Ming W. Lau

Designer synthetic media for studying microbial-catalyzed biofuel production

BackgroundThe fermentation inhibition of yeast or bacteria by lignocellulose-derived degradation products, during hexose/pentose co-fermentation, is a major bottleneck for cost-effective lignocellulosic biorefineries. To engineer microbial strains for improved performance, it is critical to understand the mechanisms of inhibition that affect fermentative organisms in the presence of major components of a lignocellulosic hydrolysate. The development of a synthetic lignocellulosic hydrolysate (SH) media with a composition similar to the actual biomass hydrolysate will be an important advancement to facilitate these studies. In this work, we characterized the nutrients and plant-derived decomposition products present in AFEX™ pretreated corn stover hydrolysate (ACH). The SH was formulated based on the ACH composition and was further used to evaluate the inhibitory effects of various families of decomposition products during Saccharomyces cerevisiae 424A (LNH-ST) fermentation.ResultsThe ACH contained high levels of nitrogenous compounds, notably amides, pyrazines, and imidazoles. In contrast, a relatively low content of furans and aromatic and aliphatic acids were found in the ACH. Though most of the families of decomposition products were inhibitory to xylose fermentation, due to their abundance, the nitrogenous compounds showed the most inhibition. From these compounds, amides (products of the ammonolysis reaction) contributed the most to the reduction of the fermentation performance. However, this result is associated to a concentration effect, as the corresponding carboxylic acids (products of hydrolysis) promoted greater inhibition when present at the same molar concentration as the amides.Due to its complexity, the formulated SH did not perfectly match the fermentation profile of the actual hydrolysate, especially the growth curve. However, the SH formulation was effective for studying the inhibitory effect of various compounds on yeast fermentation.ConclusionsThe formulation of SHs is an important advancement for future multi-omics studies and for better understanding the mechanisms of fermentation inhibition in lignocellulosic hydrolysates. The SH formulated in this work was instrumental for defining the most important inhibitors in the ACH. Major AFEX decomposition products are less inhibitory to yeast fermentation than the products of dilute acid or steam explosion pretreatments; thus, ACH is readily fermentable by yeast without any detoxification.

Cellulosic ethanol production from AFEX-treated corn stover using Saccharomyces cerevisiae 424A(LNH-ST)

Current technology using corn stover (CS) as feedstock, Ammonia Fiber Expansion (AFEX) as the pretreatment technology, and Saccharomyces cerevisiae 424A(LNH-ST) as the ethanologenic strain in Separate Hydrolysis and Fermentation was able to achieve 191.5 g EtOH/kg untreated CS, at an ethanol concentration of 40.0 g/L (5.1 vol/vol%) without washing of pretreated biomass, detoxification, or nutrient supplementation. Enzymatic hydrolysis at high solids loading was identified as the primary bottleneck affecting overall ethanol yield and titer. Degradation compounds in AFEX-pretreated biomass were shown to increase metabolic yield and specific ethanol production while decreasing the cell biomass generation. Nutrients inherently present in CS and those resulting from biomass processing are sufficient to support microbial growth during fermentation. This platform offers the potential to improve the economics of cellulosic ethanol production by reducing the costs associated with raw materials, process water, and capital equipment.

Comparing the fermentation performance of Escherichia coli KO11, Saccharomyces cerevisiae 424A(LNH-ST) and Zymomonas mobilis AX101 for cellulosic ethanol production

