Itaconic acid (IA) is a platform chemical, derived from non-petroleum sources, produced through the fermentation of glucose by Aspergillus terreus. However, producing IA from alternative sugar sources (e.g., lignocellulose) has been shown to be problematic, requiring post-hydrolysis mitigation to allow growth and IA production by the fungus. It is well known that the side products of lignocellulosic biomass conversion to sugars act as microbial growth inhibitors. An uncommon feature of fungal organic acid fermentations is production inhibition caused by mineral ions in biomass hydrolysate after pretreatment and enzymatic hydrolysis. To minimize mineral introduction during pretreatment and hydrolysis, we determined the sources of growth and production inhibitors at each of these steps. Biomass demineralization and four pretreatment strategies were evaluated for inhibitor introduction. Dilution assays determined the approximate degree of inhibition for each hydrolysate. An ammonium hydroxide pretreatment of demineralized wheat straw presented the lowest concentration of inhibitors and concomitant lowest inhibition: subsequent fermentations produced 35 g L−1 IA from wheat straw hydrolysate (91 g L−1 sugar) without post-hydrolysis mitigation.

In this study, the ability of a novel strain of Clostridium tyrobutyricum NRRL 67062 to produce butyric acid during glucose fermentation was evaluated. The strain was evaluated for substrate and product inhibition in batch experiments using anaerobic tubes. To characterize glucose inhibition, initial glucose concentrations ranging from 60 to 250 g L−1 were used, and it was demonstrated that a glucose concentration of 250 g L−1 exerted strong inhibition on cell growth and fermentation. To evaluate butyric acid inhibition, the culture was challenged with 5–50 g L−1 of butyric acid at an initial pH of 6.5. These experiments were performed without pH control. When challenged with a butyric acid concentration of 50 g L−1, cell growth was slow; however, it produced 8.25 g L−1 of butyric acid. This suggested that the butyric acid tolerance of the culture was 58 g L−1. In a scaled-up batch experiment, which was performed in a 2.5 L fermentor with an initial glucose concentration of 100 g L−1, the pH was controlled at 6.5. In this experiment, the strain produced 57.86 g L−1 of butyric acid and 12.88 g L−1 of acetic acid, thus producing 70.74 g L−1 of total acids with a productivity of 0.69 g·L−1·h−1. A concentration of 70.74 g L−1 of acids equates to a yield of 0.71 g of acid per g consumed glucose. The maximum cell concentration was 3.80 g L−1, which may have been the reason for high productivity in the batch culture. Finally, corn steep liquor (CSL; a commercial nutrient solution) provided greater growth and acid production than the refined medium.

Itaconic acid (IA), is a versatile platform chemical that is produced by Aspergillus terreus using glucose as carbon source. Lignocellulosic biomass can be used as a low cost source of sugars for IA production. However, A. terreus does not produce IA even after 100 fold dilution from enzymatically hydrolyzed wheat straw pretreated with dilute acid. In addition to organic inhibitors, Mn2+ strongly inhibits IA production. Mn2+ was present in the hydrolysate at a level 230 times over the inhibitory limit (50 ppb). Recently, PO43- limitation was shown to decrease the inhibitory effect of Mn2+ on IA production and a medium was formulated in which A. terreus produced IA very efficiently in the presence of Mn2+ up to 100 ppm. Here, the effectiveness of this medium was demonstrated at pilot scale using a stainless steel bioreactor and glucose as the substrate. The productions of IA from corn stover cellulosic hydrolysate in a biorefinery concept and mitigated wheat straw hydrolysate using the Mn2+ tolerant medium were also demonstrated at laboratory scale. The fungal strain produced 74% IA from cellulosic hydrolysate and 98% IA from wheat straw hydrolysate in comparison with respective mock sugar solution. This is the first report on using Mn2+ tolerant medium to achieve IA production by A. terreus on hydrolysates.

