Optimization of xylitol production from xylose by a novel arabitol limited co-producing Barnettozyma populi NRRL Y-12728.

Xylitol is a widely marketed sweetener with good functionality and health-promoting properties. It can be synthetized by many yeast species in a one-step reduction of xylose. Arabinose is a common contaminant found in xylose and there is ongoing interest in finding biocatalysts that selectively produce xyltiol. From a screen of 99 yeasts, Barnettozyma populi Y-12728 was found to selectively produce xylitol from both mixed sugars and corn stover hemicellulosic hydrolysate. Here, fermentation conditions for xylitol production from xylose by B. populi were optimized. The medium for xylitol production was optimized through response surface methodology. The yeast produced 31.2 ± 0.4 g xylitol from xylose (50 g L-1) in 62 h using the optimized medium. The optimal pH for xylitol production was 6.0. Glucose (10 g L-1), acetic acid (6.0 g L-1), HMF (4 mM) and ethanol (2.0 g L-1) inhibited the xylitol production. The glucose inhibition was entirely mitigated by using a 2-stage aeration strategy, indicating that the yeast was inhibited by ethanol produced from glucose under low aeration. This culture strategy will greatly benefit xylitol production from hemicellulosic hydrolysates, which often contain glucose. This is the first report on optimization of xylitol production by a Barnettozyma species.

Efficient itaconic acid production by Aspergillus terreus: Overcoming the strong inhibitory effect of manganese.

Itaconic acid (IA), a building block platform chemical, is produced industrially by Aspergillus terreus utilizing glucose. Lignocellulosic biomass can serve as a low cost source of sugars for IA production. However, the fungus could not produce IA from dilute acid pretreated and enzymatically saccharified wheat straw hydrolyzate even at 100-fold dilution. Furfural, hydroxymethyl furfural and acetic acid were inhibitory, as is typical, but Mn2+ was particularly problematic for IA production. It was present in the hydrolyzate at a level that was 230 times over the inhibitory limit (50 ppb). Recently, it was found that PO43- limitation decreased the inhibitory effect of Mn2+ on IA production. In the present study, a novel medium was developed for production of IA by varying PO43- , Fe3+ and Cu2+ concentrations using response surface methodology, which alleviated the strong inhibitory effect of Mn2+ . The new medium contained 0.08 g KH2 PO4 , 3 g NH4 NO3 , 1 g MgSO4 ·7H2 O, 5 g CaCl2 ·2 H2 O, 0.83 mg FeCl3 ·6H2 O, 8 mg ZnSO4 ·7H2 O, and 45 mg CuSO4 ·5H2 O per liter. The fungus was able to produce IA very well in the presence of Mn2+ up to 100 ppm in the medium. This medium will be extremely useful for IA production in the presence of Mn2+ . This is the first report on the development of Mn2+ tolerant medium for IA production by A. terreus.

Valorization of egg shell as a detoxifying and buffering agent for efficient polymalic acid production by Aureobasidium pullulans NRRL Y-2311-1 from barley straw hydrolysate.

Stepwise formulation of a versatile and cost-effective medium based on barley straw hydrolysate and egg shell for efficient polymalic acid production by A. pullulans NRRL Y-2311-1 was carried out for the first time. The strain did not grow and produce polymalic acid when dilute acid pretreated barley straw hydrolysate (total fermentable sugars: 94.60 g/L; furfural: 1.01 g/L; hydroxymethylfurfural: 0.55 g/L; acetic acid: 5.06 g/L) was directly used in medium formulation without detoxification (e.g. charcoal pretreatment). When CaCO3 in the medium formulation was substituted with egg shell powder, efficient production of polymalic acid was achieved without a detoxification step. Utilization of 40 g/L of egg shell powder led to 43.54 g polymalic acid production per L with the productivity of 0.30 g/L/h and yield of 0.48 g/g. The bioprocess strategy used in this study can also be utilized for mass production of several other industrially important microbial organic acids and biomaterials.

Factors Affecting Production of Itaconic Acid from Mixed Sugars by Aspergillus terreus.

