Category Archives: Biotechnology

Background:
Lignin is embedded in the plant cell wall matrix, and impedes the enzymatic saccharification of lignocellulosic feedstocks. To investigate whether enzymatic digestibility of cell wall materials can be improved by altering the relative abundance of the two major lignin monomers, guaiacyl (G) and syringyl (S) subunits, we compared the degradability of cell wall material from wild-type Arabidopsis thaliana with a mutant line and a genetically modified line, the lignins of which are enriched in G and S subunits, respectively.
Results:
Arabidopsis tissue containing G- and S-rich lignins had the same saccharification performance as the wild type when subjected to enzyme hydrolysis without pretreatment. After a 24-hour incubation period, less than 30% of the total glucan was hydrolyzed. By contrast, when liquid hot water (LHW) pretreatment was included before enzyme hydrolysis, the S-lignin-rich tissue gave a much higher glucose yield than either the wild-type or G-lignin-rich tissue. Applying a hot-water washing step after the pretreatment did not lead to a further increase in final glucose yield, but the initial hydrolytic rate was doubled.
Conclusions:
Our analyses using the model plant A. thaliana revealed that lignin composition affects the enzymatic digestibility of LHW pretreated plant material. Pretreatment is more effective in enhancing the saccharification of A. thaliana cell walls that contain S-rich lignin. Increasing lignin S monomer content through genetic engineering may be a promising approach to increase the efficiency and reduce the cost of biomass to biofuel conversion.

Background:
Degradation of the toxic compounds generated in the harsh pretreatment of lignocellulose is an inevitable step in reducing the toxin level for conducting practical enzymatic hydrolysis and ethanol fermentation processes. Various detoxification methods have been tried and many negative outcomes were found using these methods, such as the massive freshwater usage and wastewater generation, loss of the fine lignocellulose particles and fermentative sugars and incomplete removal of inhibitors. An alternate method, biodetoxification, which degrades the toxins as part of their normal metabolism, was considered a promising option for the removal of toxins without causing the above problems.
Results:
A kerosene fungus strain, Amorphotheca resinae ZN1, was isolated from the microbial community growing on the pretreated corn stover material. The degradation of the toxins as well as the lignocelluloses-derived sugars was characterized in different ways, and the results show that A. resinae ZN1 utilized each of these toxins and sugars as the sole carbon sources efficiently and grew quickly on the toxins. It was found that the solid-state culture of A. resinae ZN1 on various pretreated lignocellulose feedstocks such as corn stover, wheat straw, rice straw, cotton stalk and rape straw degraded all kinds of toxins quickly and efficiently. The consequent simultaneous saccharification and ethanol fermentation was performed at the 30% (wt/wt) solid loading of the detoxified lignocellulosic feedstocks without a sterilization step, and the ethanol titer in the fermentation broth reached above 40 g/L using food crop residues as feedstocks.
Conclusions:
The advantages of the present biodetoxification by A. resinae ZN1 over the known detoxification methods include zero energy input, zero wastewater generation, complete toxin degradation, processing on solid pretreated material, no need for sterilization and a wide lignocellulose feedstock spectrum. These advantages make it possible for industrial applications with fast and efficient biodetoxification to remove toxins generated during intensive lignocellulose pretreatment.

Background:
Cell wall resistance represents the main barrier for the production of second generation biofuels. The deconstruction of lignocellulose can provide sugars for the production of fuels or other industrial products through fermentation. Understanding the biochemical basis of the recalcitrance of cell walls to digestion will allow development of more effective and cost efficient ways to produce sugars from biomass. One approach is to identify plant genes that play a role in biomass recalcitrance, using association genetics. Such an approach requires a robust and reliable high throughput (HT) assay for biomass digestibility, which can be used to screen the large numbers of samples involved in such studies.
Results:
We developed a HT saccharification assay based on a robotic platform that can carry out in a 96-well plate format the enzymatic digestion and quantification of the released sugars. The handling of the biomass powder for weighing and formatting into 96 wells is performed by a robotic station, where the plant material is ground, delivered to the desired well in the plates and weighed with a precision of 0.1 mg. Once the plates are loaded, an automated liquid handling platform delivers an optional mild pretreatment (< 100°C) followed by enzymatic hydrolysis of the biomass. Aliquots from the hydrolysis are then analyzed for the release of reducing sugar equivalents. The same platform can be used for the comparative evaluation of different enzymes and enzyme cocktails. The sensitivity and reliability of the platform was evaluated by measuring the saccharification of stems from lignin modified tobacco plants, and the results of automated and manual analyses compared.
Conclusions:
The automated assay systems are sensitive, robust and reliable. The system can reliably detect differences in the saccharification of plant tissues, and is able to process large number of samples with a minimum amount of human intervention. The automated system uncovered significant increases in the digestibility of certain lignin modified lines in a manner compatible with known effects of lignin modification on cell wall properties. We conclude that this automated assay platform is of sufficient sensitivity and reliability to undertake the screening of the large populations of plants necessary for mutant identification and genetic association studies.