Butanol was produced commercially from cornstarch and sugarcane molasses (renewable resources) until 1983, when production of these plants was forced to cease because of unfavorable economics of production caused in part by escalating prices of these feedstocks. During recent years, the focus of research has been on the use of economically available agricultural biomass and residues and cutting-edge science and technology to make butanol production a commercially viable process again. In this study, we produced butanol from sweet sorghum bagasse (SSB) by employing high concentrations of SSB solids and integrated process technology through which simultaneous saccharification, fermentation, and recovery (SSFR) were conducted as one unit operation. The concentrated SSB (16–22% dry wt. basis or 160–220 gL−1) was used to reduce reactor size and potentially reduce fixed and operational costs. Indeed, ABE productivity and yield of 0.21 gL−1h−1 and 0.39 were obtained, respectively, when 160 gL−1 SSB (16%, dry wt.) was used in the SSFR process. In nonintegrated systems, use of >90 gL−1 solid loading is improbable and has not been done until this study.

Xylitol is a natural food sweetener, dental caries reducer and sugar substitute for diabetics. During production of xylitol by fermentation, arabinose present in hemicellulosic hydrolysates is converted to arabitol which is difficult to separate in a cost-effective way. Ninety-nine yeast strains were evaluated for production of arabitolfree xylitol from a mixture of xylose and arabinose. Among these yeast strains, five best xylitol producers with least arabitol production were: Barnettozyma populi Y-12728, Bar. californica Y-1680, Bar. californica Y-11750, Cyberlindnera mrakii Y-1364 and Bar. salicaria NRRL Y-6780. These five strains were evaluated for xylitol production from undetoxified corn stover hemicellulosic hydrolysate. Bar. populi Y-12728 produced xylitol without arabitol production from the undetoxified hydrolysate. This is the first report on the evaluation of naturally isolated yeast strains for production of xylitol from mixed sugars of xylose and arabinose and corn stover hemicellulosic hydrolysate without arabitol co-production.

BACKGROUND Butanol, a superior biofuel to ethanol, can be produced by fermentation of renewable feedstocks such as corn or molasses. Since costs of these feedstocks are high, this biofuel should be produced from economic substrates. Recent studies have identified yellow top (YT) (Physaria fendleri) as an economical feedstock which is currently used for landfill, thus causing environmental pollution. In addition, cutting edge science and technology should be used, such as application of concentrated substrates. This study used concentrated YT ranging from 80 to 150 g L-1. RESULTS At a YT concentration of 125 g L-1, the culture produced 28.8 g L-1 acetone-butanol-ethanol (ABE) as compared with 18.68 g L-1 using glucose (control) as substrate, which is 154% of the control fermentation. The control fermentation resulted in a productivity of 0.42 g L-1 h(-1), while YT resulted in a productivity of 0.70 g L-1 h(-1). In YT fermentation, specific ABE productivity was 543% of that obtained in the control run. CONCLUSION A YT concentration of 125 g L-1 is beneficial for ABE fermentation. At this concentration, ABE production level, productivity and specific productivity were greatly enhanced. Published 2019. This article is a U.S. Government work and is in the public domain in the USA.

Development of bioprocess routes for production of value-added materials from food waste is very attractive in terms of bioprocess economics and environmental protection. In this study, production of 2-keto-d-gluconic acid, a significant platform chemical, from food waste was performed by Pseudomonas reptilivora NRRL B-6 for the first time. The nitrogen source preference of the strain for 2-keto-d-gluconic acid production under different glucose concentrations in synthetic medium has been elucidated. At higher glucose concentrations, utilization of organic nitrogen source (yeast extract) led to higher product formation while at lower glucose concentration, utilization of inorganic nitrogen source (ammonium chloride) was also very feasible. The results indicated that favorable nitrogen source for 2-keto-d-gluconic acid production can change depending on the glucose concentration. The waste bread hydrolysate (glucose 181.43 g/L, protein 1.21% (w/v)) prepared by sequential application of α-amylase, amyloglucosidase, and protease was utilized in medium formulation at different glucose concentrations. Bread hydrolysate without additional supplements provided higher product formation than the optimum medium prepared with the hydrolysate. Utilization of 50 g/L CaCO3 led the maximum product formation. The maximum 2-keto-d-gluconic acid production from waste bread was 142.81 g/L with the productivity of 3.02 g/L/h and molar yield of 0.95. The proposed approach in this study confirms that waste bread as a sole source of nutrients can be valorized for microbial production of various industrially relevant platform chemicals.

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.

