Establishing an itaconic acid production process with Ustilago species on the low-cost substrate starch.

Ustilago maydis and Ustilago cynodontis are natural producers of a broad range of valuable molecules including itaconate, malate, glycolipids, and triacylglycerols. Both Ustilago species are insensitive toward medium impurities, and have previously been engineered for efficient itaconate production and stabilized yeast-like growth. Due to these features, these strains were already successfully used for the production of itaconate from different alternative feedstocks such as molasses, thick juice, and crude glycerol. Here, we analyzed the amylolytic capabilities of Ustilago species for metabolization of starch, a highly abundant and low-cost polymeric carbohydrate widely utilized as a substrate in several biotechnological processes. Ustilago cynodontis was found to utilize gelatinized potato starch for both growth and itaconate production, confirming the presence of extracellular amylolytic enzymes in Ustilago species. Starch was rapidly degraded by U. cynodontis, even though no α-amylase was detected. Further experiments indicate that starch hydrolysis is caused by the synergistic action of glucoamylase and α-glucosidase enzymes. The enzymes showed a maximum activity of around 0.5 U ml-1 at the fifth day after inoculation, and also released glucose from additional substrates, highlighting potential broader applications. In contrast to U. cynodontis, U. maydis showed no growth on starch accompanied with no detectable amylolytic activity.

Second generation Pichia pastoris strain and bioprocess designs.

Yeast was the first microorganism used by mankind for biotransformation processes that laid the foundations of industrial biotechnology. In the last decade, Pichia pastoris has become the leading eukaryotic host organism for bioproduct generation. Most of the P. pastoris bioprocess operations has been relying on toxic methanol and glucose feed. In the actual bioeconomy era, for sustainable value-added bioproduct generation, non-conventional yeast P. pastoris bioprocess operations should be extended to low-cost and renewable substrates for large volume bio-based commodity productions. In this review, we evaluated the potential of P. pastoris for the establishment of circular bioeconomy due to its potential to generate industrially relevant bioproducts from renewable sources and waste streams in a cost-effective and environmentally friendly manner. Furthermore, we discussed challenges with the second generation P. pastoris platforms and propose novel insights for future perspectives. In this regard, potential of low cost substrate candidates, i.e., lignocellulosic biomass components, cereal by-products, sugar industry by-products molasses and sugarcane bagasse, high fructose syrup by-products, biodiesel industry by-product crude glycerol, kitchen waste and other agri-food industry by products were evaluated for P. pastoris cell growth promoting effects and recombinant protein production. Further metabolic pathway engineering of P. pastoris to construct renewable and low cost substrate utilization pathways was discussed. Although, second generation P. pastoris bioprocess operations for valorisation of wastes and by-products still in its infancy, rapidly emerging synthetic biology tools and metabolic engineering of P. pastoris will pave the way for more sustainable environment and bioeconomy. From environmental point of view, second generation bioprocess development is also important for waste recycling otherwise disposal of carbon-rich effluents creates environmental concerns. P. pastoris high tolerance to toxic contaminants found in lignocellulosic biomass hydrolysate and industrial waste effluent crude glycerol provides the yeast with advantages to extend its applications toward second generation P. pastoris strain design and bioprocess engineering, in the years to come.

Development of low-cost formulations of plant growth-promoting bacteria to be used as inoculants in beneficial agricultural technologies.

Phosphorus is one of the main macronutrients for plant development. Despite its large deposits in soils, it is scarcely available for plants. Phosphate-solubilizing bacteria, belonging to the group of plant growth-promoting rhizobacteria (PGPR), are capable of mobilizing deposits of insoluble phosphates in the soil. The use of PGPR as inoculants provides an environmentally sustainable approach to increase crop production. The effectiveness of inoculants depends on their proper production, formulation and storage in order to ensure the application of the required number of viable microbial cells. In order to develop inexpensive technology, low-cost compounds for biomass production and protection should be used. After the biomass production process, the product should be formulated in a liquid or a solid form, taking into account required storage time, use of protectors/carriers, storage conditions (temperature, humidity, etc.), ease of application and maintenance of beneficial effects on crops. Careful determination of these optimal conditions would ensure a low-cost efficient inoculant that would promote the growth and yield of various crops.

The Dark Side of Microalgae Biotechnology: A Heterotrophic Biorefinery Platform Directed to ω-3 Rich Lipid Production.

Microbial oils have been considered a renewable feedstock for bioenergy not competing with food crops for arable land, freshwater and biodiverse natural landscapes. Microalgal oils may also have other purposes (niche markets) besides biofuels production such as pharmaceutical, nutraceutical, cosmetic and food industries. The polyunsaturated fatty acids (PUFAs) obtained from oleaginous microalgae show benefits over other PUFAs sources such as fish oils, being odorless, and non-dependent on fish stocks. Heterotrophic microalgae can use low-cost substrates such as organic wastes/residues containing carbon, simultaneously producing PUFAs together with other lipids that can be further converted into bioenergy, for combined heat and power (CHP), or liquid biofuels, to be integrated in the transportation system. This review analyses the different strategies that have been recently used to cultivate and further process heterotrophic microalgae for lipids, with emphasis on omega-3 rich compounds. It also highlights the importance of studying an integrated process approach based on the use of low-cost substrates associated to the microalgal biomass biorefinery, identifying the best sustainability methodology to be applied to the whole integrated system.

Response surface statistical optimization of bacterial nanocellulose fermentation in static culture using a low-cost medium.

This work aimed at the optimization of bacterial nanocellulose (BNC) production by static culture, using Komagataeibacter xylinus BPR 2001 (K. xylinus). Response surface methodology - central composite design was used to evaluate the effect of inexpensive and widely available nutrient sources, namely molasses, ethanol, corn steep liquor (CSL) and ammonium sulphate, on BNC production yield. The optimized parameters for maximum BNC production were % (m/v): molasses 5.38, CSL 1.91, ammonium sulphate 0.63, disodium phosphate 0.270, citric acid 0.115 and ethanol 1.38% (v/v). The experimental and predicted maximum BNC production yields were 7.5 ± 0.54 g/L and 6.64 ± 0.079 g/L, respectively and the experimental and predicted maximum BNC productivity were 0.829 ± 0.046 g/L/day and 0.734 ± 0.079 g/L/day, after 9 days of static culture fermentation, at 30 °C. The effect of surface area and culture medium depth on production yield and productivity were also studied. BNC dry mass production increased linearly with surface area, medium depth and fermentation time. So long as nutrients were still available in the culture media, BNC mass productivity was constant. The results show that a high BNC production yield can be obtained by static culture of K. xylinus BPR 2001 using a low-cost medium. These are promising conditions for the static industrial scale BNC production, since as compared to agitated bioreactors, higher productivities may be reached, while avoiding high capital and operating costs.

From low-cost substrates to Single Cell Oils synthesized by oleaginous yeasts.

As new feedstock for biofuels, microbial oils have received worldwide attentions due to their environmentally-friendly characters. Microbial oil production based on low-cost raw materials is significantly attractive to the current biodiesel refinery industry. In terms of SCOs production, oleaginous yeast has numerous advantages over bacteria, molds and microalgae based on their high growth rate and lipid yield. Numerous efforts have been made on the competitive lipid production combining the use of cheap raw materials as substrates by yeasts. In this paper, we provided an overview of lipid metabolism in yeast cells. New advances using oleaginous yeast as a cell factory for high-value lipid production from various low-cost substrates are also reviewed, and the enhanced strategies based on synergistic effects of oleaginous yeast and microalgae in co-culture are discussed in details.