Background:
Enzymes for plant cell wall deconstruction are a major cost in the production of ethanol from lignocellulosic biomass. The goal of this research was to develop optimized synthetic mixtures of enzymes for multiple pretreatment/substrate combinations using our high-throughput biomass digestion platform, GENPLAT, which combines robotic liquid handling, statistical experimental design, and automated Glc and Xyl assays. Proportions of six core fungal enzymes (CBH1, CBH2, EG1, beta-glucosidase, a GH10 endo-beta-1,4-xylanase, and beta-xylosidase) were optimized at a fixed enzyme loading of 15 mg/g glucan for release of Glc and Xyl from all combinations of five biomass feedstocks (corn stover, switchgrass, Miscanthus, dried distillers' grains plus solubles [DDGS], and poplar) subjected to three alkaline pretreatments (AFEX, dilute base [0.25% NaOH], and alkaline peroxide [AP]). A 16-component mixture, comprising the core set plus 10 accessory enzymes, was optimized for three pretreatment/substrate combinations. Results were compared to the performance of two commercial enzymes (Accellerase 1000 and Spezyme CP) at the same protein loadings.
Results:
When analyzed with GENPLAT, corn stover gave the highest yields of Glc with commercial enzymes and with the core set with all pretreatments, whereas corn stover, switchgrass, and Miscanthus gave comparable Xyl yields. With commercial enzymes and with the core set, yields of Glc and Xyl were highest for grass stovers pretreated by AP compared to AFEX or dilute base. Corn stover, switchgrass, and DDGS pretreated with AFEX and digested with the core set required a higher proportion of endo-beta1,4-xylanase (EX3) and a lower proportion of endo-beta-1,4-glucanase (EG1) compared to the same materials pretreated with dilute base or AP. An optimized enzyme mixture containing 16 components (by addition of alpha-glucuronidase, a GH11 endo-xylanase [EX2], Cel5A, Cel61A, Cip1, Cip2, beta-mannanase, amyloglucosidase, alpha-arabinosidase, and Cel12A to the core set) was determined for AFEX-pretreated corn stover, DDGS, and AP-pretreated corn stover. The optimized mixture for AP-corn stover contained more exo-beta-1,4-glucanase (i.e., the sum of CBH1 + CBH2) and less endo-beta-1,4-glucanase (EG1 + Cel5A) than the optimal mixture for AFEX-corn stover. Amyloglucosidase and beta-mannanase were the two most important enzymes for release of Glc from DDGS but were not required (i.e., 0% optimum) for corn stover subjected to AP or AFEX. As a function of enzyme loading over the range 0 to 30 mg/g glucan, Glc release from AP-corn stover reached a plateau of 60-70% Glc yield at a lower enzyme loading (5-10 mg/g glucan) than AFEX-corn stover. Accellerase 1000 was superior to Spezyme CP, the core set, or the 16-component mixture for Glc yield at 6 h, but the 16-component set was as effective as the commercial enzyme mixtures at 48 h.
Conclusion:
The results in this paper demonstrate that GENPLAT can be used to rapidly produce enzyme cocktails for specific pretreatment/biomass combinations. Pretreatment conditions and feedstock source both influence the Glc and Xyl yields as well as optimal enzyme proportions. It is predicted that it will be possible to improve synthetic enzyme mixtures further by the addition of additional accessory enzymes.

Background:
Overexpression of the PGM2 gene encoding phosphoglucomutase (Pgm2p) has been shown to improve galactose utilization both under aerobic and under anaerobic conditions. Similarly, xylose utilization has been improved by overexpression of genes encoding xylulokinase (XK), enzymes from the non-oxidative pentose phosphate pathway (non-ox PPP) and deletion of the endogenous aldose reductase GRE3 gene in engineered Saccharomyces cerevisiae strains carrying either fungal or bacterial xylose pathways. In the present study, we investigated how the combination of these traits affect xylose and galactose utilization in the presence or absence of glucose in S. cerevisiae strains engineered with the xylose reductase (XR)-xylitol dehydrogenase (XDH) pathway.
Results:
In the absence of PGM2 overexpression, the combined overexpression of XK, the non-ox PPP and deletion of the GRE3 gene significantly delayed aerobic growth on galactose, whereas no difference was observed between the control strain and the xylose-engineered strain when the PGM2 gene was overexpressed. Under anaerobic conditions, the overexpression of the PGM2 gene increased the ethanol yield and the xylose consumption rate in medium containing xylose as the only carbon source. The possibility of Pgm2p acting as a xylose isomerase (XI) could be excluded by measuring the XI activity in both strains. The additional copy of the PGM2 gene also resulted in a shorter fermentation time during the co-consumption of galactose and xylose. However, the effect was lost upon addition of glucose to the growth medium.
Conclusions:
PGM2 overexpression was shown to benefit xylose and galactose fermentation, alone and in combination. In contrast, galactose fermentation was impaired in the engineered xylose-utilizing strain harbouring extra copies of the non-ox PPP genes and a deletion of the GRE3 gene, unless PGM2 was overexpressed. These cross-reactions are of particular relevance for the fermentation of mixed sugars from lignocellulosic feedstock.

Background:
Replacing the energy-intensive evaporation of stillage by anaerobic digestion is one way of decreasing the energy demand of the lignocellulosic biomass to the ethanol process. The biogas can be upgraded and sold as transportation fuel, injected directly into the gas grid or be incinerated on-site for combined heat and power generation. A techno-economic evaluation of the spruce-to-ethanol process, based on SO2-catalysed steam pretreatment followed by simultaneous saccharification and fermentation, has been performed using the commercial flow-sheeting program Aspen Plus™. Various process configurations of anaerobic digestion of the stillage, with different combinations of co-products, have been evaluated in terms of energy efficiency and ethanol production cost versus the reference case of evaporation.
Results:
Anaerobic digestion of the stillage showed a significantly higher overall energy efficiency (87-92%), based on the lower heating values, than the reference case (81%). Although the amount of ethanol produced was the same in all scenarios, the production cost varied between 4.00 and 5.27 Swedish kronor per litre (0.38-0.50 euro/L), including the reference case.
Conclusions:
Higher energy efficiency options did not necessarily result in lower ethanol production costs. Anaerobic digestion of the stillage with biogas upgrading was demonstrated to be a favourable option for both energy efficiency and ethanol production cost. The difference in the production cost of ethanol between using the whole stillage or only the liquid fraction in anaerobic digestion was negligible for the combination of co-products including upgraded biogas, electricity and district heat.