Green seaweeds (Ulva fasciata sp.) as nitrogen source for fungal cellulase production.

In developing countries, local enzyme production can help decrease the dependency of imported enzymes for bioconversion of e.g. cellulosic feedstocks, but the use of conventional nitrogen sources contributes significantly to such enzyme production cost. Use of local resources is therefore important to consider. Green seaweeds are marine macroalgae that are rich in nitrogen, but not exploited for their nitrogen content. Cellulase production was accomplished by using cocoa pod husk (CPH) and green seaweed (GS) (Ulva fasciata sp.) as growth substrates for Polyporus ciliatus CBS 366.74 in submerged cultivation. The nitrogen concentration of GS was comparable to that of CPH with 0.6% w/v peptone at 4% w/v substrate concentration. A decline of cellulase activity in peptone supplemented GS growth media indicated nitrogen sufficiency of GS to serve as a potential nitrogen source for the fungal growth and cellulase production. Comparison of enzyme production on CPH growth media supplemented with either GS or peptone based on equivalent carbon to nitrogen ratios was done for two Polyporus strains namely; P. ciliatus CBS 366.74 and P. brumalis CBS 470.77. Peptone could be substituted by up to 0.6% w/v with GS at inclusion levels of 50-100% of substrate concentration to attain satisfactory cellulase productivity. However, the cellulase productivity response varied among the two Polyporus species. This study demonstrated that green seaweeds may be used as alternative nitrogen sources for fungal cellulase production.

Cellulase production by white-rot basidiomycetous fungi: solid-state versus submerged cultivation.

White-rot basidiomycetous (WRB) fungi are a group of wood-decaying fungi that are known to be endowed with the ability to secrete enzymes that can catalyze decomposition of a range of plant cell wall polysaccharides, including cellulose and lignin. Expression of these enzymes is induced by the substrate and the enzyme yields obtained depend on the growth of the fungi and thus the mode of cultivation. In order to exploit WRB fungi for local enzyme production for converting lignocellulosic materials in biorefinery processes, the fungi can principally be cultivated in either solid-state (SSC) or submerged cultivation (SmC) systems. In this review, we quantitatively assess the data available in the literature on cellulase production yields by WRB fungi cultivated by SSC or SmC. The review also assesses cellulolytic enzyme production rates and enzyme recovery when WRB fungi are cultivated on different biomass residues in SSC or SmC systems. Although some variation in cellulase production yields have been reported for certain substrates, the analysis convincingly shows that SmC is generally more efficient than SSC for obtaining high cellulase production yields and high cellulase production rates on the substrate used. However, the cultivation method also affects the enzyme activity profile obtained, and the resulting enzyme titers and significant dilution of the enzymes usually occurs in SmC. The review also highlights some future approaches, including sequential cultivations and co-cultivation of WRB fungi for improved enzyme expression, as well as on-site approaches for production of enzyme blends for industrial biomass conversion. The quantitative comparisons made have implications for selection of the most appropriate cultivation method for WRB fungi for attaining maximal cellulase production.

Enhanced production of succinic acid from methanol-organosolv pretreated Strophanthus preussii by recombinant Escherichia coli.

A biorefinery process for high yield production of succinic acid from biomass sugars was investigated using recombinant Escherichia coli. The major problem been addressed is utilization of waste biomass for the production of succinic acid using metabolic engineering strategy. Here, methanol extract of Strophanthus preussii was used for fermentation. The process parameters were optimized. Glucose (9 g/L), galactose (4 g/L), xylose (6 g/L) and arabinose (0.5 g/L) were the major sugars present in the methanol extract of S. preussii. E. coli K3OS with overexpression of soluble nucleotide pyridine transhydrogenase sthA and mutation of lactate dehydrogenase A (ldhA), phosphotransacetylase acetate kinase A (pta-ackA), pyruvate formate lyase B (pflB), pyruvate oxidase B (poxB), produced a final succinic acid concentration of 14.40 g/L and yield of 1.10 mol/mol total sugars after 72 h dual-phase fermentation in M9 medium. Here, we show that the maximum theoretical yield using methanol extracts of S. preussii was 64%. Hence, methanol extract of S. preussii could be used for the production of biochemicals such as succinate, malate and pyruvate.

