Mingjie Jin

Evaluation of ammonia fibre expansion (AFEX) pretreatment for enzymatic hydrolysis of switchgrass harvested in different seasons and locations.

BackgroundWhen producing biofuels from dedicated feedstock, agronomic factors such as harvest time and location can impact the downstream production. Thus, this paper studies the effectiveness of ammonia fibre expansion (AFEX) pretreatment on two harvest times (July and October) and ecotypes/locations (Cave-in-Rock (CIR) harvested in Michigan and Alamo harvested in Alabama) for switchgrass (Panicum virgatum).ResultsBoth harvest date and ecotype/location determine the pretreatment conditions that produce maximum sugar yields. There was a high degree of correlation between glucose and xylose released regardless of the harvest, pretreatment conditions, or enzyme formulation. Enzyme formulation that produced maximum sugar yields was the same across all harvests except for the CIR October harvest. The least mature sample, the July harvest of CIR switchgrass, released the most sugars (520 g/kg biomass) during enzymatic hydrolysis while requiring the least severe pretreatment conditions. In contrast, the most mature harvest released the least amount of sugars (410 g/kg biomass). All hydrolysates were highly fermentable, although xylose utilisation in the July CIR hydrolysate was poor.ConclusionsEach harvest type and location responded differently to AFEX pretreatment, although all harvests successfully produced fermentable sugars. Thus, it is necessary to consider an integrated approach between agricultural production and biochemical processing in order to insure optimal productivity.

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.

Alkali‐based AFEX pretreatment for the conversion of sugarcane bagasse and cane leaf residues to ethanol

Sugarcane is one of the major agricultural crops cultivated in tropical climate regions of the world. Each tonne of raw cane production is associated with the generation of 130 kg dry weight of bagasse after juice extraction and 250 kg dry weight of cane leaf residue postharvest. The annual world production of sugarcane is ∼1.6 billion tones, generating 279 MMT tones of biomass residues (bagasse and cane leaf matter) that would be available for cellulosic ethanol production. Here, we investigated the production of cellulosic ethanol from sugar cane bagasse and sugar cane leaf residue using an alkaline pretreatment: ammonia fiber expansion (AFEX). The AFEX pretreatment improved the accessibility of cellulose and hemicelluloses to enzymes during hydrolysis by breaking down the ester linkages and other lignin carbohydrate complex (LCC) bonds and the sugar produced by this process is found to be highly fermentable. The maximum glucan conversion of AFEX pretreated bagasse and cane leaf residue by cellulases was ∼85%. Supplementation with hemicellulases during enzymatic hydrolysis improved the xylan conversion up to 95–98%. Xylanase supplementation also contributed to a marginal improvement in the glucan conversion. AFEX‐treated cane leaf residue was found to have a greater enzymatic digestibility compared to AFEX‐treated bagasse. Co‐fermentation of glucose and xylose, produced from high solid loading (6% glucan) hydrolysis of AFEX‐treated bagasse and cane leaf residue, using the recombinant Saccharomyces cerevisiae (424A LNH‐ST) produced 34–36 g/L of ethanol with 92% theoretical yield. These results demonstrate that AFEX pretreatment is a viable process for conversion of bagasse and cane leaf residue into cellulosic ethanol. Biotechnol. Bioeng. 2010;107: 441–450.

Comparative genomics of xylose-fermenting fungi for enhanced biofuel production

Cellulosic biomass is an abundant and underused substrate for biofuel production. The inability of many microbes to metabolize the pentose sugars abundant within hemicellulose creates specific challenges for microbial biofuel production from cellulosic material. Although engineered strains of Saccharomyces cerevisiae can use the pentose xylose, the fermentative capacity pales in comparison with glucose, limiting the economic feasibility of industrial fermentations. To better understand xylose utilization for subsequent microbial engineering, we sequenced the genomes of two xylose-fermenting, beetle-associated fungi, Spathaspora passalidarum and Candida tenuis. To identify genes involved in xylose metabolism, we applied a comparative genomic approach across 14 Ascomycete genomes, mapping phenotypes and genotypes onto the fungal phylogeny, and measured genomic expression across five Hemiascomycete species with different xylose-consumption phenotypes. This approach implicated many genes and processes involved in xylose assimilation. Several of these genes significantly improved xylose utilization when engineered into S. cerevisiae, demonstrating the power of comparative methods in rapidly identifying genes for biomass conversion while reflecting on fungal ecology.

Microbial lipid-based lignocellulosic biorefinery: feasibility and challenges

Although single-cell oil (SCO) has been studied for decades, lipid production from lignocellulosic biomass has received substantial attention only in recent years as biofuel research moves toward producing drop-in fuels. This review gives an overview of the feasibility and challenges that exist in realizing microbial lipid production from lignocellulosic biomass in a biorefinery. The aspects covered here include biorefinery technologies, the microbial oil market, oleaginous microbes, lipid accumulation metabolism, strain development, process configurations, lignocellulosic lipid production, technical hurdles, lipid recovery, and technoeconomics. The lignocellulosic SCO-based biorefinery will be feasible only if a combination of low- and high-value lipids are coproduced, while lignin and protein are upgraded to high-value products.

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.

Consolidated bioprocessing (CBP) performance of Clostridium phytofermentans on AFEX‐treated corn stover for ethanol production

Consolidated bioprocessing (CBP) is believed to be a potentially cost‐efficient and commercially viable way to produce cellulosic biofuels. In this study, we have evaluated the performance of the CBP organism Clostridium phytofermentans (ATCC 700394) on AFEX‐treated corn stover (AFEX‐CS). Fermentation conditions including temperature, inoculation size, nutrients, and initial pH were investigated. At optimal conditions with 0.5% (w/w) glucan loading of AFEX‐CS, C. phytofermentans hydrolyzed 76% of glucan and 88.6% of xylan in 10 days. These values reached 87% and 102% of those obtained by simultaneous saccharification and co‐fermentation (SSCF) using commercial enzymes and S. cerevisiae 424A. Ethanol titer for CBP was found to be 2.8 g/L which was 71.8% of that yielded by SSCF (3.9 g/L). Decomposition products from AFEX‐CS helped to increase ethanol yield somewhat during CBP. Particle size played a crucial role in the enhancement of sugar conversion by CBP. Biotechnol. Bioeng. 2011; 108:1290–1297.

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.

Consolidated bioprocessing (CBP) of AFEX™‐pretreated corn stover for ethanol production using Clostridium phytofermentans at a high solids loading

Consolidated bioprocessing (CBP) using Clostridium phytofermentans (ATCC 700394) on ammonia fiber expansion (AFEX™)‐treated corn stover (AFEX™‐CS) at a low solids loading showed promising results [Jin et al. (2011) Biotechnol Bioeng 108(6): 1290–1297]. However, industrial relevant process requires high solids loading. Therefore, we studied high solids loading CBP performance on AFEX™‐CS. The factors potentially affecting the performance including solids loading, CBP products acetate and ethanol, and degradation products resulting from pretreatment were investigated. At 4% (w/w) glucan loading, C. phytofermentans performed well on AFEX™‐CS with no nutrients supplementation and reached similar sugar conversions as a fermentation with nutrients supplementation. A glucan conversion of 48.9% and a xylan conversion of 77.9% were achieved after 264 h with 7.0 g/L ethanol and 8.8 g/L acetate produced. Relatively high concentrations of acetate produced at high solids loading was found to be the major factor limiting the CBP performance. Degradation products in AFEX™‐CS helped enhance ethanol production. Biotechnol. Bioeng. 2012; 109:1929–1936.

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.