PUF-Immobilized Bjerkandera adusta DSM 3375 as a Tool for Bioremediation of Creosote Oil Contaminated Soil.

Creosote oil, a byproduct of coal distillation, is primarily composed of aromatic compounds that are difficult to degrade, such as polycyclic aromatic hydrocarbons, phenolic compounds, and N-, S-, and O-heterocyclic compounds. Despite its toxicity and carcinogenicity, it is still often used to impregnate wood, which has a particularly negative impact on the condition of the soil in plants that impregnate wooden materials. Therefore, a rapid, effective, and eco-friendly technique for eliminating the creosote in this soil must be developed. The research focused on obtaining a preparation of Bjerkandera adusta DSM 3375 mycelium immobilized in polyurethane foam (PUF). It contained mold cells in the amount of 1.10 ± 0.09 g (DW)/g of the carrier. The obtained enzyme preparation was used in the bioremediation of soil contaminated with creosote (2% w/w). The results showed that applying the PUF-immobilized mycelium of B. adusta DSM 3375 over 5, 10, and 15 weeks of bioremediation, respectively, removed 19, 30, and 35% of creosote from the soil. After 15 weeks, a 73, 79, and 72% level of degradation of fluoranthene, pyrene, and fluorene, respectively, had occurred. The immobilized cells have the potential for large-scale study, since they can degrade creosote oil in soil.

Metabolic Potential of Halophilic Filamentous Fungi-Current Perspective.

Salty environments are widely known to be inhospitable to most microorganisms. For centuries salt has been used as a food preservative, while highly saline environments were considered uninhabited by organisms, and if habited, only by prokaryotic ones. Nowadays, we know that filamentous fungi are widespread in many saline habitats very often characterized also by other extremes, for example, very low or high temperature, lack of light, high pressure, or low water activity. However, fungi are still the least understood organisms among halophiles, even though they have been shown to counteract these unfavorable conditions by producing multiple secondary metabolites with interesting properties or unique biomolecules as one of their survival strategies. In this review, we focused on biomolecules obtained from halophilic filamentous fungi such as enzymes, pigments, biosurfactants, and osmoprotectants.

Polycistronic Expression System for Pichia pastoris Composed of Chitino- and Chitosanolytic Enzymes.

Chitin is one of the most abundant biopolymers. Due to its recalcitrant nature and insolubility in accessible solvents, it is often considered waste and not a bioresource. The products of chitin modification such as chitosan and chitooligosaccharides are highly sought, but their preparation is a challenging process, typically performed with thermochemical methods that lack specificities and generate hazardous waste. Enzymatic treatment is a promising alternative to these methods, but the preparation of multiple biocatalysts is costly. In this manuscript, we biochemically characterised chitin deacetylases of Mucor circinelloides IBT-83 and utilised one of them for the construction of the first eukaryotic, polycistronic expression system employing self-processing 2A sequences. The three chitin-processing enzymes; chitin deacetylase of M. circinelloides IBT-83, chitinase from Thermomyces lanuginosus, and chitosanase from Aspergillus fumigatus were expressed under the control of the same promoter in methylotrophic yeast Pichia pastoris and characterised for their synergistic action towards their respective substrates.

When Salt Meddles Between Plant, Soil, and Microorganisms.

In extreme environments, the relationships between species are often exclusive and based on complex mechanisms. This review aims to give an overview of the microbial ecology of saline soils, but in particular of what is known about the interaction between plants and their soil microbiome, and the mechanisms linked to higher resistance of some plants to harsh saline soil conditions. Agricultural soils affected by salinity is a matter of concern in many countries. Soil salinization is caused by readily soluble salts containing anions like chloride, sulphate and nitrate, as well as sodium and potassium cations. Salinity harms plants because it affects their photosynthesis, respiration, distribution of assimilates and causes wilting, drying, and death of entire organs. Despite these life-unfavorable conditions, saline soils are unique ecological niches inhabited by extremophilic microorganisms that have specific adaptation strategies. Important traits related to the resistance to salinity are also associated with the rhizosphere-microbiota and the endophytic compartments of plants. For some years now, there have been studies dedicated to the isolation and characterization of species of plants' endophytes living in extreme environments. The metabolic and biotechnological potential of some of these microorganisms is promising. However, the selection of microorganisms capable of living in association with host plants and promoting their survival under stressful conditions is only just beginning. Understanding the mechanisms of these processes and the specificity of such interactions will allow us to focus our efforts on species that can potentially be used as beneficial bioinoculants for crops.