Acetate is a superior substrate for microbial fuel cell initiation preceding bioethanol effluent utilization.

This study assessed cell voltage development, electricity recovery, and microbial community composition in response to initial substrate including acetate, xylose, acetate/xylose 1:1 mixture (ace/xyl), and bioethanol effluent (BE) during microbial fuel cell (MFC) operation at 1000 Ω external resistance. The BE mainly contained 20.5 g/L xylose, 1.8 g/L arabinose, and 2.5 g/L propionic acid. The MFCs initially fed with acetate showed shorter initiation time (1 day), higher average cell voltage (634 ± 9 mV), and higher coulombic efficiency (31.5 ± 0.5 %) than those initially fed with ace/xyl or xylose. However, BE-initiated MFCs only generated 162 ± 1 mV. The acetate-initiated MFCs exhibited longer adaptation time (21 h) and lower cell voltage (645 ± 10 mV) when the substrate was switched to xylose, whereas substrate switching to BE produced the highest voltage (656 mV), maximum power density (362 ± 27 mW/m(2)), maximum current density (709 ± 27 mA/m(2)), and coulombic efficiency (25 ± 0.5 %) in the acetate-initiated MFCs. The microbial community in acetate-initiated MFCs was less diverse and contained more electrogenic bacteria (13.9 ± 0.4 %) including Geobacter sulfurreducens and Desulfuromonas acetexigen than the MFCs initially fed with ace/xyl, xylose, and BE. After switching the substrate to xylose and subsequently to BE, the microbial community in the acetate-initiated MFCs became more diverse, while no significant changes were observed in ace/xyl-, xylose-, and BE-initiated MFCs. The results showed that initial substrate affected the power generation and the capability to adapt to the substrate alteration in MFCs. Acetate-initiated MFCs showed best performance in utilizing BE.

Thermostability enhancement of an endo-1,4-ß-galactanase from Talaromyces stipitatus by site-directed mutagenesis.

Enzymatic conversion of pectinaceous biomasses such as potato and sugar beet pulp at high temperatures is advantageous as it gives rise to lower substrate viscosity, easier mixing, and increased substrate solubility and lowers the risk of contamination. Such high-temperature processing requires development of thermostable enzymes. Talaromyces stipitatus was found to secrete endo-1,4-ß-galactanase when grown on sugar beet pectin as sole carbon source. The mature protein contained 353 AA and the MW was estimated to 36.5 kDa. It was subjected to codon optimization and produced in Pichia pastoris in 2 l scale yielding 5.3 g. The optimal reaction condition for the endo-1,4-ß-galactanase was determined to be 46 °C at pH 4.5 at which the specific activity was estimated to be 6.93 µmol/min/mg enzyme with half-lives of 13 and 2 min at 55 and 60 °C, respectively. For enhancement of the half-life of TSGAL, nine single amino acid residues were selected for site-directed mutagenesis on the basis of semi-rational design. Of these nine mutants, G305A showed half-lives of 114 min at 55 °C and 15 min at 60 °C, respectively. This is 8.6-fold higher than that of the TSGAL at 55 °C, whereas the other mutants displayed moderate positive to negative changes in their half-lives.

The significance of the initiation process parameters and reactor design for maximizing the efficiency of microbial fuel cells.

Microbial fuel cells (MFCs) can be used for electricity generation via bioconversion of wastewater and organic waste substrates. MFCs also hold potential for production of certain chemicals, such as H2 and H2O2. The studies of electricity generation in MFCs have mainly focused on the microbial community formation, substrate effect on the anode reaction, and the cathode's catalytic properties. To improve the performance of MFCs, the initiation process requires more investigation because of its significant effect on the anodic biofilm formation. This review explores the factors which affect the initiation process, including inoculum, substrate, and reactor configuration. The key messages are that optimal performance of MFCs for electricity production requires (1) understanding of the electrogenic bacterial biofilm formation, (2) proper substrates at the initiation stage, (3) focus on operational conditions affecting initial biofilm formation, and (4) attention to the reactor configuration.