Itaconic acid (IA; a building block platform chemical) is currently produced industrially from glucose by fermentation with Aspergillus terreus. In order to expand the use of IA, its production cost must be lowered. Lignocellulosic biomass has the potential to serve as low-cost source of sugars for IA production. It was found that the fungus cannot produce IA from dilute acid pretreated and enzymatically saccharified wheat straw hydrolysate even at 100-fold dilution. The effects of typical compounds (acetic acid, furfural, HMF and Mn2+, enzymes, CaSO4), culture conditions (initial pH, temperature, aeration), and medium components (KH2PO4, NH4NO3, CaCl2·2H2O, FeCl3·6H2O) on growth and IA production by A. terreus NRRL 1972 using mixed sugar substrate containing glucose, xylose, and arabinose (4:3:1, 80 g L-1) mimicking the wheat straw hydrolysate were investigated. Acetic acid, furfural, Mn2+, and enzymes were strong inhibitors to both growth and IA production from mixed sugars. Optimum culture conditions (pH 3.1, 33 °C, 200 rpm) and medium components (0.8 g KH2PO4, 3 g NH4NO3, 2.0 g CaCl2·2H2O, 0.83-3.33 mg FeCl3·6H2O per L) as well as tolerable levels of inhibitors (0.4 g acetic acid, < 0.1 g furfural, 100 mg HMF, < 5.0 ppb Mn2+, 24 mg CaSO4 per L) for mixed sugar utilization were established. The results will be highly useful for developing a bioprocess technology for IA production from lignocellulosic feedstocks.

Ninety six well microtiter plate as microbioreactors for production of itaconic acid by six Aspergillus terreus strains.

Itaconic acid (IA) is a building block platform chemical that is currently produced industrially from glucose by fermentation with Aspergillus terreus. However, lignocellulosic biomass has the potential to serve as low cost source of sugars for production of IA. Previously, 100 A. terreus strains were evaluated for production of IA from pentose sugars in shake-flasks. Six selected strains were then investigated for IA production in shake-flasks. But none of the strains grew and produced IA using biomass hydrolyzates. In order to study the factors inhibiting fungal growth and IA production, we have evaluated these six strains for sugar utilization and IA production from glucose, xylose, arabinose, mixed sugars, and both dilute acid and liquid hot water pretreated wheat straw hydrolyzates in microtiter plate (MTP) microbioreactors at 100µL scale. The results clearly indicate that MTP is very useful as a convenient, reliable and affordable platform to investigate the reasons for inhibition of growth and IA production by the A. terreus strains and should greatly aid in strain development and optimization of IA production by the fungal strains.

Production of itaconic acid from pentose sugars by Aspergillus terreus.

Itaconic acid (IA), an unsaturated 5-carbon dicarboxylic acid, is a building block platform chemical that is currently produced industrially from glucose by fermentation with Aspergillus terreus. However, lignocellulosic biomass has potential to serve as low-cost source of sugars for production of IA. Research needs to be performed to find a suitable A. terreus strain that can use lignocellulose-derived pentose sugars and produce IA. One hundred A. terreus strains were evaluated for the first time for production of IA from xylose and arabinose. Twenty strains showed good production of IA from the sugars. Among these, six strains (NRRL strains 1960, 1961, 1962, 1972, 66125, and DSM 23081) were selected for further study. One of these strains NRRL 1961 produced 49.8 ± 0.3, 38.9 ± 0.8, 34.8 ± 0.9, and 33.2 ± 2.4 g IA from 80 g glucose, xylose, arabinose and their mixture (1:1:1), respectively, per L at initial pH 3.1 and 33°C. This is the first report on the production of IA from arabinose and mixed sugar of glucose, xylose, and arabinose by A. terreus. The results presented in the article will be very useful in developing a process technology for production of IA from lignocellulosic feedstocks. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1059-1067, 2017.

Biological pretreatment of corn stover with Phlebia brevispora NRRL-13108 for enhanced enzymatic hydrolysis and efficient ethanol production.

Biological pretreatment of lignocellulosic biomass by white-rot fungus can represent a low-cost and eco-friendly alternative to harsh physical, chemical, or physico-chemical pretreatment methods to facilitate enzymatic hydrolysis. In this work, solid-state cultivation of corn stover with Phlebia brevispora NRRL-13018 was optimized with respect to duration, moisture content and inoculum size. Changes in composition of pretreated corn stover and its susceptibility to enzymatic hydrolysis were analyzed. About 84% moisture and 42 days incubation at 28°C were found to be optimal for pretreatment with respect to enzymatic saccharification. Inoculum size had little effect compared to moisture level. Ergosterol data shows continued growth of the fungus studied up to 57 days. No furfural and hydroxymethyl furfural were produced. The total sugar yield was 442 ± 5 mg/g of pretreated corn stover. About 36 ± 0.6 g ethanol was produced from 150 g pretreated stover per L by fed-batch simultaneous saccharification and fermentation (SSF) using mixed sugar utilizing ethanologenic recombinant Eschericia coli FBR5 strain. The ethanol yields were 32.0 ± 0.2 and 38.0 ± 0.2 g from 200 g pretreated corn stover per L by fed-batch SSF using Saccharomyces cerevisiae D5A and xylose utilizing recombinant S. cerevisiae YRH400 strain, respectively. This research demonstrates that P. brevispora NRRL-13018 has potential to be used for biological pretreatment of lignocellulosic biomass. This is the first report on the production of ethanol from P. brevispora pretreated corn stover. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:365-374, 2017.

