Continuous aryl alcohol oxidase production under growth-limited conditions using a trickle bed reactor.

An A. nidulans strain with a pyridoxine marker was used for continuous production of aryl alcohol oxidase (AAO) in a trickle bed reactor (TBR). Modified medium with reduced zinc, no copper, and 5 g/L ascorbic acid that reduced melanin production and increased AAO productivity under growth limited conditions was used. Two air flow rates, 0.11 L/min (0.1 vvm) and 1.1 L/min (1.0 vvm) were tested. More melanin formation and reduced protein productivity were observed with air flow rate of 1.1 L/min. Three random packings were used as support for the fungus inside the TBR column, two of which were hydrophobic and one which was hydrophilic, and three different dilution rates were tested. The use of GEA BCN 030 hydrophobic packing resulted in greater AAO yield and productivity than the other packings. Increasing dilution rates favored melanin formation and citric, lactic and succinic acid accumulation, which decreased AAO yield and productivity.

Process simulation of ethanol production from biomass gasification and syngas fermentation.

The hybrid gasification-syngas fermentation platform can produce more bioethanol utilizing all biomass components compared to the biochemical conversion technology. Syngas fermentation operates at mild temperatures and pressures and avoids using expensive pretreatment processes and enzymes. This study presents a new process simulation model developed with Aspen Plus® of a biorefinery based on a hybrid conversion technology for the production of anhydrous ethanol using 1200tons per day (wb) of switchgrass. The simulation model consists of three modules: gasification, fermentation, and product recovery. The results revealed a potential production of about 36.5million gallons of anhydrous ethanol per year. Sensitivity analyses were also performed to investigate the effects of gasification and fermentation parameters that are keys for the development of an efficient process in terms of energy conservation and ethanol production.

Prevention of melanin formation during aryl alcohol oxidase production under growth-limited conditions using an Aspergillus nidulans cell factory.

An Aspergillus nidulans cell factory was genetically engineered to produce an aryl alcohol oxidase (AAO). The cell factory initiated production of melanin when growth-limited conditions were established using stationary plates and shaken flasks. This phenomenon was more pronounced when the strain was cultured in a trickle bed reactor (TBR). This study investigated different approaches to reduce melanin formation in fungal mycelia and liquid medium in order to increase the enzyme production yield. Removal of copper from the medium recipe reduced melanin formation in agar cultures and increased enzyme activities by 48% in agitated liquid cultures. Copper has been reported as a key element for tyrosinase, an enzyme responsible for melanin production. Ascorbic acid (0.44g/L) stopped melanin accumulation, did not affect growth parameters and resulted in AAO activity that was more than two-fold greater than a control treatment with no ascorbic acid.

High-yield production of aryl alcohol oxidase under limited growth conditions in small-scale systems using a mutant Aspergillus nidulans strain.

Aryl alcohol oxidase (MtGloA) is an enzyme that belongs to the ligninolytic consortium and can play an important role in the bioenergy industry. This study investigated production of an MtGloA client enzyme by a mutant strain of Aspergillus nidulans unable to synthesize its own pyridoxine. Pyridoxine limitation can be used to control cell growth, diverting substrate to protein production. In agitated culture, enzyme production was similar when using media with 1 mg/L and without pyridoxine (26.64 ± 6.14 U/mg mycelia and 26.14 ± 8.39 U/mg mycelia using media with and without pyridoxine, respectively). However, the treatment lacking pyridoxine had to be supplemented with pyridoxine after 156 h of fermentation to sustain continued enzyme production. Use of extremely diluted pyridoxine levels allowed reduced fungal growth while maintaining steady enzyme production. Concentrations of 9 and 13.5 µg/L pyridoxine allowed MtGloA production with a growth rate of only 5% of that observed when using the standard 1 mg/L pyridoxine media.

Butanol production from hydrothermolysis-pretreated switchgrass: Quantification of inhibitors and detoxification of hydrolyzate.

The present study evaluated butanol production from switchgrass based on hydrothermolysis pretreatment. The inhibitors present in the hydrolyzates were measured. Results showed poor butanol production (1g/L) with non-detoxified hydrolyzate. However, adjusting the pH of the non-detoxified hydrolyzate to 6 and adding 4 g/L CaCO3 increased butanol formation to about 6g/L. There was about 1g/L soluble lignin content (SLC), and various levels of furanic and phenolic compounds found in the non-detoxified hydrolyzate. Detoxification of hydrolyzates with activated carbon increased the butanol titer to 11 g/L with a total acetone, butanol and ethanol (ABE) concentration of 17 g/L. These results show the potential of butanol production from hydrothermolysis pretreated switchgrass.

Process development for biological production of butanol from Eastern redcedar.

Eastern redcedar is an invasive softwood species in Oklahoma and across grasslands in the Central Plains of the United States and potential feedstock for butanol production. Butanol has higher energy content than ethanol and can be upgraded to jet and diesel fuels. The objective of this study was to develop a process for production of butanol from redcedar. Results showed that Clostridium acetobutylicum ATCC 824 and Clostridium beijerinckii NCIMB 8052 did not grow in fermentation medium with citrate buffer. However, both strains grew in the medium with acetate buffer, resulting in 3-4g/L greater butanol than without acetate. Detoxification of redcedar hydrolyzate was required to increase butanol concentration from 1 to 13g/L. Hydrolyzate was detoxified by activated carbon to remove inhibitors. Fermentations in detoxified redcedar hydrolyzate reached 13g/L butanol and 19g/L total ABE, comparable to glucose control. This shows the potential for redcedar use in butanol production.

Simultaneous saccharification and fermentation of Eastern redcedar heartwood and sapwood using a novel size reduction technique.

This study investigated the effect of two wood zones (sapwood versus heartwood) and size reduction techniques [Crumbles® (Crumbles® is a registered trademark of Forest Concepts, LLC, Auburn, WA, USA) particles versus ground particles] on wood glucan-to-ethanol yield after acid bisulfite pretreatment and simultaneous saccharification and fermentation (SSF) of Eastern redcedar. SSFs were conducted at 8% solids loading (w/w dry basis) using Accellerase® 1500 at a loading of 46FPU/g glucan and Saccharomyces cerevisiae D5A for ethanol fermentation. The size reduction technique had no effect on ethanol yield. However, sapwood glucan-to-ethanol yields were significantly greater than heartwood yields. The highest wood glucan-to-ethanol yield of 187L/dryMg (95% of theoretical) was achieved with sapwood crumbled particles in 240h. Ground sapwood, crumbled heartwood and ground heartwood achieved ethanol yields of 89%, 81% and 80% of theoretical in 240h, respectively. Preliminary mass balances showed 100% glucan recovery with crumbled sapwood and extensive (72%) delignification.