Poly(hydroxyalkanoate) production by Cupriavidus necator from fatty waste can be enhanced by phaZ1 inactivation

PHA production from waste oils or fats requires microorganisms that should be both excellent PHA producers and equipped with enzymatic activities allowing hydrolysation of triglycerides. Unfortunately, microbes with the combination of substrate-utilization and PHA production are not currently available, and the strategies to be adopted are the use of costly commercial enzymes, or genetic modification of microorganisms exhibiting high PHA product yields. In the present work, after a general investigation on the ability of Cupriavidus necator to grow on a number of fatty substrates, the possibility to enhance PHA production by limiting intracellular depolymerisation, was investigated. By knocking out the related phaZ1 gene, the construction of C. necator recombinant strains impaired in depolymerase (PhaZ1) activity was achieved. The polymer yield of the recombinant strain was finally compared to that of the parental C. necator DSM 545

Innately robust yeast strains isolated from grape marc have a great potential for lignocellulosic ethanol production

Bioethanol from lignocellulose is an attractive alternative to fossil fuels, and Saccharomyces cerevisiae is the most important ethanol producer. However, yeast cells are challenged by various environmental stresses during ethanol production on an industrial scale, and robust strains with a high tolerance to inhibitors, temperature and osmolality are needed for the effective feasibility of lignocellulosic ethanol. To search for such innately more resistant yeast, we selected grape marc as an extreme environment due to limited nutrients, exposure to solar radiation, temperature fluctuations, weak acids and ethanol. Using a temperature of 40 A degrees C as the key selection criterion, we isolated 120 novel S. cerevisiae strains from grape marc and found high ethanol yields (up to 92 % of the theoretical maximum) when inoculated at 40 A degrees C in minimal media with a high sugar concentration. For the first time, this work assessed yeast tolerance to inhibitors at 40 A degrees C, and the newly isolated yeast strains displayed interesting abilities to withstand increasing levels of single inhibitors or cocktails containing a mixture of inhibitory compounds. The newly isolated strains showed significantly higher fermentative abilities and tolerance to inhibitors than the industrial and commercial benchmark S. cerevisiae strains. The strong physiological robustness and fitness of a few of these S. cerevisiae yeast strains support their potential industrial application and encourage further studies in genetic engineering to enhance their ethanol performance in terms of rate and yield through the co-fermentation of all available carbon sources

Production of bioethanol from multiple waste streams of rice milling

This work describes the feasibility of using rice milling by-products as feedstock for bioethanol. Starch-rich residues (rice bran, broken, unripe and discolored rice) were individually fermented (20% w/v) through Consolidated Bioprocessing by two industrial engineered yeast secreting fungal amylases. Rice husk (20% w/v), mainly composed by lignocellulose, was pre-treated at 55 degrees C with alkaline peroxide, saccharified through optimized dosages of commercial enzymes (Cellic (R) CTec2) and fermented by the recombinant strains. Finally, a blend of all the rice by-products, formulated as a mixture (20% w/v) according to their proportions at milling plants, were co-processed to ethanol by optimized pre-treatment, saccharification and fermentation by amylolytic strains. Fermenting efficiency for each by-product was high (above 88% of the theoretical) and further confirmed on the blend of residues (nearly 52 g/L ethanol). These results demonstrated for the first time that the co-conversion of multiple waste streams is a promising option for second generation ethanol production

Utilisation of wheat bran as a substrate for bioethanol production using recombinant cellulases and amylolytic yeast

Wheat bran, generated from the milling of wheat, represents a promising feedstock for the production of bioethanol. This substrate consists of three main components: starch, hemicellulose and cellulose. The optimal conditions for wheat bran hydrolysis have been determined using a recombinant cellulase cocktail (RCC), which contains two cellobiohydrolases, an endoglucanase and a beta-glucosidase. The 10% (w/v, expressed in terms of dry matter) substrate loading yielded the most glucose, while the 2% loading gave the best hydrolysis efficiency (degree of saccharification) using unmilled wheat bran. The ethanol production of two industrial amylolytic Saccharomyces cerevisiae strains, MEL2[TLG1-SFA1] and M2n [TLG1-SFA1], were compared in a simultaneous saccharification and fermentation (SSF) for 10% wheat bran loading with or without the supplementation of optimised RCC. The recombinant yeasts. cerevisiae MEL2[TLG1-SFA1] and M2n[TLG1-SFA1] completely hydrolysed wheat bran's starch producing similar amounts of ethanol (5.3 +/- 0.14 g/L and 5.0 +/- 0.09 g/L, respectively). Supplementing SSF with RCC resulted in additional ethanol production of about 2.0 g/L. Scanning electron microscopy confirmed the effectiveness of both RCC and engineered amylolytic strains in terms of cellulose and starch depolymerisatio

