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A detailed investigation of choline chloride-lactic acid (ChCl-LA)-based deep eutectic solvent (DES) extraction of lignin nanoparticles from herbaceous biomass (wheat straw) was conducted. It was f...

Nitrogen supply is critical to the synthesis of intracellular PHA in various bacteria. However, the specific role of the nitrogen in synthesizing PHA from benzoate, a lignin model compound use for the study of bacteria catabolism of aromatics, is still not clear. In this study, two culture conditions were maintained for Pseudomonas putida KT2440 to produce PHA using benzoate as a carbon source. Under nitrogen-limited and surplus conditions, the accumulation of PHA was to 37.3% and 0.25% of cell dry weight, respectively. A model fit to the kinetics of biomass growth and PHA accumulation showed good agreement with data. GC–MS and NMR showed that PHA contained six hydroxyl fatty acid monomers under nitrogen-limited conditions, while two monomers were identified under nitrogen surplus conditions. The average molecular weight of PHA increased after the nitrogen source was exhausted. These results provide a promising strategy for optimization of lignin to PHA yields.

Lignin is a major part of the recalcitrant fraction of lignocellulose and in nature its degradation occurs through oxidative enzymes along with microbes mediated oxidative chemical actions. Oxygen assisted wet-explosion pretreatment promotes lignin solubility and leads to an increase biodegradation of lignin during anaerobic digestion processes. The pretreatment of feedlot manure was performed in a 10 L reactor at 170 C for 25 min using 4 bars oxygen and the material was fed to a continuous stirred tank reactoroperated at 55 C for anaerobic digestion. Methane yield of untreated and pretreated material was 70 ± 27 and 320 ± 36 L/kg-VS/day, respectively, or 4.5 times higher yield as a result of the pretreatment. Aliphatic acids formed during the pretreatment were utilized by microbes. 44.4% lignin in pretreatedmaterial was actually converted in the anaerobic digestion process compared to 12.6% for untreated material indicating the oxygen assisted explosion promoted lignin degradation.

A commercial laccase from Trametes versicolor was conjugated with biopolymer chitosan using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) as the cross-linking agent. Laccase-chitosan conjugation strategies were tested using different molar ratios of glucosamine monomer/protein with different molar excess ratios of EDC relative to laccase. Immobilization techniques were developed to improve the stability against thermal and chemical denaturation, storage and reusability of this biocatalyst. The conjugation resulted in a solid biocatalyst with an apparent laccase activity of ±626 U/g, 12 and 60 folds higher in the conjugation efficiency of biocatalyst relative to the immobilized and free laccase activity respectively when compared with zero EDC/laccase ratio used in conjugation solution. The conjugated laccases formed successfully eliminated the emerging pollutant triclosan (TCS) from aqueous solutions, having a higher potential to transform TCS than free laccase. UPLC-QTOF results indicate the formation of TCS oligomers. Furthermore, they are the first evidence of direct dechlorination of TCS mediated by the oxidative action of laccases.

With the aim to produce cellulases and to study the effect of mechanical agitation, a 35 L draft-tube airlift bioreactor equipped with a mechanical impeller was developed and validated to grow Trichoderma reesei RUT-C30 in a cellulose culture medium with lactose and lactobionic acid as fed batch. Cultures carried out without mechanical agitation resulted in higher volumetric enzyme productivity (200 U L−1 h−1), filter paper activity (17 U mL−1), carboxymethyl cellulase activity (11.8 U mL−1) and soluble proteins (3.2 mg mL−1) when compared to those with agitation. Stereo and polarized light microscopy analyses reveal that mechanical agitation resulted in shorter mycelial hyphae and larger numbers of tips.

