Prachand Shrestha

Sequential saccharification of corn fiber and ethanol production by the brown rot fungus Gloeophyllum trabeum

Degradation of lignocellulosic biomass to sugars through a purely biological process is a key to sustainable biofuel production. Hydrolysis of the corn wet-milling co-product-corn fiber-to simple sugars by the brown rot fungus Gloeophyllum trabeum was studied in suspended-culture and solid-state fermentations. Suspended-culture experiments were not effective in producing harvestable sugars from the corn fiber. The fungus consumed sugars released by fungal extracellular enzymes. Solid-state fermentation demonstrated up to 40% fiber degradation within 9days. Enzyme activity assays on solid-state fermentation filtrates confirmed the involvement of starch- and cellulose-degrading enzymes. To reduce fungal consumption of sugars and to accelerate enzyme activity, 2- and 3-d solid-state fermentation biomasses (fiber and fungus) were submerged in buffer and incubated at 37 degrees C without shaking. This anaerobic incubation converted up to almost 11% of the corn fiber into harvestable reducing sugars. Sugars released by G. trabeum were fermented to a maximum yield of 3.3g ethanol/100g fiber. This is the first report, to our knowledge, of G. trabeum fermenting sugar to ethanol. The addition of Saccharomyces cerevisiae as a co-culture led to more rapid fermentation to a maximum yield of 4.0g ethanol/100g fiber. The findings demonstrate the potential for this simple fungal process, requiring no pretreatment of the corn fiber, to produce more ethanol by hydrolyzing and fermenting carbohydrates in this lignocellulosic co-product.

Solid-Substrate Fermentation of Corn Fiber by Phanerochaete chrysosporium and Subsequent Fermentation of Hydrolysate into Ethanol

The goal of this study was to develop a fungal process for ethanol production from corn fiber. Laboratory-scale solid-substrate fermentation was performed using the white-rot fungus Phanerochaete chrysosporium in 1 L polypropylene bottles as reactors via incubation at 37 degrees C for up to 3 days. Extracellular enzymes produced in situ by P. chrysosporium degraded lignin and enhanced saccharification of polysaccharides in corn fiber. The percentage biomass weight loss and Klason lignin reduction were 34 and 41%, respectively. Anaerobic incubation at 37 degrees C following 2 day incubation reduced the fungal sugar consumption and enhanced the in situ cellulolytic enzyme activities. Two days of aerobic solid-substrate fermentation of corn fiber with P. chrysosporium, followed by anaerobic static submerged-culture fermentation resulted in 1.7 g of ethanol/100 g of corn fiber in 6 days, whereas yeast ( Saccharomyces cerevisiae) cocultured with P. chrysosporium demonstrated enhanced ethanol production of 3 g of ethanol/100 g of corn fiber. Specific enzyme activity assays suggested starch and hemi/cellulose contribution of fermentable sugar.

Ultrasound improved ethanol fermentation from cassava chips in cassava-based ethanol plants.

The effects of ultrasound and heat pretreatments on ethanol yields from cassava chips were investigated. Cassava slurries were sonicated for 10 and 30 s at the amplitudes of 80, 160, and 320 microm(pp) (peak to peak amplitude in microm) corresponding to low, medium, and high power levels, respectively. The sonicated and non-sonicated (control) samples were then subjected to simultaneous liquefaction-saccharification and ethanol fermentation. Cassava starch-to-ethanol conversion efficiencies showed that higher ethanol yields were directly related to sonication times, but not to power levels. Significantly higher ethanol yields were observed only for sonicated samples at the high power level. The ethanol yield from the sonicated sample was 2.7-fold higher than yield from the control sample. Starch-to-ethanol conversion rates from sonicated cassava chips were also significantly higher; the fermentation time could be reduced by nearly 24 h for sonicated samples to achieve the same ethanol yield as control samples. Thus, ultrasound pretreatment enhanced both the overall ethanol yield and fermentation rate. When compared to heat-treated samples, the sonicated samples produced nearly 29% more ethanol yield. Combined heat and ultrasound treatment had no significant effect on overall ethanol yields from cassava chips. Ultrasound is also preferable to heat pretreatment because of lower energy requirements, as indicated by energy balances. Integration of ultrasound application in cassava-based ethanol plants can significantly improve ethanol yields and reduce the overall production costs.