Upgrading of straw hydrolysate for production of hydrogen and phenols in a microbial electrolysis cell (MEC).

In a microbial electrolysis cell (MEC), hydrolysate produced by hydrothermal treatment of wheat straw was used for hydrogen production during selective recovery of phenols. The average H2 production rate was 0.61 m³ H2/m³ MEC·day and equivalent to a rate of 0.40 kg COD/m³ MEC·day. The microbial community in the anode biofilm was adapted by establishment of xylose-degrading bacteria of the Bacteriodetes phylum (16%) and Geobacter sulfurreducens (49%). During the process, 61% of the chemical oxygen demand was removed as hydrogen at 64% yield. The total energy production yield was 78% considering the energy content in the consumed compounds and the cell voltage of 0.7 V. The highest hydrogen production was equivalent to 0.8 kg COD/m³ MEC·day and was obtained at pH 7-8 and 25°C. Accumulation of 53% w/v phenolic compounds in the liquor was obtained by stepwise addition of the hydrolysate during simultaneous production of hydrogen from consumption of 95% for the hemicellulose and 100% of the fatty acids. Final calculations showed that hydrolysate produced from 1 kg wheat straw was upgraded by means of the MEC to 22 g hydrogen (266 L), 8 g xylan, and 9 g polyphenolics for potential utilization in biobased materials.

Valorization of municipal organic waste into purified lactic acid.

Municipal organic waste (biowaste) consists of food derived starch, protein and sugars, and lignocellulose derived cellulose, hemicellulose, lignin and pectin. Proper management enables nutrient recycling and sustainable production of platform chemicals such as lactic acid (LA). This review gathers the most important information regarding use of biowaste for LA fermentation covering pre-treatment, enzymatic hydrolysis, fermentation and downstream processing to achieve high purity LA. The optimal approach was found to treat the two biowaste fractions separately due to different pre-treatment and enzyme needs for achieving enzymatic hydrolysis and to do continues fermentation to achieve high cell density and high LA productivity up to 12 g/L/h for production of both L and D isomers. The specific productivity was 0.4 to 0.5 h-1 but with recalcitrant biomass, the enzymatic hydrolysis was rate limiting. Novel purification approaches included reactive distillation and emulsion liquid membrane separation yielding purities sufficient for polylactic acid production.

Identification and characterization of fermentation inhibitors formed during hydrothermal treatment and following SSF of wheat straw.

A pilot plant for hydrothermal treatment of wheat straw was compared in reactor systems of two steps (first, 80 degrees C; second, 190-205 degrees C) and of three steps (first, 80 degrees C; second, 170-180 degrees C; third, 195 degrees C). Fermentation (SSF) with Sacharomyces cerevisiae of the pretreated fibers and hydrolysate from the two-step system gave higher ethanol yield (64-75%) than that obtained from the three-step system (61-65%), due to higher enzymatic cellulose convertibility. At the optimal conditions (two steps, 195 degrees C for 6 min), 69% of available C6-sugar could be fermented into ethanol with a high hemicellulose recovery (65%). The concentration of furfural obtained during the pretreatment process increased versus temperature from 50 mg/l at 190 degrees C to 1,200 mg/l at 205 degrees C as a result of xylose degradation. S. cerevisiae detoxified the hydrolysates by degradation of several toxic compounds such as 90-99% furfural and 80-100% phenolic aldehydes, which extended the lag phase to 5 h. Acetic acid concentration increased by 0.2-1 g/l during enzymatic hydrolysis and 0-3.4 g/l during fermentation due to hydrolysis of acetyl groups and minor xylose degradation. Formic acid concentration increased by 0.5-1.5 g/l probably due to degradation of furfural. Phenolic aldehydes were oxidized to the corresponding acids during fermentation reducing the inhibition level.

Effective production of succinic acid from coconut water (Cocos nucifera) by metabolically engineered Escherichia coli with overexpression of Bacillus subtilis pyruvate carboxylase.

