Methylene blue as a lignin surrogate in manganese peroxidase reaction systems.

Manganese peroxidase (MnP) is associated with lignin degradation and is thus relevant to lignocellulosic-utilization technologies. Technological applications require reaction mixture optimization. A surrogate substrate can facilitate this if its susceptibility to degradation is easily monitored and mirrors that of lignin. The dye methylene blue (MB) was evaluated in these respects as a surrogate substrate by testing its reactivity in reaction mixtures containing relevant redox mediators (dicarboxylic acids, fatty acids). Relative rates of MB degradation were compared to available literature reports of lignin degradation under similar conditions, and suggest that MB can be a useful lignin surrogate in MnP systems.

Furfural and 5-hydroxymethyl-furfural degradation using recombinant manganese peroxidase.

Biomass pretreatment-derived degradation compounds, such as furfural and 5-hydroxymethyl-furfural (HMF), inhibit the growth of fermentation microorganisms that utilize biomass to produce fuels and chemicals. Here we report that recombinant manganese peroxidase (rMnP) produced from the yeast Pichia pastoris can degrade furfural and HMF making them less toxic to microorganisms. Treatment with rMnP or manganese(III) acetate reduced furfural and HMF concentrations in a dose-dependent manner. Furfural disappearance was accompanied by malonate disappearance and accumulation of four distinct degradation products. Furfural was more readily degraded by rMnP and manganese(III) acetate than HMF. Growth assays using Saccharomyces cerevisiae indicated that rMnP treatment reduced the toxicity of furfural and HMF. This work provides an avenue to use rMnP to increase the growth of fermentation microorganisms that are inhibited by toxic compounds derived from pretreatment of biomass.

Cloning, expression and characterization of xylose isomerase from the marine bacterium Fulvimarina pelagi in Escherichia coli.

Production of a xylose isomerase (XI) with high tolerance to the inhibitors xylitol and calcium, and high activity at the low pH and temperature conditions characteristic of yeast fermentations, is desirable for a simultaneous isomerization/fermentation process for cellulosic ethanol production. A putative XI gene (xylA) from the marine bacterium Fulvimarina pelagi was identified by sequence analysis of the F. pelagi genome, and was PCR amplified, cloned, and expressed in Escherichia coli. The rXI was produced in shake flask and fed-batch fermentations using glucose as the growth substrate. The optimum pH for rXI was approximately 7, although activity was evident at pH as low as 5.5. The purified rXI had a molecular weight in 160 kDA, a Vmax of 0.142 U/mg purified rXI, and a KM for xylose in the range of 1.75-4.17 mM/L at pH 6.5 and a temperature of 35°C. The estimated calcium and xylitol KI values for rXI in cell-free extracts were 2,500 mg/L and >50 mM, respectively. The low KM of the F. pelagi xylose isomerase is consistent with the low nutrient conditions of the pelagic environment. These results indicate that Ca2+ and xylitol are not likely to be inhibitory in applications employing the rXI from F. pelagi to convert xylose to xylulose in fermentations of complex biomass hydrolysates. A higher Vmax at low pH (<6) and temperature (30°C) would be preferable for use in biofuels production. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1230-1237, 2016.

Assessing wastewater metal toxicity with bacterial bioluminescence in a bench-scale wastewater treatment system.

The effectiveness of a previously developed toxicity monitoring method for activated sludge wastewater treatment employing a bioluminescent bacterium (Shk1) was evaluated in batch experiments and a bench-scale activated sludge system exposed to heavy metals (Cu, Zn, Ni, and Cd). Influent wastewater (primary clarifier supernatant) and activated sludge from a municipal wastewater treatment plant were used in both batch experiments and in the bench-scale wastewater treatment system. Shk1 bioluminescence was most sensitive to Cd and Zn, followed by Cu, and then Ni in order of decreasing sensitivity. In contrast, activated sludge specific oxygen uptake rate was most sensitive to Cu, followed by Cd and Zn, and finally Ni. The same pattern of sensitivity was observed in batch and bench-scale evaluations. Batch experiments examining the effect of metal adsorption were performed. The adsorption of metals to activated sludge and reduction in bioavailability due to chelation by soluble organics or by precipitation in wastewater was found to be an important effect in mediating differences in toxicity response between bioluminescence and respirometry. Batch adsorption experiments indicated that the activated sludge adsorption capacity was highest for Cu, followed by Cd, Ni, and then Zn. A simple mathematical model for the soluble metal concentration in the aeration basin and clarifier was developed utilizing metal distribution coefficients determined from the batch adsorption experiments. Model predictions compared well with results from the bench-scale activated sludge experiments.

Kinetic analysis of bacterial bioluminescence.

Bioluminescence from the lux-based bacterial reporter Pseudomonas fluorescens HK44 was experimentally investigated under growth substrate-rich and limiting conditions in batch, continuous stirred tank (CSTR), and turbidostat reactors. A mechanistically based, mathematical model was developed to describe bioluminescence based on 1) production and decay of catalytic enzymes, and 2) reactant cofactor availability. In the model, bioluminescence was a function of inducer, growth substrate, and biomass concentration. A saturational dependence on growth substrate concentration accommodated dependence on cofactor availability and inducer concentration to accommodate enzyme production was incorporated in the model. Under growth substrate and inducer limiting conditions in the batch reactor and CSTR, bioluminescence was found to decrease in response to cellular energy limitations. The effective lux system enzyme decay rate was determined in independent measurements to be 0.35 hr(-1) and the model captured most of the bioluminescent behavior, except at long growth times and high cell density.