Autotrophic production of polyhydroxyalkanoates using acidogenic-derived H2 and CO2 from fruit waste

[Abstract] The environmental concerns regarding fossil plastics call for alternative biopolymers such as polyhydroxyalkanoates (PHAs) whose manufacturing costs are however still too elevated. Autotrophic microbes like Cupriavidus necator, able to convert CO2 and H2 into PHAs, offer an additional strategy. Typically, the preferred source for CO2 and H2 are expensive pure gases or syngas, which has toxic compounds for most PHAs-accumulating strains. In this work, for the first time, H2 and CO2 originating from an acidogenic reactor were converted autotrophically into poly(3-hydroxybutyrate) P(3HB). During the first stage, a mixed microbial community continuously catabolized melon waste into H2 (26.7 %) and CO2 (49.2 %) that were then used in a second bioreactor by C. necator DSM 545 to accumulate 1.7 g/L P(3HB). Additionally, the VFAs (13 gCOD/L) produced during acidogenesis were processed into 2.7 g/L of P(3HB-co-3HV). This is the first proof-of-concept of using acidogenic-derived H2 and CO2 from fruit waste to produce PHAs.This work was funded by Università degli Studi di Padova through BIRD210708/21 and DOR2352129/23 and Xunta de Galicia (Spain) through the Competitive Reference Research Groups grant (ED431C 2021/55 project). The authors thank FRANCESCON OP SOC.AGR. SOC.CONS. a.r.l. for the donation of the melon waste. Additionally, the authors thank Professor Veiga and Professor Kennes’s team for the technical support with the maintenance of bioreactors.Xunta de Galicia; ED431C 2021/55Italia. Università degli Studi di Padova; BIRD210708/21Italia. Università degli Studi di Padova; DOR2352129/2

Bacterial Production of PHAs from Lipid-Rich by-Products

Background and Objective: Due to oil shortage and environmental problems, synthetic plastics will surely be replaced by alternative, biodegradable materials. A possible good example could be polyhydroxyalkanoates, and the inexpensive agricultural fatty byproducts could be usefully converted to polyhydroxyalkanoates by properly selected and/or developed microbes.Material and Methods: Among the more common by-products available, a variety of lipid-rich residues have been explored as substrate, such as crude glycerol from biodiesel, biodiesel obtained from fatty residues, and, from slaughterhouse, bacon rind, udder and tallow. In this paper, several new isolates and collection PHA-producing microbes have been screened for both lipolytic activities and polyhydroxyalkanoates production. The soil proved to be the most promising mining place to find new interesting microbial species, even better than more specific and selective environments such as slaughterhouses.Results and Conclusion: Remarkably, two of the collection strains used here, known to be polyhydroxyalkanoates producers, resulted as really promising, being able to grow directly on all the substrates tested and to produce variable amounts of the polymer, including the co-polymers P (3HB-co-3HV).Conflict of interest: The authors declare no conflict of interest

MICROBIAL PROCESSING OF ORGANIC WASTE STREAMS INTO PHAs AND OTHER HIGH VALUE BIO-PRODUCTS

In the last years, economic and environmental concerns arose for oil shortage and climate change; for these reasons the scientific community focused on possible oil substitutes. In this perspectives, the production of new energy, materials and chemicals of non-fossil origin, could be based on biological resources such as biomasses. The efforts of the microbiology group of DAFNAE are mainly devoted to the exploitation of waste and residual biomasses for the production of high value bio-products such as polyhydroxyalkanoates (PHAs), bioethanol and biohydrogen. PHAs are today considered among the most promising substitutes for petrol-based plastics nevertheless their substitution over the conventional plastics is limited by their expensive manufacturing because of the costly raw materials used as carbon sources and the complex downstream phase of PHAs recovery from bacterial cells. Possible solutions could be i) the utilization of cheap wastes of agro-food origin as carbon sources and ii) the simplification of downstream purification processes. To these aims, Cupriavidus necator DSM545, a well-known PHAs accumulator, has been genetically modified in order to acquire the ability of metabolizing lactose from whey (dairy industry) or lipids (from slaughterhouse) and the capacity to produce nuclease to facilitate downstream processes. In the first case, the modified strains resulted able to grow using whey or lipids as carbon sources, accumulating up to 30 and 60% of PHAs, respectively. In the second case, the recombinant C. necator DSM 545 resulted in an effective decrease of viscosity of bacterial cells lysates, thus avoiding the use of costly commercial nucleases for an efficient downstream. Bioethanol is a fuel obtained from renewable resources and it could be a promising alternative to petrol fuels. First generation bioethanol is mainly produced from corn and sugarcane, thus conflicting with food and feed production . On the contrary, bioethanol from residual and lignocellulosic biomass has environmental impact lower than fossil fuels and would not threaten food supplies. Unfortunately, Saccharomyces cerevisiae, the yeast used for industrial bioethanol production, is not equipped with suitable hydrolytic activities and thus cannot directly utilize starchy and lignocellulosic wastes as feedstock without the use of commercial enzymes. Recent studies were focused to develop a \u201cConsolidated bioprocessing\u201d (CBP), approach where a single yeast is able to hydrolyse starch and lignocellulose and ferment the resulting sugars into ethanol. . With this purpose, novel and robust S. cerevisiae strains were recently engineered at DAFNAE to secrete efficient cellulases and amylases for the efficient saccharification and fermentation of starchy and cellulosic by-products up to 65 g/L ethanol

