Ruohang Wang

A wheat biorefining strategy based on solid-state fermentation for fermentative production of succinic acid

In this study, a novel generic feedstock production strategy based on solid-state fermentation (SSF) has been developed and applied to the fermentative production of succinic acid. Wheat was fractionated into bran, gluten and gluten-free flour by milling and gluten extraction processes. The bran, which would normally be a waste product of the wheat milling industry, was used to produce glucoamylase and protease enzymes via SSF using Aspergillus awamori and Aspergillus oryzae, respectively. The resulting solutions were separately utilised for the hydrolysis of gluten-free flour and gluten to generate a glucose-rich stream of over 140gl(-1) glucose and a nitrogen-rich stream of more than 3.5gl(-1) free amino nitrogen. A microbial feedstock consisting of these two streams contained all the essential nutrients required for succinic acid fermentations using Actinobacillus succinogenes. In a fermentation using only the combined hydrolysate streams, around 22gl(-1) succinic acid was produced. The addition of MgCO3 into the wheat-derived medium improved the succinic acid production further to more than 64gl(-1). These results demonstrate the SSF-based strategy is a successful approach for the production of a generic feedstock from wheat, and that this feedstock can be efficiently utilised for succinic acid production.

Cereal-based biorefinery development: Utilisation of wheat milling by-products for the production of succinic acid

A novel wheat-based bioprocess for the production of a nutrient-complete feedstock for the fermentative succinic acid production by Actinobacillus succinogenes has been developed. Wheat was fractionated into bran, middlings and flour. The bran fraction, which would normally be a waste product of the wheat milling industry, was used as the sole medium in two solid-state fermentations (SSF) of Aspergillus awamori and Aspergillus oryzae that produce enzyme complexes rich in amylolytic and proteolytic enzymes, respectively. The resulting fermentation solids were then used as crude enzyme sources, by adding directly to an aqueous suspension of milled bran and middlings fractions (wheat flour milling by-products) to generate a hydrolysate containing over 95g/L glucose, 25g/L maltose and 300mg/L free amino nitrogen (FAN). This hydrolysate was then used as the sole medium for A. succinogenes fermentations, which led to the production of 50.6g/L succinic acid. Supplementation of the medium with yeast extract did not significantly improve succinic acid production though increasing the inoculum concentration to 20% did result in the production of 62.1g/L succinic acid. Results indicated that A. succinogenes cells were able to utilise glucose and maltose in the wheat hydrolysate for cell growth and succinic acid production. The proposed process could be potentially integrated into a wheat-milling process to upgrade the wheat flour milling by-products (WFMB) into succinic acid, one of the future platform chemicals of a sustainable chemical industry.

Chemical transformations of succinic acid recovered from fermentation broths by a novel direct vacuum distillation-crystallisation method

A novel alternative methodology (direct crystallisation) to the traditional calcium precipitation to obtain succinic acid (SA) from defined and wheat-based fermentation broths is reported. SA crystals were successfully recovered from fermentation broths (FB) using this method. A higher SA crystal purity (95%) and yield (70%) were obtained in the direct crystallisation method compared to a slightly modified traditional calcium precipitation method (90% and 24%, respectively). Chemical transformations (e.g. esterifications) to high-added value derivatives of both recovered SA crystals were then investigated using a range of solid acids including our acidic tunable mesoporous carbonaceous materials denoted as Starbon® acids. Results showed that SA crystals could be successfully converted into mono- and diesters in high yields and selectivities employing solid acids regardless of the reaction conditions. The order of reactivity was found to be: pure SA crystals > SA crystals from defined FB > FB SA crystals. Results demonstrate that SA can be effectively purified from actual fermentation broths, showing the importance of integrating the fermentation and downstream processing to optimise the fermentative production of SA and its chemical transformations to produce high-added value derivatives.

Influence of malt, wheat, and barley extracts on the bile tolerance of selected strains of lactobacilli

The bile tolerance of three strains of lactobacilli, selected on the basis of their ability to ferment cereal substrates and tolerate simulated gastric transit was examined. Lactobacillus reuteri (NCIMB 1195), L. acidophilus (NCIMB 8821), and L. plantarum (NCIMB 8826) were exposed to 2% bile for 4 h in a phosphate-saline buffer at pH 7. The effects of supplementing the assays with cereal extracts, increasing concentrations of glucose, and free amino nitrogen was also examined. In the absence of any supplements L. reuteri showed the greatest resistance to bile, whilst L. acidophilus displayed the greatest sensitivity. Addition of cereal extracts improved the tolerance of all three strains, barley and wheat extracts imparted similar levels of enhancement in viability, whilst the addition of malt showed a greater positive influence. The extent of improvement was related to the concentration of soluble sugars and FAN present in the cereals, this was illustrated further by the addition of glucose to the assays, as the viability of the organisms was progressively improved with increasing concentrations. The presence of free amino nitrogen also increased bacterial resistance to bile but to a lesser extent both glucose and cereal extracts.