Systematic search for cultivatable fungi that best deconstruct cell walls of Miscanthus and sugarcane in the field

ABSTRACT The goals of our project were to document the diversity and distributions of cultivable fungi associated with decaying Miscanthus and sugarcane plants in nature and to further assess biodegradation of host plant cell walls by these fungi in pure cultures. Late in 2008 and early in 2009 we collected decaying Miscanthus and Saccharum from 8 sites in Illinois and 11 sites in Louisiana, respectively. To recover fungi that truly decay plants and to recover slow-growing fungi, we washed the plant material repeatedly to remove spores and cultivated fungi from plant fragments small enough to harbor at most one mycelium. We randomly selected 950 fungal colonies out of 4,560 microwell colonies and used molecular identification to discover that the most frequently recovered fungal species resided in Hypocreales (Sordariomycetes), Pleosporales (Dothideomycetes), and Chaetothryiales (Eurotiomycetes) and that only a few weedy species were recovered. We were particularly interested in Pleosporales and Chaetothyriales, groups that have not been mined for plant decay fungi. To confirm that we had truly recovered fungi that deconstruct plant cell walls, we assayed the capacity of the fungi to consume whole, alkali-pretreated, ground Miscanthus. Solid substrate cultures of the nine most commonly encountered Ascomycota resulted in Miscanthus weight loss of 8 to 13% over 4 weeks. This is the first systematic, high-throughput, isolation and biodegradation assessment of fungi isolated from decaying bioenergy grasses.

Ethanol production via in situ fungal saccharification and fermentation of mild alkali and steam pretreated corn fiber.

The effect of mild alkali and steam pretreatments on fungal saccharification and sequential simultaneous-saccharification and fermentation (SSF) of corn fiber to ethanol was studied. The corn fiber was pretreated with: (i) 2% NaOH (w/w) at 30 degrees C for 2h and (ii) steaming at 100 degrees C for 2h. Ethanol yields were 2.6g, 2.9g and 5.5g ethanol/100g of corn fiber, respectively, for Phanerochaete chrysosporium, Gloeophyllum trabeum and Trichoderma reesei saccharification and sequential SSFs. SSF with commercial cellulase enzyme - Spezyme-CP had 7.7g ethanol/100g corn fiber. Mild alkali pretreatment resulted in higher glucose yields following fungal saccharification of corn fiber. However, the ethanol yields were comparatively similar for untreated and mild alkali pretreated corn fiber. Solid-substrate fermentation of corn fiber with fungi can be improved to either eliminate or reduce the dosage of commercial cellulase enzymes during SSF.

Enzyme production by wood-rot and soft-rot fungi cultivated on corn fiber followed by simultaneous saccharification and fermentation.

This research aims at developing a biorefinery platform to convert lignocellulosic corn fiber into fermentable sugars at a moderate temperature (37 °C) with minimal use of chemicals. White-rot (Phanerochaete chrysosporium), brown-rot (Gloeophyllum trabeum), and soft-rot (Trichoderma reesei) fungi were used for in situ enzyme production to hydrolyze cellulosic and hemicellulosic components of corn fiber into fermentable sugars. Solid-substrate fermentation of corn fiber by either white- or brown-rot fungi followed by simultaneous saccharification and fermentation (SSF) with coculture of Saccharomyces cerevisiae has shown a possibility of enhancing wood rot saccharification of corn fiber for ethanol fermentation. The laboratory-scale fungal saccharification and fermentation process incorporated in situ cellulolytic enzyme induction, which enhanced overall enzymatic hydrolysis of hemi/cellulose components of corn fiber into simple sugars (mono-, di-, and trisaccharides). The yeast fermentation of the hydrolyzate yielded 7.8, 8.6, and 4.9 g ethanol per 100 g corn fiber when saccharified with the white-, brown-, and soft-rot fungi, respectively. The highest ethanol yield (8.6 g ethanol per 100 g initial corn fiber) is equivalent to 35% of the theoretical ethanol yield from starch and cellulose in corn fiber. This research has significant commercial potential to increase net ethanol production per bushel of corn through the utilization of corn fiber. There is also a great research opportunity to evaluate the remaining biomass residue (enriched with fungal protein) as animal feed.