Worldwide, there are various feedstocks such as straws, corn stover, sugarcane bagasse, sweet sorghum bagasse (SSB), grasses, leaves, whey permeate, household organic waste, and food waste (FW) that can be converted to valuable biofuels such as butanol. For the present studies, an economic analysis was performed to compare butanol production from three feedstocks (SSB; FW; and yellow top presscake, YTP or YT) using a standard process and an advanced integrated process design. The total plant capacity was set at 170,000–171,000 metric tons of total acetone butanol ethanol (ABE) per year (99,300 tons of just butanol per year). Butanol production from SSB typically requires pretreatment, separate hydrolysis, fermentation, and product recovery (SHFR). An advanced process was developed in which the last three steps were combined into a single unit operation for simultaneous saccharification, fermentation, and recovery (SSFR). For the SHFR and SSFR plants, the total capital investments were estimated as $213.72 × 106 and $198.16 × 106, respectively. It was further estimated that the minimum butanol selling price (using SSB as a feedstock) for the two processes were $1.14/kg and $1.05/kg. Therefore, SSFR lowered the production cost markedly compared to that of the base case. Butanol made using FW had an estimated minimum selling price of only $0.42/kg. This low selling price is because the FW to butanol process does not require pretreatment, hydrolysis, and cellulolytic enzymes. For this plant, the total capital investment was projected to be $107.26 × 106. The butanol selling price using YTP as a feedstock was at $0.73/kg and $0.79/kg with total capital investments for SSFR and SHFR of $122.58 × 106 and $132.21 × 106, respectively. In the Results and Discussion section, the availability of different feedstocks in various countries such as Brazil, the European Union, New Zealand, Denmark, and the United States are discussed. Additionally, the use of various microbial strains and product recovery technologies are also discussed.

Yellow Top (Physaria fendleri) is a plant that belongs to the mustard family. This plant is used to produce seeds that are rich in hydroxy oil. After extraction of oil, the presscake is land filled. The seedcake is rich in polymeric sugars and can be used for various bioconversions. For the present case, the seedcake or presscake was hydrolyzed with dilute (0.50% [v/v]) H2SO4 and enzymes to release sugars including glucose, xylose, galactose, arabinose, and mannose. Then, the hydrolyzate was used to produce acetone–butanol–ethanol (ABE). Using 100 gL−1 presscake (prior to pretreatment), 19.22 gL−1 of ABE was successfully produced of which butanol was the major product. In this process, an ABE productivity of 0.48 gL−1 h−1 was obtained. These results are superior to glucose fermentation to produce ABE in which an ABE productivity of 0.42 gL−1 h−1 was obtained. Use of Yellow Top to produce butanol has the following advantages: (i) it is an economic feedstock and is expected to produce butanol economically; (ii) it avoids pollution concerns when not land filled; and (iii) rate of ABE production is not inhibited when fermented this substrate. It is suggested that the potential of this feedstock be further explored by optimizing process parameters for this valuable fermentation. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2767, 2019.

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.

Lignocellulosic biomass has the potential to serve as a low-cost source of sugars for production of itaconic acid (IA, a building block platform chemical) by fermentation with Aspergillus terreus. However, the IA production from biomass hydrolysate was severely inhibited. Mn2+ was found to inhibit the IA production strongly. The effect of Mn2+ on each medium component (KH2PO4, NH4NO3, MgSO4·7H2O, CaCl2·2H2O, FeCl3·6H2O, ZnSO4·7H2O, and CuSO4·7H2O) was evaluated for sugar utilization and IA production by A. terreus NRRL 1972. Both K+ and PO4−3 were necessary for IA production. Low PO4−3 in the medium greatly alleviated the inhibitory effect of Mn2+ on IA production. However, high PO4−3 (K+) was detrimental for IA production in the presence of Mn2+. The inhibitory effect of Mn2+ on IA production was partly eliminated by increasing the CuSO4·7H2O level in the medium. This is the first report on the effect of phosphate limitation to alleviate the inhibition of IA production by Mn2+ and on the relationship of Mn2+ on the medium components for utilization of sugar and production of IA.

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 bio-materials.

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 100 μL 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.