BackgroundFermentations using Escherichia coli KO11, Saccharomyces cerevisiae 424A(LNH-ST), and Zymomonas mobilis AX101 are compared side-by-side on corn steep liquor (CSL) media and the water extract and enzymatic hydrolysate from ammonia fiber expansion (AFEX)-pretreated corn stover.ResultsThe three ethanologens are able produce ethanol from a CSL-supplemented co-fermentation at a metabolic yield, final concentration and rate greater than 0.42 g/g consumed sugars, 40 g/L and 0.7 g/L/h (0-48 h), respectively. Xylose-only fermentation of the tested ethanologenic bacteria are five to eight times faster than 424A(LNH-ST) in the CSL fermentation.All tested strains grow and co-ferment sugars at 15% w/v solids loading equivalent of ammonia fiber explosion (AFEX)-pretreated corn stover water extract. However, both KO11 and 424A(LNH-ST) exhibit higher growth robustness than AX101. In 18% w/w solids loading lignocellulosic hydrolysate from AFEX pretreatment, complete glucose fermentations can be achieved at a rate greater than 0.77 g/L/h. In contrast to results from fermentation in CSL, S. cerevisiae 424A(LNH-ST) consumed xylose at the greatest extent and rate in the hydrolysate compared to the bacteria tested.ConclusionsOur results confirm that glucose fermentations among the tested strains are effective even at high solids loading (18% by weight). However, xylose consumption in the lignocellulosic hydrolysate is the major bottleneck affecting overall yield, titer or rate of the process. In comparison, Saccharomyces cerevisiae 424A(LNH-ST) is the most relevant strains for industrial production for its ability to ferment both glucose and xylose from undetoxified and unsupplemented hydrolysate from AFEX-pretreated corn stover at high yield.

Two-step SSCF to convert AFEX-treated switchgrass to ethanol using commercial enzymes and Saccharomyces cerevisiae 424A(LNH-ST)

It is well known that simultaneous saccharification and co-fermentation (SSCF) reduces cellulosic ethanol production cost compared to separate hydrolysis and fermentation (SHF). However, the traditional SSCF process of converting Ammonia Fiber Expansion (AFEX) pretreated switchgrass to ethanol using both commercial enzymes and Saccharomyces cerevisiae 424A(LNH-ST) gave reduced ethanol yield due to lower xylose consumption. To overcome this problem we have developed a two-step SSCF process, in which xylan was hydrolyzed and fermented first followed by the hydrolysis and fermentation of glucan. Important parameters, such as temperature, cellulases loading during xylan hydrolysis and fermentation, initial OD(600) for inoculation of S. cerevisiae 424A(LNH-ST), and pH, were studied for best performance. Compared with traditional SSCF, the two-step SSCF showed higher xylose consumption and higher ethanol yield. The sugar conversion was also enhanced from 70% by enzymatic hydrolysis to 82% by two-step SSCF. One important finding is that the residue from enzymatic hydrolysis plays a significant role in reducing xylose consumption and ethanol metabolic yield during SSCF.

The impacts of pretreatment on the fermentability of pretreated lignocellulosic biomass: a comparative evaluation between ammonia fiber expansion and dilute acid pretreatment

BackgroundPretreatment chemistry is of central importance due to its impacts on cellulosic biomass processing and biofuels conversion. Ammonia fiber expansion (AFEX) and dilute acid are two promising pretreatments using alkaline and acidic pH that have distinctive differences in pretreatment chemistries.ResultsComparative evaluation on these two pretreatments reveal that (i) AFEX-pretreated corn stover is significantly more fermentable with respect to cell growth and sugar consumption, (ii) both pretreatments can achieve more than 80% of total sugar yield in the enzymatic hydrolysis of washed pretreated solids, and (iii) while AFEX completely preserves plant carbohydrates, dilute acid pretreatment at 5% solids loading degrades 13% of xylose to byproducts.ConclusionThe selection of pretreatment will determine the biomass-processing configuration, requirements for hydrolysate conditioning (if any) and fermentation strategy. Through dilute acid pretreatment, the need for hemicellulase in biomass processing is negligible. AFEX-centered cellulosic technology can alleviate fermentation costs through reducing inoculum size and practically eliminating nutrient costs during bioconversion. However, AFEX requires supplemental xylanases as well as cellulase activity. As for long-term sustainability, AFEX has greater potential to diversify products from a cellulosic biorefinery due to lower levels of inhibitor generation and lignin loss.

Simultaneous saccharification and co-fermentation (SSCF) of AFEXTM pretreated corn stover for ethanol production using commercial enzymes and Saccharomyces cerevisiae 424A(LNH-ST).

