Zymomonas mobilis as an emerging biotechnological chassis for the production of industrially relevant compounds

Zymomonas mobilis is a well-recognized ethanologenic bacterium with outstanding characteristics which make it a promising platform for the biotechnological production of relevant building blocks and fine chemicals compounds. In the last years, research has been focused on the physiological, genetic, and metabolic engineering strategies aiming at expanding Z. mobilis ability to metabolize lignocellulosic substrates toward biofuel production. With the expansion of the Z. mobilis molecular and computational modeling toolbox, the potential of this bacterium as a cell factory has been thoroughly explored. The number of genomic, transcriptomic, proteomic, and fluxomic data that is becoming available for this bacterium has increased. For this reason, in the forthcoming years, systems biology is expected to continue driving the improvement of Z. mobilis for current and emergent biotechnological applications. While the existing molecular toolbox allowed the creation of stable Z. mobilis strains with improved traits for pinpointed biotechnological applications, the development of new and more flexible tools is crucial to boost the engineering capabilities of this bacterium. Novel genetic toolkits based on the CRISPR-Cas9 system and recombineering have been recently used for the metabolic engineering of Z. mobilis. However, they are mostly at the proof-of-concept stage and need to be further improved. Graphical Abstract

Evaluation of different lignocellulosic substrates for cultivation of medicinal mushroom Ganoderma lucidum

Aim: The present study was formulated with an aim to evaluate different locally available residues from trees such as mixed saw dust, saw dust of coconut wood log, coconut leaf stalks/petiole, coconut coir waste, saw dust of areca nut wood log for cultivation of medicinal mushroom Ganoderma lucidum. Methodology: Locally available agro residues viz., mixed saw dust, saw dust of coconut wood log, chopped coconut leaf stalks/petiole, coconut coir waste, saw dust of areca nut wood log were mixed with 20% wheat bran as supplement and packed in bags at 175 g/bag, autoclaved and aseptically inoculated with grain spawn of G. lucidum and provided with different temperature and humidity conditions for production of fruiting bodies. Results: Among the substrates, coconut wood log saw dust supported early spawn run within 46.5 days and early pinhead production on day 54 followed by pinhead expansion in 62.3 days and first harvest within 70.5 days. The average number of fruiting bodies were also higher (5.75 numbers/bag) with an average weight of 13.5 g/fruiting body that gave significantly higher yield of 77.5 g/175 g substrate with bioefficiency of 44.3% in a cropping cycle of 100.5 days compared to other substrates. Interpretation: The results show that coconut wood log saw dust substrate offers great scope for artificial cultivation of G. lucidum with a significant bioefficiency of 44.3 %.

Production and characterization of cellulase from mushroom (Pleurotus ostreatus) for effective degradation of cellulose

Cellulases are a group of hydrolytic enzymes capable of hydrolyzing the most abundant organic polymer that means cellulose to smaller sugar components including glucose subunits. The aim of this study was to screen cellulase producing oyster mushroom collected from Eucalyptus tree bark to evaluate the in vitro production of cellulase by Pleurotus ostreatus using different lignocellulosic substrates, and to characterize the cellulase produced with respect to changes in pH, temperature, and concentration of substrates. A total of ten mushroom specimens were randomly collected from Eucalyptus tree bark in the premise of Holetta Agricultural Research Center campus. All of the collected mushroom specimens were identified morphologically and biochemically as Pleurotus ostreatus and also screened for their ability to produce cellulase by detecting and measuring zone of hydrolysis on commercial media containing Carbxymethyl Cellulose (CMC) as the sole carbon source. These mushroom specimens were cultivated using both solid state fermentation and submerged fermentation systems supplemented with different lignocellulosic substrates (wheat straw, teff straw, bean straw, wood fiber and Eucalyptus tree bark) to identify the most suitable medium for the production of cellulase. The highest enzyme production was obtained on bean straw and wheat straw which resulted in 0.191 U/ml, 0.868 U/ml and 0.389 U/ml; and 0.216 U/ml, 0.444 U/ml, and 0.245 U/ml of FPase, CMCase, and β-glucosidase in solid state fermentation. The lowest values were, however, obtained in media containing wood fiber in both solid state fermentation and submerged fermentation. Comparison of the lignocellulosic substrates revealed that wheat straw was selected for further growth parameter optimization. The production of cellulase was higher at the 5th day of incubation period, and the optimum pH and incubation temperature required for maximum cellulase production were 4 and 30°C, respectively. Sucrose and Yeast extract at 1% concentration were found to be the most preferred carbon and nitrogen sources for cellulase production by Pleurotus ostreatus. The optimum pH and temperature for cell_free cellulase activity on were found to be 4 and 50°C, respectively. Generally the cellulases produced by Pleurotus ostreatus were stable and active at temperatures ranging from 20-50°C. These characteristics hopefully would make this enzyme potentially attractive in a variety of industrial applications including animal feed treatments. There was a linear relationship between cellulase and its substrate concentration for there was an increase in activity with increase in substrate concentration. The relationship between rate of reaction and substrate concentration depended on the affinity of the enzyme for its substrate. Finally the cellulase was tested for its ability to saccharify agricultural wastes and the results showed the highest release of sugars from wheat straw.

Biodiesel Production From Lignocellulosic Biomass Using Oleaginous Microbes: Prospects for Integrated Biofuel Production

Biodiesel is an eco-friendly, renewable, and potential liquid biofuel mitigating greenhouse gas emissions. Biodiesel has been produced initially from vegetable oils, non-edible oils, and waste oils. However, these feedstocks have several disadvantages such as requirement of land and labor and remain expensive. Similarly, in reference to waste oils, the feedstock content is succinct in supply and unable to meet the demand. Recent studies demonstrated utilization of lignocellulosic substrates for biodiesel production using oleaginous microorganisms. These microbes accumulate higher lipid content under stress conditions, whose lipid composition is similar to vegetable oils. In this paper, feedstocks used for biodiesel production such as vegetable oils, non-edible oils, oleaginous microalgae, fungi, yeast, and bacteria have been illustrated. Thereafter, steps enumerated in biodiesel production from lignocellulosic substrates through pretreatment, saccharification and oleaginous microbe-mediated fermentation, lipid extraction, transesterification, and purification of biodiesel are discussed. Besides, the importance of metabolic engineering in ensuring biofuels and biorefinery and a brief note on integration of liquid biofuels have been included that have significant importance in terms of circular economy aspects.

A multi-omics approach to lignocellulolytic enzyme discovery reveals a new ligninase activity from Parascedosporium putredinis NO1

Lignocellulose, the structural component of plant cells, is a major agricultural byproduct and the most abundant terrestrial source of biopolymers on Earth. The complex and insoluble nature of lignocellulose limits its conversion into value-added commodities, and currently, efficient transformation requires expensive pretreatments and high loadings of enzymes. Here, we report on a fungus from the Parascedosporium genus, isolated from a wheat-straw composting community, that secretes a large and diverse array of carbohydrate-active enzymes (CAZymes) when grown on lignocellulosic substrates. We describe an oxidase activity that cleaves the major β-ether units in lignin, thereby releasing the flavonoid tricin from monocot lignin and enhancing the digestion of lignocellulose by polysaccharidase mixtures. We show that the enzyme, which holds potential for the biorefining industry, is widely distributed among lignocellulose-degrading fungi from the Sordariomycetes phylum.