through simultaneous fermentation and dyeing (SFD), by incubating the fabric in the sonicated bacterial culture after fermentation and by using cell-free extract containing violacein. Maximum color change (ΔE) and color strength (K/S) obtained for SFD fabrics were 74.81 and 22.01, respectively, while no alteration of fastness and staining of dye at acid and alkaline perspiration or at water was indicated. The dyed fabrics presented significant antifungal activity against Candida albicans, C. parapsilosis, and C. krusei, as well as antibacterial properties against Escherichia coli, Staphylococcus aureus, and the S. aureus MRSA. We have shown that J. lividum cultures can be successfully used for violacein production and for simultaneous dying of fabrics resulting in dyed fabrics with antimicrobial properties without utilization of organic solvents.

Studies on the catalytic behavior of a membrane-bound lipolytic enzyme from the microalgae Nannochloropsis oceanica CCMP1779.

The catalytic behavior of a membrane-bound lipolytic enzyme (MBL-Enzyme) from the microalgae Nannochloropsis oceanica CCMP1779 was investigated. The biocatalyst showed maximum activity at 50 °C and pH 7.0, and was stable at pH 7.0 and temperatures from 40 to 60 °C. Half-lives at 60 °C, 70 °C and 80 °C were found 866.38, 150.67 and 85.57 min respectively. Thermal deactivation energy was 68.87 kJ mol-1. The enzyme's enthalpy (ΔΗ*), entropy (ΔS*) and Gibb's free energy (ΔG*) were in the range of 65.86-66.27 kJ mol-1, 132.38-140.64 J mol-1 K-1 and 107.80-115.81 kJ mol-1, respectively. Among p-nitrophenyl esters of fatty acids tested, MBL-Enzyme exhibited the highest hydrolytic activity against p-nitrophenyl palmitate (pNPP). The Km and Vmax values were found 0.051 mM and of 0.054 mmole pNP mg protein-1 min-1, respectively with pNPP as substrate. The presence of Mn2+ increased lipolytic activity by 68.25%, while Fe3+ and Cu2+ ions had the strongest inhibitory effect. MBL-Enzyme was stable in the presence of water miscible (66% of the initial activity in ethanol) and water immiscible (71% of the initial activity in n-octane) solvents. Myristic acid was found to be the most efficient acyl donor in esterification reactions with ethanol. Methanol was the best acyl acceptor among the primary alcohols tested.

Toxicity and metabolism of p-chlorophenol in the marine microalga Tetraselmis marina.

Toxicity and metabolism of para-chlorophenol (p-CP) in the marine microalga Tetraselmis marina have been studied. The inhibition constant EC(50) for p-CP was 272+/-17 microM (34.8+/-2.2 mg L(-1)) under the experimental conditions. Two metabolites were detected in the growth medium in the presence of p-CP by reverse phase HPLC and their concentrations increased at the expense of p-CP. The two metabolites, which were found to be more polar than p-CP, were isolated by a C18 column. They were identified as p-chlorophenyl-beta-D-glucopyranoside (p-CPG) and p-chlorophenyl-beta-D-(6-O-malonyl)-glucopyranoside (p-CPGM) by electrospray ionization-mass spectrometric analysis in a negative ion mode. The molecular structures of p-CPG and p-CPGM were further confirmed by enzymatic and alkaline hydrolyses. Treatment with beta-glucosidase released free p-CP and glucose from p-CPG, whereas p-CPGM was completely resistant. Alkaline hydrolysis completely cleaved the esteric bond of the malonylated glucoconjugate and yielded p-CPG and malonic acid. It was concluded that the pathway of p-CP metabolism in T. marina involves an initial conjugation of p-CP to glucose to form p-chlorophenyl-beta-d-glucopyranoside, followed by acylation of the glucoconjugate to form p-chlorophenyl-beta-D-(6-O-malonyl)-glucopyranoside. The metabolism of p-CP in T. marina was mainly driven by photosynthesis, and to a lesser extent by anabolic metabolism in the dark. Accordingly, the detoxification rate under light was about seven times higher than in the darkness. This work provides the first evidence that microalgae can adopt a combined glucosyl transfer and malonyl transfer process as a survival strategy for detoxification of such xenobiotics as p-CP.

Cell bound and extracellular glucose oxidases from Aspergillus niger BTL: evidence for a secondary glycosylation mechanism.

Two glucose oxidase (GOX) isoforms where purified to electrophoretic homogeneity from the mycelium extract (GOXI) and the extracellular medium (GOXII) of Aspergillus niger BTL cultures. Both enzymes were found to be homodimers with nonreduced molecular masses of 148 and 159 kDa and pI values of 3.7 and 3.6 for GOXI and GOXII, respectively. The substrate specificity and the kinetic characteristics of the two GOX forms, as expressed through their apparent K m values on glucose, as well as pH and T activity optima, were almost identical. The only structural difference between the two enzymes was in their degrees of glycosylation, which were determined equal to 14.1 and 20.8% (w/w) of their molecular masses for GOXI and GOXII, respectively. The above difference in the carbohydrate content between the two enzymes seems to influence their pH and thermal stabilities. GOXII proved to be more stable than GOXI at pH values 2.5, 3.0, 8.0, and 9.0. Half-lives of GOXI at pH 3.0 and 8.0 were 8.9 and 17.5 h, respectively, whereas the corresponding values for GOXII were 13.5 and 28.1 h. As far as the thermal stability is concerned, GOXII was also more thermostable than GOXI as judged by the deactivation constants determined at various temperatures. More specifically, the half-lives of GOXI and GOXII, at 45 degrees C, were 12 and 49 h, respectively. These results suggest A. niger BTL probably possesses a secondary glycosylation mechanism that increases the stability of the excreted GOX.