Process design and optimization of novel wheat-based continuous bioethanol production system

A novel design of a wheat‐based biorefinery for bioethanol production, including wheat milling, gluten extraction as byproduct, fungal submerged fermentation for enzyme production, starch hydrolysis, fungal biomass autolysis for nutrient regeneration, yeast fermentation with recycling integrated with a pervaporation membrane for ethanol concentration, and fuel‐grade ethanol purification by pressure swing distillation (PSD), was optimized in continuous mode using the equation‐based software General Algebraic Modelling System (GAMS). The novel wheat biorefining strategy could result in a production cost within the range of

Proliferation of Lactobacillus plantarum in Solid-State Fermentation of Oats

0.96–0.50 gal−1 ethanol (

The application of a generic feedstock from wheat for microbial fermentations.

0.25–0.13 L−1 ethanol) when the production capacity of the plant is within the range of 10–33.5 million gal y−1 (37.85–126.8 million L y−1). The production of value‐added byproducts (e.g., bran‐rich pearlings, gluten, pure yeast cells) was identified as a crucial factor for improving the economics of fuel ethanol production from wheat. Integration of yeast fermentation with pervaporation membrane could result in the concentration of ethanol in the fermentation outlet stream (up to 40 mol %). The application of a PSD system that consisted of a low‐pressure and a high‐pressure column and employing heat integration between the high‐ and low‐pressure columns resulted in reduced operating cost (up to 44%) for fuel‐grade ethanol production.

Development of a Generic Fermentation Feedstock from Whole Wheat Flour

In an attempt to introduce probiotic functionalities to breakfast cereals and similar food products, the technique of solid‐state fermentation (SSF) was applied to cultivate Lactobacillus plantarum (NCIMB 8826) on oat bran and spent oats after lipid extraction by supercritical CO2 extraction. When compared to the frequently favored submerged processes for bacterium incubation, SSF presents not only the potential of simple downstream processing but also a more natural growth environment for the target bacterium. Preliminary studies confirmed that oat bran contained balanced nutrients to support a 25‐fold bacterium propagation within a range of moisture content from 50% to 58% after a 36‐h cultivation. Limited hydrolysis of the raw materials by the enzyme complex from submerged incubation of Aspergillus awamori and A. oryzae to increase nutrient accessibility extended the exponential growth phase and enhanced bacterial growth by over 183‐fold. The process with the most potential, however, was to simultaneously grow both fungi aerobically on the raw materials in solid state to achieve sufficient hydrolysis, followed by controlled fungal autolysis at 65 °C prior to anaerobic bacterium incubation. Following this process bacterium population reached a maximum of 7.3 × 109 cells in each gram of the fermented solids, corresponding to a 1653‐fold increase from the point of inoculation.

Optimization and cost estimation of novel wheat biorefining for continuous production of fermentation feedstock.

The feasibility of a generic fermentation feedstock produced from wheat flour has been confirmed in several fermentations of yeasts, bacterium, and filamentous fungus for the production of commodity chemicals. Saccharomyces cerevisiae was incubated for the observation of yeast growth and ethanol production, Pichia farinosa for glycerol production, Monascus purpureus for fungal growth and pigment production, and Lactobacillus bulgaricus for bacteria growth and lactic acid production. The results confirmed that the feedstock contained no inhibitory components to the strains tested. Similar or higher metabolite yields were obtained in comparison with other studies carried in commonly used media.

Modelling studies of a process to produce a generic bioconversion feedstock from wheat

There is an inevitability that one day much of the finite resource we currently use as feedstock for the production of chemicals and energy will have been consumed (or will no longer be available) and industry will be obliged to turn to renewable resources as replacement raw materials. Of the alternatives available, cereals offer amongst the best potential, being energy intense and environmentally benign. However, whatever other factors influence the choice, the real driving force for the adoption of new raw materials is the relative economics associated with obtaining and processing those materials compared with traditional feedstocks.