Succinic acid is an important acid which is used in medicine and pharmaceutical companies. Metabolically engineered Escherichia coli strain was used for the effective production of succinic acid using Cocos nucifera water, which contained 5.00 ± 0.02 g/L glucose, 6.10 ± 0.01 g /L fructose and 6.70 ± 0.02 g /L sucrose. Fermentation of C. nucifera water with E. coli M6PM produced a final concentration of 11.78 ± 0.02 g/L succinic acid and yield of 1.23 ± 0.01 mol/mol, 0.66 ± 0.01 g/g total sugars after 72 h dual-phase fermentation in M9 medium while modeled sugar was 0.38 ± 0.02 mol/mol total sugars. It resulted in 72% of the maximum theoretical yield of succinic acid. Here we show that novel substrate of C. nucifera water resulted in effective production of succinic acid. These investigations unveil the importance of C. nucifera water as a substrate for the production of biochemicals.

Hydrothermal treatment of wheat straw at pilot plant scale using a three-step reactor system aiming at high hemicellulose recovery, high cellulose digestibility and low lignin hydrolysis.

A pilot plant (IBUS) consisting of three reactors was used for hydrothermal treatment of wheat straw (120-150 kg/h) aiming at co-production of bioethanol (from sugars) and electricity (from lignin). The first reactor step was pre-soaking at 80 degrees C, the second extraction of hemicellulose at 170-180 degrees C and the third improvement of the enzymatic cellulose convertibility at 195 degrees C. Water added to the third reactor passed countercurrent to straw. The highest water addition (600 kg/h) gave the highest hemicellulose recovery (83%). With no water addition xylose degradation occurred resulting in low hemicellulose recovery (33%) but also in high glucose yield in the enzymatic hydrolysis (72 g/100g glucose in straw). Under these conditions most of the lignin was retained in the fibre fraction, which resulted in a lignin rich residue with high combustion energy (up to 31 MJ/kg) after enzymatic hydrolysis of cellulose and hemicellulose.

Efficient One-Step Fusion PCR Based on Dual-Asymmetric Primers and Two-Step Annealing.

Gene splicing by fusion PCR is a versatile and widely used methodology, especially in synthetic biology. We here describe a rapid method for splicing two fragments by one-round fusion PCR with a dual-asymmetric primers and two-step annealing (ODT) method. During the process, the asymmetric intermediate fragments were generated in the early stage. Thereafter, they were hybridized in the subsequent cycles to serve as template for the target full-length product. The process parameters such as primer ratio, elongation temperature and cycle numbers were optimized. In addition, the fusion products produced with this method were successfully applied in seamless genome editing. The fusion of two fragments by this method takes less than 0.5 day. The method is expected to facilitate various kinds of complex genetic engineering projects with enhanced efficiency.

Green synthesis of gold and silver nanoparticles from Cannabis sativa (industrial hemp) and their capacity for biofilm inhibition.

BACKGROUND: Cannabis sativa (hemp) is a source of various biologically active compounds, for instance, cannabinoids, terpenes and phenolic compounds, which exhibit antibacterial, antifungal, anti-inflammatory and anticancer properties. With the purpose of expanding the auxiliary application of C. sativa in the field of bio-nanotechnology, we explored the plant for green and efficient synthesis of gold nanoparticles (AuNPs) and silver nanoparticles (AgNPs). METHODS AND RESULTS: The nanoparticles were synthesized by utilizing an aqueous extract of C. sativa stem separated into two different fractions (cortex and core [xylem part]) without any additional reducing, stabilizing and capping agents. In the synthesis of AuNPs using the cortex enriched in bast fibers, fiber-AuNPs (F-AuNPs) were achieved. When using the core part of the stem, which is enriched with phenolic compounds such as alkaloids and cannabinoids, core-AuNPs (C-AuNPs) and core-AgNPs (C-AgNPs) were formed. Synthesized nanoparticles were character-ized by UV-visible analysis, transmission electron microscopy, atomic force microscopy, dynamic light scattering, Fourier transform infrared, and matrix-assisted laser desorption/ionization time-of-flight. In addition, the stable nature of nanoparticles has been shown by thermogravimetric analysis and inductively coupled plasma mass spectrometry (ICP-MS). Finally, the AgNPs were explored for the inhibition of Pseudomonas aeruginosa and Escherichia coli biofilms. CONCLUSION: The synthesized nanoparticles were crystalline with an average diameter between 12 and 18 nm for F-AuNPs and C-AuNPs and in the range of 20-40 nm for C-AgNPs. ICP-MS analysis revealed concentrations of synthesized nanoparticles as 0.7, 4.5 and 3.6 mg/mL for F-AuNPs, C-AuNPs and C-AgNPs, respectively. Fourier transform infrared spectroscopy revealed the presence of flavonoids, cannabinoids, terpenes and phenols on the nanoparticle surface, which could be responsible for reducing the salts to nanoparticles and further stabilizing them. In addition, the stable nature of synthesized nanoparticles has been shown by thermogravimetric analysis and ICP-MS. Finally, the AgNPs were explored for the inhibition of P. aeruginosa and E. coli biofilms. The nanoparticles exhibited minimum inhibitory concentration values of 6.25 and 5 µg/mL and minimum bactericidal concentration values of 12.5 and 25 µg/mL against P. aeruginosa and E. coli, respectively.