In these studies, liquid hot water (LHW) pretreated and enzymatically hydrolyzed Sweet Sorghum Bagasse (SSB) hydrolyzates were fermented in a fed‐batch reactor. As reported in the preceding paper, the culture was not able to ferment the hydrolyzate I in a batch process due to presence of high level of toxic chemicals, in particular acetic acid released from SSB during the hydrolytic process. To be able to ferment the hydrolyzate I obtained from 250 g L−1 SSB hydrolysis, a fed‐batch reactor with in situ butanol recovery was devised. The process was started with the hydrolyzate II and when good cell growth and vigorous fermentation were observed, the hydrolyzate I was slowly fed to the reactor. In this manner the culture was able to ferment all the sugars present in both the hydrolyzates to acetone butanol ethanol (ABE). In a control batch reactor in which ABE was produced from glucose, ABE productivity and yield of 0.42 g L−1 h−1 and 0.36 were obtained, respectively. In the fed‐batch reactor fed with SSB hydrolyzates, these productivity and yield values were 0.44 g L−1 h−1 and 0.45, respectively. ABE yield in the integrated system was high due to utilization of acetic acid to convert to ABE. In summary we were able to utilize both the hydrolyzates obtained from LHW pretreated and enzymatically hydrolyzed SSB (250 g L−1) and convert them to ABE. Complete fermentation was possible due to simultaneous recovery of ABE by vacuum. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:967–972, 2018

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

In these studies, we pretreated sweet sorghum bagasse (SSB) using liquid hot water (LHW) or dilute H2 SO4 (2 g L-1 ) at 190°C for zero min (as soon as temperature reached 190°C, cooling was started) to reduce generation of sugar degradation fermentation inhibiting products such as furfural and hydroxymethyl furfural (HMF). The solids loading were 250-300 g L-1 . This was followed by enzymatic hydrolysis. After hydrolysis, 89.0 g L-1 sugars, 7.60 g L-1 acetic acid, 0.33 g L-1 furfural, and 0.07 g L-1 HMF were released. This pretreatment and hydrolysis resulted in the release of 57.9% sugars. This was followed by second hydrolysis of the fibrous biomass which resulted in the release of 43.64 g L-1 additional sugars, 2.40 g L-1 acetic acid, zero g L-1 furfural, and zero g L-1 HMF. In both the hydrolyzates, 86.3% sugars present in SSB were released. Fermentation of the hydrolyzate I resulted in poor acetone-butanol-ethanol (ABE) fermentation. However, fermentation of the hydrolyzate II was successful and produced 13.43 g L-1 ABE of which butanol was the main product. Use of 2 g L-1 H2 SO4 as a pretreatment medium followed by enzymatic hydrolysis resulted in the release of 100.6-93.8% (w/w) sugars from 250 to 300 g L-1 SSB, respectively. LHW or dilute H2 SO4 were used to economize production of cellulosic sugars from SSB. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:960-966, 2018.

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. Softwood has the potential to serve as low cost source of sugars for its production. Effective utilization of all softwood derived sugars such as glucose, mannose and galactose by the fungus for production of IA will lower the cost of its production. In this work, 20 A. terreus strains were evaluated for the first time for IA production from mannose and galactose in shake-flasks at initial pH of 3·1, 33°C and 200 rev min-1 for 7 days. Strain NRRL 1971 possesses the unique ability to produce high concentrations of IA from mannose. It produced 36·4 ± 0·2 g IA from 80 g mannose per litre with a yield of 0·46 g g-1 mannose (highest titre reported so far). This strain has the potential to be used for IA production from softwood. The maximum (1·1 ± 0·2 g) IA was produced by strain DSM 23081 from 80 g galactose per litre utilizing only 9·1 ± 0·3 g. Galactose was not suitable for IA production by these strains. This is the first detailed report on the production of IA from mannose and galactose. SIGNIFICANCE AND IMPACT OF THE STUDY:Itaconic acid (IA) is a building block platform chemical which 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. Softwood has the potential to serve as low cost source of sugars for its production. In this work, 20 A. terreus strains were evaluated for the first time for production of IA from mannose and galactose, sugars derived from softwood. A novel strain was found that gave the highest IA titre reported so far. Galactose was a poor substrate for IA production by A. terreus.

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.