Biochemical and catalytic properties of an endoxylanase purified from the culture filtrate of Sporotrichum thermophile.

An endo-beta-1,4-xylanase (1,4-beta-D-xylan xylanoxydrolase, EC 3.2.1.8) present in culture filtrates of Sporotrichum thermophile ATCC 34628 was purified to homogeneity by Q-Sepharose and Sephacryl S-200 column chromatographies. The enzyme has a molecular mass of 25,000 Da, an isoelectric point of 6.7, and is optimally active at pH 5 and at 70 degrees C. Thin-layer chromatography (TLC) analysis showed that endo-xylanase liberates mainly xylose (Xyl) and xylobiose (Xyl2) from beechwood 4-O-methyl-D-glucuronoxylan, O-acetyl-4-O-methylglucuronoxylan and rhodymenan (a beta-(1-->4)-beta(1-->3)-xylan). Also, the enzyme releases an acidic xylo-oligosaccharide from 4-O-methyl-D-glucuronoxylan, and an isomeric xylotetraose and an isomeric xylopentaose from rhodymenan. Analysis of reaction mixtures by high performance liquid chromatography (HPLC) revealed that the enzyme cleaves preferentially the internal glycosidic bonds of xylooligosaccharides, [1-3H]-xylooligosaccharides and xylan. The enzyme also hydrolyses the 4-methylumbelliferyl glycosides of beta-xylobiose and beta-xylotriose at the second glycosidic bond adjacent to the aglycon. The endoxylanase is not active on pNPX and pNPC. The enzyme mediates a decrease in the viscosity of xylan associated with a release of only small amounts of reducing sugar. The enzyme is irreversibly inhibited by series of omega-epoxyalkyl glycosides of D-xylopyranose. The results suggest that the endoxylanase from S. thermophile has catalytic properties similar to the enzymes belonging to family 11.

Mode of action of family 10 and 11 endoxylanases on water-unextractable arabinoxylan.

Microbial endo-beta-1,4-xylanases (EXs, EC 3.2.1.8) belonging to glycanase families 10 and 11 differ in their action on water-unextractable arabinoxylan (WU-AX). WU-AX was incubated with different levels of a Thermoascus aurantiacus family 10 and a Sporotrichum thermophile family 11 endoxylanases. At 10 g l(-1) arabinoxylan, enzyme concentrations (KE values) needed to obtain half-maximal hydrolysis rates (V(max) values) were 4.4 nM for the xylanase from T. aurantiacus and 7.1 nM for the xylanase from S. thermophile. Determination of Vmax/KE revealed that the family 10 enzyme hydrolysed two times more efficiently WU-AX than the family 11 enzyme. Molecular weights of the products formed were assessed and separation of feruloyl-oligosaccharides was achieved by anion-exchange and size-exclusion chromatography (SEC). The main difference between the feruloylated products by xylanases of family 10 and 11 concerned the length of the products containing feruloyl-arabinosyl substitution. The xylanase from T. aurantiacus liberated from WU-AX a feruloyl arabinoxylodisaccharide (FAX2) as the shortest feruloylated fragment in contrast with the enzyme from S. thermophile, which liberated a feruloyl arabinoxylotrisaccharide (FAX3). These results indicated that different factors govern WU-AX breakdown by the two endoxylanases.

Utilization of household food waste for the production of ethanol at high dry material content.

BACKGROUND: Environmental issues and shortage of fossil fuels have turned the public interest to the utilization of renewable, environmentally friendly fuels, such as ethanol. In order to minimize the competition between fuels and food production, researchers are focusing their efforts to the utilization of wastes and by-products as raw materials for the production of ethanol. household food wastes are being produced in great quantities in European Union and their handling can be a challenge. Moreover, their disposal can cause severe environmental issues (for example emission of greenhouse gasses). On the other hand, they contain significant amounts of sugars (both soluble and insoluble) and they can be used as raw material for the production of ethanol. RESULTS: Household food wastes were utilized as raw material for the production of ethanol at high dry material consistencies. A distinct liquefaction/saccharification step has been included to the process, which rapidly reduced the viscosity of the high solid content substrate, resulting in better mixing of the fermenting microorganism. This step had a positive effect in both ethanol production and productivity, leading to a significant increase in both values, which was up to 40.81% and 4.46 fold, respectively. Remaining solids (residue) after fermentation at 45% w/v dry material (which contained also the unhydrolyzed fraction of cellulose), were subjected to a hydrothermal pretreatment in order to be utilized as raw material for a subsequent ethanol fermentation. This led to an increase of 13.16% in the ethanol production levels achieving a final ethanol yield of 107.58 g/kg dry material. CONCLUSIONS: In conclusion, the ability of utilizing household food waste for the production of ethanol at elevated dry material content has been demonstrated. A separate liquefaction/saccharification process can increase both ethanol production and productivity. Finally, subsequent fermentation of the remaining solids could lead to an increase of the overall ethanol production yield.