Xylose consumption by Saccharomyces cerevisiae 424A(LNH-ST) during simultaneous saccharification and co-fermentation (SSCF) of AFEX(TM) pretreated switchgrass was inhibited by unhydrolyzed solids. Such inhibitory effects were not found in unhydrolyzed solids from AFEX(TM) pretreated corn stover (AFEX(TM)-CS). However, the xylose consumption was still unsatisfactory during 6h pre-hydrolysis SSCF. By extending the pre-hydrolysis time to 24h or longer, the xylose consumption was improved significantly. In order to better understand the reasons for such improvement, the hydrolysate slurries after 6h pre-hydrolysis and 24h pre-hydrolysis were studied and compared. We found that the glucose concentration after pre-hydrolysis was the critical factor that determined cell viability and hence xylose consumption during SSCF. Low temperature (30°C) and ethanol inhibition were shown to be the factors limiting hydrolysis rate and hence productivity during SSCF.

An integrated paradigm for cellulosic biorefineries: utilization of lignocellulosic biomass as self-sufficient feedstocks for fuel, food precursors and saccharolytic enzyme production

Simultaneously achieving economic, environmental and social sustainability is a major challenge for the emerging renewable fuel industry. We approach this problem by demonstrating a cellulosic biorefinery paradigm which produces ethanol and food precursors using lignocellulosic biomass as the exclusive source for carbohydrates and minerals. Enzymatic hydrolysate from Ammonia Fiber Expansion (AFEX)-pretreated corn stover at 18% w/w solids loading was found to be nutrient-rich. This hydrolysate was fermented completely within 48 h in two stages to produce ethanol and native yeast cells. An in-house saccharolytic enzyme production using AFEX-pretreated corn stover as carbohydrate source greatly reduces the dependence on commercial enzymes. The inducer mixture is 2.5–7 times more potent than lactose, a common enzyme inducer. Economic analysis indicates that the proposed paradigm is substantially more cost-effective relative to the 2005 NREL model. This improvement is largely attributed to the native yeast cells co-production and the reduction of enzyme cost through the in-house production.

Effect of primary degradation-reaction products from Ammonia Fiber Expansion (AFEX)-treated corn stover on the growth and fermentation of Escherichia coli KO11.

The primary degradation-reaction products (DRP) identified in Ammonia Fiber Expansion (AFEX)-pretreated corn stover are acetate, lactate, 4-hydroxybenzaldehyde (4HBD) and acetamide. The effects of these products at a broad concentration range were tested on Escherichia coli KO11, a strain engineered for cellulosic ethanol production. Fermentations using glucose or xylose as the sole carbohydrate source and a sugar mixture of glucose and xylose were conducted to determine how these products and sugar selection affected fermentation performance. Co-fermentation of the sugar mixture exhibited the lowest overall ethanol productivity compared to single-sugar fermentations and was more susceptible to inhibition. Metabolic ethanol yield increased with the increasing initial concentration of acetate. Although these degradation-reaction products (with exception of acetamide) are generally perceived to be inhibitory, organic acids and 4-hydroxybenzaldehyde at low levels stimulated fermentation. Adaptation of cells to these products prior to fermentation increased overall fermentation rate.

Comparative lipidomic profiling of xylose-metabolizing S. cerevisiae and its parental strain in different media reveals correlations between membrane lipids and fermentation capacity

Phospholipids (PLs) serve as the foundation for structure and function in most cell membranes. In order to reveal the correlations between PLs composition and fermentation performance of cells, a comparative lipidomics study was carried out using a recombinant xylose fermenting yeast strain Saccharomyces cerevisiae 424A(LNH‐ST) and its parental strain 4124. Profiling of yeast lipids was performed using ultra performance liquid chromatography (UPLC)‐MS/MS, leading to identification of 123 PL species. PL compositions were determined for both strains grown in rich medium (yeast extract peptone), limited medium (yeast nitrogen base), and ammonia fiber expansion pretreated corn stover hydrolysate. Principal component analysis of lipidomic data revealed that the PL profile for both strains varied significantly depending upon cultivating media composition. Further analysis of different classes of PLs revealed that the phosphatidylinositol/phosphatidylserine (PI/PS) ratio was closely related to cell growth rates. Both strains possessed higher phosphatidylcholine (PC) levels at an expense of phosphatidylethanolamine (PE) levels when entering stationary phase and the PC/PE ratios showed consistency with glucose utilization rates. Interestingly, PI synthesis lagged behind when available nutrients were limited, and PI levels were closely correlated with xylose metabolism. Biotechnol. Bioeng. 2011; 108:12–21.