For the complete hydrolysis of cellulose, the cellulolytic fungi produce a whole set of commercially important enzymes called cellulases. The aim of this work was to investigate an approach to enhance the production of these enzymes by co-culturing Trichoderma reesei and Aspergillus niger in a bioreactor to convert cellulose substrate into soluble sugars through a synergetic action of enzyme complex simultaneously produced by these two fungi. The experiments were conducted as fed batch growth on a Cellulose–Yeast extract medium. A mixture of lactose and lactobionic acid was added into the bioreactor as cellulase inducers. The results of mixed culture experiments exhibited a highly significant increase in the production of volumetric enzyme activity (98.4 U L−1 h−1), filter paper activity (7.1 U mL−1), carboxymethyl cellulase activity (4.7 U mL−1), soluble proteins (2.1 mg mL−1), dry biomass (21.4 g−1 L−1), and percentage of utilized cellulose (89.4%) as compared with A. niger monocultures.

Filamentous fungi Trichoderma reesei are considered to be one of the most efficient hyper producers of cellulase that is used in industry. Cellulase production by T. reesei was carried out using high concentration of cellulose to substitute glucose with the aim to improve cellulase production while trying to reduce production costs. The experiments were conducted separately as fed batch growth with T. reesei cultured using four media in a 7 L stirred tank bioreactor. A mixture of lactose and lactobionic acid was added into the bioreactor as cellulase inducers. The use of a cellulose–yeast extract culture medium yielded the highest enzyme and cell production with a volumetric enzyme activity of 69.8 U L−1 h−1, a filter paper activity of 5.02 U mL−1, a CMCase activity of 4.2 U mL−1, and a fungal biomass of 14.7 g L−1. The biomass concentration as a function of time was constant with relatively rapid, early growth on easily metabolized growth medium components (yeast extract), followed by a second slower growth phase due to hydrolysis of cellulose, which follow cellulase concentration augmentation. The costs to produce 1 L of production medium in laboratory-scale experiments were calculated to compare the tested media.

The filamentous fungus, Asperigillus carbonarius, is able to produce a series of hydrocarbons in liquid culture using lignocellulosic biomasses, such as corn stover and switch grass as carbon source. The hydrocarbons produced by the fungus show similarity to jet fuel composition and might have industrial application. The production of hydrocarbons was found to be dependent on type of media used. Therefore, ten different carbon sources (oat meal, wheat bran, glucose, carboxymethyl cellulose, avicel, xylan, corn stover, switch grass, pretreated corn stover, and pretreated switch grass) were tested to identify the maximum number and quantity of hydrocarbons produced. Several hydrocarbons were producedinclude undecane, dodecane, tetradecane, hexadecane 2,4-dimethylhexane, 4-methylheptane, 3-methyl-1-butanol, ethyl benzene, o-xylene. Oatmeal was found to be the carbon source resulting in the largest amounts of hydrocarbon products. The production of fungal hydrocarbons, especially from lignocellulosic biomasses, holds a great potential for future biofuel production whenever our knowledge on regulators and pathways increases.

Aspergillus strains are being considered as potential hosts for recombinant heterologous protein production because of their excellent extracellular enzyme production characteristics. However, Aspergillus proteases are problematic in that they modify and degrade the heterologous proteins in the extracellular medium. In previous studies we observed that media adjustments and maintenance of a filamentous morphology greatly reduced protease activity and that a low concentration of the aspartic protease inhibitor pepstatin inhibited the latter protease activity to the extent of approximately 90%. In this paper we report that when the serine protease inhibitor chymostatin is used in combination with pepstatin 99-100% of total protease activity in Aspergillus cultures is inhibited. In protease assays a concentration of 30 microM chymostatin combined with 0.075 microM pepstatin was required for maximum inhibition. Inhibitor concentrations of chymostatin and pepstatin of 120 and 0.3 microM, respectively, when added to Aspergillus cultures, has no significant effect on biomass production, glucose utilization or culture pH pattern. The potential of using these protease inhibitors in cultures of recombinant Aspergillus strains producing heterologous proteins will now be investigated to determine if the previously observed recombinant protein denaturing effects of Aspergillus proteases can be negated.