Enhancement of xylose utilization from corn stover by a recombinant Escherichia coli strain for ethanol production.

Effects of substrate-selective inoculum prepared by growing on glucose, xylose, arabinose, GXA (glucose, xylose, arabinose, 1:1:1) and corn stover hydrolyzate (dilute acid pretreated and enzymatically hydrolyzed, CSH) on ethanol production from CSH by a mixed sugar utilizing recombinant Escherichia coli (strain FBR5) were investigated. The initial ethanol productivity was faster for the seed grown on xylose followed by GXA, CSH, glucose and arabinose. Arabinose grown seed took the longest time to complete the fermentation. Delayed saccharifying enzyme addition in simultaneous saccharification and fermentation of dilute acid pretreated CS by the recombinant E. coli strain FBR5 allowed the fermentation to finish in a shorter time than adding the enzyme simultaneously with xylose grown inoculum. Use of substrate selective inoculum and fermenting pentose sugars first under glucose limited condition helped to alleviate the catabolite repression of the recombinant bacterium on ethanol production from lignocellulosic hydrolyzate.

Pilot scale conversion of wheat straw to ethanol via simultaneous saccharification and fermentation.

The production of ethanol from wheat straw (WS) by dilute acid pretreatment, bioabatement of fermentation inhibitors by a fungal strain, and simultaneous saccharification and fermentation (SSF) of the bio-abated WS to ethanol using an ethanologenic recombinant bacterium was studied at a pilot scale without sterilization. WS (124.2g/L) was pretreated with dilute H2SO4 in two parallel tube reactors at 160°C. The inhibitors were bio-abated by growing the fungus aerobically. The maximum ethanol produced by SSF of the bio-abated WS by the recombinant Escherichia coli FBR5 at pH 6.0 and 35°C was 36.0g/L in 83h with a productivity of 0.43gL(-1)h(-1). This value corresponds to an ethanol yield of 0.29g/g of WS which is 86% of the theoretical ethanol yield from WS. This is the first report on the production of ethanol by the recombinant bacterium from a lignocellulosic biomass at a pilot scale.

Dilute sulfuric acid pretreatment of corn stover for enzymatic hydrolysis and efficient ethanol production by recombinant Escherichia coli FBR5 without detoxification.

A pretreatment strategy for dilute H2SO4 pretreatment of corn stover was developed for the purpose of reducing the generation of inhibitory substances during pretreatment so that a detoxification step is not required prior to fermentation while maximizing sugar yield. The optimal conditions for pretreatment of corn stover (10%, w/v) were: 0.75% H2SO4, 160°C, and 0-5 min holding time. The conditions were chosen based on maximum glucose release after enzymatic hydrolysis, minimum loss of pentose sugars and minimum formation of sugar degradation products such as furfural and hydroxymethyl furfural. The pretreated corn stover after enzymatic saccharification generated 63.2 ± 2.2 and 63.7 ± 2.3 g total sugars per L at 0 and 5 min holding time, respectively. Furfural production was 0.45 ± 0.1 and 0.87 ± 0.4 g/L, respectively. The recombinant Escherichia coli strain FBR5 efficiently fermented non-detoxified corn stover hydrolyzate if the furfural content is <0.5 g/L.

Response surface optimization of corn stover pretreatment using dilute phosphoric acid for enzymatic hydrolysis and ethanol production.

Dilute H(3)PO(4) (0.0-2.0%, v/v) was used to pretreat corn stover (10%, w/w) for conversion to ethanol. Pretreatment conditions were optimized for temperature, acid loading, and time using central composite design. Optimal pretreatment conditions were chosen to promote sugar yields following enzymatic digestion while minimizing formation of furans, which are potent inhibitors of fermentation. The maximum glucose yield (85%) was obtained after enzymatic hydrolysis of corn stover pretreated with 0.5% (v/v) acid at 180°C for 15min while highest yield for xylose (91.4%) was observed from corn stover pretreated with 1% (v/v) acid at 160°C for 10min. About 26.4±0.1g ethanol was produced per L by recombinant Escherichia coli strain FBR5 from 55.1±1.0g sugars generated from enzymatically hydrolyzed corn stover (10%, w/w) pretreated under a balanced optimized condition (161.81°C, 0.78% acid, 9.78min) where only 0.4±0.0g furfural and 0.1±0.0 hydroxylmethyl furfural were produced.