Patrik R. Lennartsson

Integration of the first and second generation bioethanol processes and the importance of by-products.

Lignocellulosic ethanol has obstacles in the investment costs and uncertainties in the process. One solution is to integrate it with the running dry mills of ethanol from grains. However, the economy of these mills, which dominate the world market, are dependent on their by-products DDGS (Distillers Dried Grains and Solubles), sold as animal feed. The quality of DDGS therefore must not be negatively influenced by the integration. This puts restraints on the choice of pretreatment of lignocelluloses and utilizing the pentose sugars by food-grade microorganisms. The proposed solution is to use food related filamentous Zygomycetes and Ascomycetes fungi, and to produce fungal biomass as a high-grade animal feed from the residues after the distillation (stillage). This also has the potential to improve the first generation process by increasing the amount of the thin stillage directly sent back into the process, and by decreasing the evaporator based problems.

Zygomycetes-based biorefinery: Present status and future prospects

Fungi of the phylum Zygomycetes fulfil all requirements for being utilized as core catalysts in biorefineries, and would be useful in creating new sustainable products. Apart from the extended use of Zygomycetes in preparing fermented foods, industrial metabolites such as lactic acid, fumaric acid, and ethanol are produced from a vast array of feedstocks with the aid of Zygomycetes. These fungi produce enzymes that facilitate their assimilation of various complex substrates, e.g., starch, cellulose, phytic acid, and proteins, which is relevant from an industrial point of view. The enzymes produced are capable of catalyzing various reactions involved in biodiesel production, preparation of corticosteroid drugs, etc. Biomass produced with the aid of Zygomycetes consists of proteins with superior amino acid composition, but also lipids and chitosan. The biomass is presently being tested for animal feed purposes, such as fish feed, as well as for lipid extraction and chitosan production. Complete or partial employment of Zygomycetes in biorefining procedures is consequently attractive, and is expected to be implemented within a near future.

A pilot study on lignocelluloses to ethanol and fish feed using NMMO pretreatment and cultivation with zygomycetes in an air-lift reactor

A complete process for the production of bioethanol and fungal biomass from spruce and birch was investigated. The process included milling, pretreatment with N-methylmorpholine-N-oxide (NMMO), washing of the pretreated wood, enzymatic hydrolysis, and cultivation of the zygomycetes fungi Mucor indicus. Investigated factors included wood chip size (0.5-16 mm), pretreatment time (1-5h), and scale of the process from bench-scale to 2m high air-lift reactor. Best hydrolysis yields were achieved from wood chips below 2mm after 5h of pretreatment. Ethanol yields (mg/g wood) of 195 and 128 for spruce, and 175 and 136 for birch were achieved from bench-scale and airlift, respectively. Fungal biomass yields (mg/g wood) of 103 and 70 for spruce, and 86 and 66 for birch from bench scale and airlift respectively were simultaneously achieved. NMMO pretreatment and cultivation with M. indicus appear to be a good alternative for ethanol production from birch and spruce.

Waste biorefineries using filamentous ascomycetes fungi : Present status and future prospects

Filamentous ascomycetes fungi have had important roles in natural cycles, and are already used industrially for e.g. supplying of citric, gluconic and itaconic acids as well as many enzymes. Faster human activities result in higher consumption of our resources and producing more wastes. Therefore, these fungi can be explored to use their capabilities to convert back wastes to resources. The present paper reviews the capabilities of these fungi in growing on various residuals, producing lignocellulose-degrading enzymes and production of organic acids, ethanol, pigments, etc. Particular attention has been on Aspergillus, Fusarium, Neurospora and Monascus genera. Since various species are used for production of human food, their biomass can be considered for feed applications and so biomass compositional characteristics as well as aspects related to culture in bioreactor are also provided. The review has been further complemented with future research avenues.

Effects of different growth forms of Mucor indicus on cultivation on dilute-acid lignocellulosic hydrolyzate, inhibitor tolerance, and cell wall composition.

The dimorphic fungus Mucor indicus was grown in different forms classified as purely filamentous, mostly filamentous, mostly yeast-like and purely yeast-like, and the relationship between morphology and metabolite production, inhibitor tolerance and the cell wall composition was investigated. Low concentrations of spores in the inoculum with subsequent aeration promoted filamentous growth, whereas higher spore concentrations and anaerobic conditions promoted yeast-like growth. Ethanol was the main metabolite with glycerol next under all conditions tested. The yields of ethanol from glucose were between 0.39 and 0.42 g g(-1) with productivities of 3.2-5.0 g l(-1) h(-1). The ethanol productivity of mostly filamentous cells was increased from 3.9 to 5.0 g l(-1) h(-1) by the presence of oxygen, whereas aeration of purely yeast-like cells showed no such effect. All growth forms were able to tolerate 4.6 g l(-1) furfural and 10 g l(-1) acetic acid and assimilate the sugars, although with different consumption rates. The cell wall content of the fungus measured as alkali insoluble materials (AIM) of the purely yeast-like cells was 26% of the biomass, compared to 8% of the pure filaments. However, the chitosan concentration of the filaments was 29% of the AIM, compared to 6% of the yeast-like cells.