Aspergillus species have been used in different industrial applications including extracellular enzyme production and as potential hosts for recombinant heterologous proteins production because of their excellent growth and fermentation properties. However, recombinant protein production levels are typically low due to the impact of Aspergillus proteases in modifying and degrading the heterologous proteins, especially in the extracellular medium. The potential of using the protease inhibitor pepstatin in filamentous Aspergillus niger culture was investigated in this study. The majority of protease activity was released into the medium as biomass content decreases after maximum biomass concentration was attained and a low concentration of pepstatin effectively inhibited the extracellular protease activity to an extent of 80–90%. The effectiveness of pepstatin in inhibiting the majority of protease activity was further demonstrated in media supplemented with both non-peptide and peptide-containing nitrogen sources. Pepstatin addition had no effect on microbial growth. Dilution of the culture filtrate released inhibition, indicating that pepstatin did not have a notable effect on protease production and that inhibition was reversible.

While Aspergillus strains are also being considered as potential hosts for production of extracellular heterologous proteins, the proteases produced by the host are highly problematic in that they typically modify and degrade the recombinant proteins. Culture-based approaches for minimization of protease activity in culture supernatants of Aspergillus niger NRRL-3 included reduction or elimination of peptide nitrogen in the medium, preferential use of a defined salts medium rather than a non-peptide nitrogen medium containing yeast-nitrogen base, supplementation of the medium with carboxymethylcellulose and cultivation at pH 6.5 rather than 7.5. In general, increased proteolytic activity was observed after maximum biomass was observed and biomass was declining suggesting the majority of protease activity was released by cell lysis. Carboxymethylcellulose shifted mycelial morphology from pelleted to filamentous. Mycelium lysis in the centre of pellets, with resultant release of intracellular proteases, would explain why filamentous cultures exhibited much lower proteolytic activity than pelleted cultures.

The effect of fluoride on the food and dietary water intake and utilisation were studied in final instars of silkworm, Bombyx mori L. race NB4D2 fed ad libitum with mulberry leaves treated with sodium fluoride at concentrations of 25 ppm, 50 ppm and 75 ppm. The larval duration increased by two days while food consumption, assimilation rate, assimilation efficiency, feeding and metabolic rates increased significantly in all the treated larvae. On the other hand, the amount of faeces produced, food conversion, conversion rate and conversion efficiencies decreased significantly in all the treated batches when compared to controls. The water intake and utilisation parameters increased significantly while rate of water loss through faeces, water retained in the body and retention efficiency decreased in all the fluoride-treated silkworms. The factors responsible for these changes in treated batches are discussed in relation to the effect of fluoride on cocoon and shell weights.

Endophytic fungi belonging to the genus Gliocladium are able to degrade plant cellulose and synthesize complex hydrocarbons under microaerophilic conditions. These fungi could thus be used to produce biofuels from cellulosics without the need for hydrolytic pretreatments. Gas chromatography-mass spectrometry-solid-phase micro-extraction (GC-MS-SPME) of head space gases from Gliocladium cultures demonstrated the production of C(6)-C(19) hydrocarbons including hexane, benzene, heptane, 3,4-dimethyl hexane, 1-octene, m-xylene, 3-methyl nonane, dodecane, tridecane, hexadecane and nonadecane directly from the cellulosic biomass. Hydrocarbon production was 100-fold higher in co-cultures of Gliocladium and Escherichia coli than in pure Gliocladium cultures. The dry mycelia weight is stable at stationary period in co-culture condition which may lead to synthesize more hydrocarbons. These fungi could potentially be developed into cost-effective biocatalysts for production of biofuels.

The objective of this study was to determine how fungal morphology influences the volumetric cellulase productivity of Trichoderma reesei cultured in four media with lactose and lactobionic acid as fed-batch in a 7 L stirred tank bioreactor. The use of a cellulose–yeast extract culture medium yielded the highest enzyme production with a volumetric enzyme activity of 69.8 U L −1 h −1 , and a maximum fungal biomass of 14.7 g L −1 . These findings were associated with the following morphological characteristics of the fungus: total mycelia was 98% of total mean projected area, mean hyphae length of 10 mm, mean hyphae volume of 45.1 mm 3 , mean hyphae diameter of 7.9 μm, number of branches 9, and number of tips per hypha 29. A positive correlation was found between the total mycelia, the number of tips and the volumetric enzyme productivity, indicating the weight of these variables on the enzyme productivity.