Comparison of traditional field retting and Phlebia radiata Cel 26 retting of hemp fibres for fibre-reinforced composites.

Classical field retting and controlled fungal retting of hemp using Phlebia radiata Cel 26 (a mutant with low cellulose degrading ability) were compared with pure pectinase treatment with regard to mechanical properties of the produced fibre/epoxy composites. For field retting a classification of the microbial evolution (by gene sequencing) and enzyme profiles were conducted. By phylogenetic frequency mapping, different types of fungi, many belonging to the Ascomycota phylum were found on the fibres during the first 2 weeks of field retting, and thereafter, different types of bacteria, notably Proteobacteria, also proliferated on the field retted fibres. Extracts from field retted fibres exhibited high glucanase activities, while extracts from P. radiata Cel 26 retted fibres showed high polygalacturonase and laccase activities. As a result, fungal retting gave a significantly higher glucan content in the fibres than field retting (77 vs. 67%) and caused a higher removal of pectin as indicated by lower galacturonan content of fibres (1.6%) after fibres were retted for 20 days with P. radiata Cel 26 compared to a galacturonan content of 3.6% for field retted fibres. Effective fibre stiffness increased slightly after retting with P. radiata Cel 26 from 65 to 67 GPa, while it decreased after field retting to 52 GPa. Effective fibre strength could not be determined similarly due to variations in fibre fracture strain and fibre-matrix adhesion. A maximum composite strength with 50 vol% fibres of 307 MPa was obtained using P. radiata Cel 26 compared to 248 MPa with field retting.

Preliminary results on optimization of pilot scale pretreatment of wheat straw used in coproduction of bioethanol and electricity.

The overall objective in this European Union-project is to develop cost and energy effective production systems for coproduction of bioethanol and electricity based on integrated biomass utilization. A pilot plan reactor for hydrothermal pretreatment (including weak acid hydrolysis, wet oxidation, and steam pretreatment) with a capacity of 100 kg/h was constructed and tested for pretreatment of wheat straw for ethanol production. Highest hemicellulose (C5 sugar) recovery and extraction of hemicellulose sugars was obtained at 190 degrees C whereas highest C6 sugar yield was obtained at 200 degrees C. Lowest toxicity of hydrolysates was observed at 190 degrees C; however, addition of H2O2 improved the fermentability and sugar recoveries at the higher temperatures. The estimated total ethanol production was 223 kg/t straw assuming utilisation of both C6 and C5 during fermentation, and 0.5 g ethanol/g sugar.

PCR-Based Seamless Genome Editing with High Efficiency and Fidelity in Escherichia coli.