Production of ethanol and biomass from thin stillage by Neurospora intermedia : A pilot study for process diversification

Dry mill ethanol processes produce ethanol and animal feed from whole grains, where the wastewater after the distillation and separation of solid materials is called “thin stillage.” In this work, similar production of ethanol (3.5 g/L) and biomass (5 g/L) from thin stillage was obtained during batch cultivation of the edible fungus Neurospora intermedia in a 2‐m high airlift reactor and bubble column. The fungal biomass, containing 50% w/w protein and 12% w/w lipids, was rich in essential amino acids and omega‐3 and ‐6 fatty acids. In a continuous mode of fermentation, dilution rates of up to 0.2 h−1 could be applied without cell washout in the bubble column at 0.5 vvm. At 0.1 h−1, around 5 g/L of ethanol and 4 g/L of biomass containing ca. 50% w/w protein were produced. The fungus was able to assimilate saccharides in the liquid fraction as well as sugar backbones such as xylan and arabinan in the solid fraction. The inclusion of the current process could potentially lead to the production of 11 000 m3 of ethanol (5.5% improvement vs. normal industrial process) and around 6300 tons of high‐quality biomass for animal feed at a typical facility producing 200 000 m3 ethanol per year.

Value-added products from dairy waste using edible fungi

Edible fungi-based biorefineries are a route to treat dairy waste. Dairy waste is converted into biomass for feed and ethanol using edible fungi. Protein-rich biomass (0.48 g/g waste) and ethanol (0.06 g/g waste) were produced. Integrating the conversion process at ethanol plants is probably the most suitable route.

Ethanol and Protein from Ethanol Plant By-Products Using Edible Fungi Neurospora intermedia and Aspergillus oryzae

Feasible biorefineries for production of second-generation ethanol are difficult to establish due to the process complexity. An alternative is to partially include the process in the first-generation plants. Whole stillage, a by-product from dry-mill ethanol processes from grains, is mostly composed of undegraded bran and lignocelluloses can be used as a potential substrate for production of ethanol and feed proteins. Ethanol production and the proteins from the stillage were investigated using the edible fungi Neurospora intermedia and Aspergillus oryzae, respectively. N. intermedia produced 4.7 g/L ethanol from the stillage and increased to 8.7 g/L by adding 1 FPU of cellulase/g suspended solids. Saccharomyces cerevisiae produced 0.4 and 5.1 g/L ethanol, respectively. Under a two-stage cultivation with both fungi, up to 7.6 g/L of ethanol and 5.8 g/L of biomass containing 42% (w/w) crude protein were obtained. Both fungi degraded complex substrates including arabinan, glucan, mannan, and xylan where reductions of 91, 73, 38, and 89% (w/v) were achieved, respectively. The inclusion of the current process can lead to the production of 44,000 m3 of ethanol (22% improvement), around 12,000 tons of protein-rich biomass for animal feed, and energy savings considering a typical facility producing 200,000 m3 ethanol/year.

Pellet formation of zygomycetes and immobilization of yeast

Pelleted growth provides many advantages for filamentous fungi, including decreased broth viscosity, improved aeration, stirring, and heat transfer. Thus, the factors influencing the probability of pellet formation of Rhizopus sp. in a defined medium was investigated using a multifactorial experimental design. Temperature, agitation intensity, Ca(2+)-concentration, pH, and solid cellulose particles, each had a significant effect on pelletization. Tween 80, spore concentration, and liquid volume were not found to have a significant effect. All of the effects were additive; no interactions were significant. The results were used to create a simple defined medium inducing pelletization, which was used for immobilization of a flocculating strain of Saccharomyces cerevisiae in the zygomycetes pellets. A flor-forming S. cerevisiae strain was also immobilized, while a non-flocculating strain colonized the pellets but was not immobilized. No adverse effects were detected as a result of the close proximity between the filamentous fungus and the yeast, which potentially allows for co-fermentation with S. cerevisiae immobilized in pellets of zygomycetes.

Bioethanol Production Processes

Ethanol, with more than 86 billion liters production in 2011, is the dominant biofuel in the global fuel market. Nowadays, ethanol is produced from starch- and sugar sources, so-called 1st generati ...