a wooded riparian strip set up for nitrogen removal can affect the water flux microbial composition

This research is part of a project aimed at verifying the potential of a specifically assessed wooded riparian zone in removing excess of combined nitrogen from the Zero river flow for the reduction of nutrient input into Venice Lagoon. Specific objectives were pursued to determine seasonal fluctuations of the microbial populations from the input water to a drainage ditch, conveying back the flux into the river after passing through the soil of the wooded riparian strip. The bacterial communities were determined by combined approaches involving cultivation, microscopic methods and DNA based techniques to determine both culturable and total microbial community in water. The results indicate that the size of the bacterial population, including the culturable fraction, increases from the river to the drainage ditch especially on the warm season. The multiple approach here adopted enabled also to demonstrate that the special condition created in the buffer strip supports the development and the metabolism of the microbial community. The nature of the bacterial population, in terms of phylotypes distribution, was investigated by 16S rDNA analysis indicating that the most represented genera belong to Gamma-proteobacteria, which is known to include an exceeding number of important pathogens. In spring, the effect of the buffer strip seems to significantly reduce such a sub-population. The changes observed for the total bacterial community composition become much evident in summer, as revealed by both denaturing gradient gel electrophoresis cluster analysis and by the diversity index calculation. The hydraulic management coupled to the suspension of farming practices and the development of the woody and herbaceous vegetation resulted in a condition suitable for the containment of undesired microbiota (mainly during the spring season) while continuing to support denitrification activity (especially throughout the summer) as verified by the total nitrogen removal

Comparison of bacteriocins production from Enterococcus faecium strains in cheese whey and optimised commercial MRS medium

The production of bacteriocins from cheap substrates could be useful for many food industrial applications. This study aimed at determining the conditions needed for optimal production of enterocins SD1, SD2, SD3 and SD4 secreted by Enterococcus faecium strains SD1, SD2, SD3 and SD4, respectively. To our knowledge, this is the first use of cheese whey—a low-cost milk by-product—as a substrate for bacteriocin production by E. faecium; skimmed milk and MRS broths were used as reference media. This cheese manufacturing residue proved to be a promising substrate for the production of bacteriocins. However, the levels of secreted antimicrobial compounds were lower than those achieved by E. faecium strains in MRS broth. Bacteriocin production was affected strongly by physical and chemical factors such as growth temperature, time of incubation, pH, and the chemical composition of the culture medium. The optimal temperature and time of incubation supporting the highest bacteriocin production was determined for each strain. Different types, sources and amounts of organic nitrogen, sugar, and inorganic salts played an essential role in bacteriocin secretion. E. faecium strains SD1 and SD2—producing high bacteriocin levels both in cheese whey and skimmed milk—could be of great interest for potential applications in cheese-making

Biodiversity and bioprospecting of fungal endophytes from the Antarctic plant Colobanthus quitensis

Microorganisms from extreme environments are considered as a new and valuable reservoir of bioactive molecules of biotechnological interest and are also utilized as tools for enhancing tolerance to (a)biotic stresses in crops. In this study, the fungal endophytic community associated with the leaves of the Antarctic angiosperm Colobanthus quitensis was investigated as a new source of bioactive molecules. We isolated 132 fungal strains and taxonomically annotated 26 representative isolates, which mainly belonged to the Basidiomycota division. Selected isolates of Trametes sp., Lenzites sp., Sistotrema sp., and Peniophora sp. displayed broad extracellular enzymatic profiles; fungal extracts from some of them showed dose-dependent antitumor activity and inhibited the formation of amyloid fibrils of α-synuclein and its pathological mutant E46K. Selected fungal isolates were also able to promote secondary root development and fresh weight increase in Arabidopsis and tomato and antagonize the growth of pathogenic fungi harmful to crops. This study emphasizes the ecological and biotechnological relevance of fungi from the Antarctic ecosystem and provides clues to the bioprospecting of Antarctic Basidiomycetes fungi for industrial, agricultural, and medical applications