Efficiency and fidelity are the key obstacles for genome editing toolboxes. In the present study, a PCR-based tandem repeat assisted genome editing (TRAGE) method with high efficiency and fidelity was developed. The design of TRAGE is based on the mechanism of repair of spontaneous double-strand breakage (DSB) via replication fork reactivation. First, cat-sacB cassette flanked by tandem repeat sequence was integrated into target site in chromosome assisted by Red enzymes. Then, for the excision of the cat-sacB cassette, only subculturing is needed. The developed method was successfully applied for seamlessly deleting, substituting and inserting targeted genes using PCR products. The effects of different manipulations including sucrose addition time, subculture times in LB with sucrose and stages of inoculation on the efficiency were investigated. With our recommended procedure, seamless excision of cat-sacB cassette can be realized in 48 h efficiently. We believe that the developed method has great potential for seamless genome editing in E. coli.

Pretreatment of the macroalgae Chaetomorpha linum for the production of bioethanol--comparison of five pretreatment technologies.

A qualified estimate for pretreatment of the macroalgae Chaetomorpha linum for ethanol production was given, based on the experience of pretreatment of land-based biomass. C. linum was subjected to hydrothermal pretreatment (HTT), wet oxidation (WO), steam explosion (STEX), plasma-assisted pretreatment (PAP) and ball milling (BM), to determine effects of the pretreatment methods on the conversion of C. linum into ethanol by simultaneous saccharification and fermentation (SSF). WO and BM showed the highest ethanol yield of 44 g ethanol/100g glucan, which was close to the theoretical ethanol yield of 57 g ethanol/100g glucan. A 64% higher ethanol yield, based on raw material, was reached after pretreatment with WO and BM compared with unpretreated C. linum, however 50% of the biomass was lost during WO. Results indicated that the right combination of pretreatment and marine macroalgae, containing high amounts of glucan and cleaned from salts, enhanced the ethanol yield significantly.

Bio-oil based biorefinery strategy for the production of succinic acid.

BACKGROUND: Succinic acid is one of the key platform chemicals which can be produced via biotechnology process instead of petrochemical process. Biomass derived bio-oil have been investigated intensively as an alternative of diesel and gasoline fuels. Bio-oil could be fractionized into organic phase and aqueous phase parts. The organic phase bio-oil can be easily upgraded to transport fuel. The aqueous phase bio-oil (AP-bio-oil) is of low value. There is no report for its usage or upgrading via biological methods. In this paper, the use of AP-bio-oil for the production of succinic acid was investigated. RESULTS: The transgenic E. coli strain could grow in modified M9 medium containing 20 v/v% AP-bio-oil with an increase in OD from 0.25 to 1.09. And 0.38 g/L succinic acid was produced. With the presence of 4 g/L glucose in the medium, succinic acid concentration increased from 1.4 to 2.4 g/L by addition of 20 v/v% AP-bio-oil. When enzymatic hydrolysate of corn stover was used as carbon source, 10.3 g/L succinic acid was produced. The obtained succinic acid concentration increased to 11.5 g/L when 12.5 v/v% AP-bio-oil was added. However, it decreased to 8 g/L when 50 v/v% AP-bio-oil was added. GC-MS analysis revealed that some low molecular carbon compounds in the AP-bio-oil were utilized by E. coli. CONCLUSIONS: The results indicate that AP-bio-oil can be used by E. coli for cell growth and succinic acid production.

Electric power generation by a submersible microbial fuel cell equipped with a membrane electrode assembly.

Membrane electrode assemblies (MEAs) were incorporated into the cathode chamber of a submersible microbial fuel cell (SMFC). A close contact of the electrodes could produce high power output from SMFC in which anode and cathode electrodes were connected in parallel. In polarization test, the maximum power density was 631 mW/m(2) at current density of 1772 mA/m(2) at 82 Ω. With 180-Ω external resistance, one set of the electrodes on the same side could generate more power density of 832±4 mW/m(2) with current generation of 1923±4 mA/m(2). The anode, inclusive a biofilm behaved ohmic, whereas a Tafel type behavior was observed for the oxygen reduction. The various impedance contributions from electrodes, electrolyte and membrane were analyzed and identified by electrochemical impedance spectroscopy. Air flow rate to the cathode chamber affected microbial voltage generation, and higher power generation was obtained at relatively low air flow less than 2 mL/min.