Industrial biotechnology

Lignocellulosic biomass is the 2nd generation feedstock for biofuel production through fermentation processes. The material has a rigid structure, which needs to be broken down by a pretreatment procedure to expose cellulose for hydrolysis. The hydrolysis products, so called biomass hydrolysates, contain next to the sugar monomers, toxic compounds released and formed during the pretreatment process. These compounds inhibit the growth of the fermenting host(s). To improve the fermentability of biomass hydrolysates, identification of these inhibitory compounds is of great importance.

To efficiently use lignocellulosic biomass hydrolysates as fermentation media for bioethanol production, besides being capable of producing significant amount of ethanol, the fermenting host should also meet the following two requirements: (1) resistant to the inhibitory compounds formed during biomass pretreatment process, (2) capable of utilizing C5 sugars, such as xylose, as carbon source. In our laboratory, a screening was conducted on microorganisms collected from environmental sources for their tolerance to hydrolysate inhibitors.

Profiling and structural elucidation of secondary metabolites produced by the filamentous fungus Penicillium chrysogenum and derived deletion strains were used to identify the various metabolites and enzymatic steps belonging to the roquefortine/meleagrin pathway. Major abundant metabolites of this pathway were identified as histidyltryptophanyldiketopiperazine (HTD), dehydrohistidyltryptophanyldi-ketopipera​zine(DHTD), roquefortine D, roquefortine C, glandicoline A, glandicoline B and meleagrin. Specific genes could be assigned to each enzymatic reaction step.

Pervaporation can be applied in ethanol production from lignocellulosic biomass. Hydrophobic pervaporation, using a commercial PDMS membrane, was employed to concentrate the ethanol produced by fermentation of lignocellulosic hydrolysate. To our knowledge, this is the first report describing this. Pervaporation carried out with three different lignocellulosic fermentation broths reduced the membrane performance by 17-20% as compared to a base case containing only 3 wt.% ethanol in water. The membrane fouling caused by these fermentation broths was irreversible.

Lignocellulosic biomass is the future feedstock for the production of biofuel and bio-based chemicals. The pretreatment-hydrolysis product of biomass, so-called hydrolysate, contains not only fermentable sugars, but also compounds that inhibit its fermentability by microbes. To reduce the toxicity of hydrolysates as fermentation media, knowledge of the identity of inhibitors and their dynamics in hydrolysates need to be obtained. In the past decade, various studies have applied targeted metabolomics approaches to examine the composition of biomass hydrolysates.

The carbohydrate composition of lignocellulosic biomass hydrolysates is highly complex. High performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD), a widely used method for carbohydrate analysis, provides limited chemical information on the detected peaks. To improve the detection and increase the chemical information of the carbohydrates, HPAEC was coupled with mass spectrometry (MS).

To compare the composition and performance of various lignocellulosic biomass hydrolysates as fermentation media, 8 hydrolysates were generated from a grass-like and a wood biomass. The hydrolysate preparation methods used were 1) dilute acid, 2) mild alkaline, 3) alkaline/peracetic acid, and 4) concentrated acid. These hydrolysates were fermented at 30°C, pH 5.0 using Saccharomyces cerevisiae CEN.PK113-7D as model strain. The growth in different hydrolysates varied in the aspects of lag-phase, growth rate, glucose consumption rate and ethanol production rate.