Biodegradation of slop oil by endophytic Bacillus cereus EN18 coupled with lipase from Rhizomucor miehei (Palatase®).

Removal of slop oil, a by-product of oil refining, also obtained in cleaning up of oil tanks and filters is a difficult issue. High content of hydrocarbons (C3-C40) and other organic compounds makes this waste difficult to eliminate from the environment. The purpose of this investigation was to combine bacterial degradation by endophytic Bacillus cereus EN18 with biotransformation performed using lipase enzyme preparation (Palatase®) to remove recalcitrant compounds present in slop oil from the environment. Endophytic B. cereus EN18 was able to biodegrade up to 40% of slop oil while supplementation with lipase improved the efficiency of contamination removal in about one third. Also the use of lipase enzyme preparation resulted in higher microbial activity of B. cereus EN18 bacterial strain, as well as higher concentration of fatty acids in the culture medium, which indicates higher degradation efficiency. Obtained results suggest that lipase preparation from Rhizomucor miehei (Palatase®) may be a useful agent to improve microbial degradation of recalcitrant pollutants, like slop oil in water environments. GC and spectrometric analysis revealed that hydrocarbons from slop oil were effectively degraded while using both microbial degradation and lipase catalysis.

Enzymatic Modifications of Chitin, Chitosan, and Chitooligosaccharides.

Chitin and its N-deacetylated derivative chitosan are two biological polymers that have found numerous applications in recent years, but their further deployment suffers from limitations in obtaining a defined structure of the polymers using traditional conversion methods. The disadvantages of the currently used industrial methods of chitosan manufacturing and the increasing demand for a broad range of novel chitosan oligosaccharides (COS) with a fully defined architecture increase interest in chitin and chitosan-modifying enzymes. Enzymes such as chitinases, chitosanases, chitin deacetylases, and recently discovered lytic polysaccharide monooxygenases had attracted considerable interest in recent years. These proteins are already useful tools toward the biotechnological transformation of chitin into chitosan and chitooligosaccharides, especially when a controlled non-degradative and well-defined process is required. This review describes traditional and novel enzymatic methods of modification of chitin and its derivatives. Recent advances in chitin processing, discovery of increasing number of new, well-characterized enzymes and development of genetic engineering methods result in rapid expansion of the field. Enzymatic modification of chitin and chitosan may soon become competitive to conventional conversion methods.

Oil accumulation and in situ trans/esterification by lipolytic fungal biomass.

The goal of this study was to increase the cost-effectiveness of oil production by an oleaginous and lipolytic strain M. circinelloides IBT-83, by optimizing both lipids accumulation in the mycelium containing intracellular lipases, and a one-step process coupling lipids extraction and enzymatic trans/esterification. In optimal conditions (culture medium composed of corn steep solids, plant oil, glucose and NO3-) over 50gd.w./dm3 of biomass containing over 60% of lipids was produced. The lipids extracted with acetone or petroleum ether contain free fatty acids and triacylglycerols. The supplementation of the second solvent with alcohol results in enzymatic trans/esterification of lipids with the yield of over 80% of esters in 1 h. To our knowledge, this is the first suggestion to convert fungal oils into esters during their extraction using intracellular lipases contained in the same fungus. What is important, it is possible to obtain a second product, lipase preparation, in this process.

Scale-up of PUF-immobilized fungal chitosanase-lipase preparation production.

Mucor circinelloides IBT-83 mycelium that exhibits both lipolytic (AL) and chitosanolytic (ACH) activities was immobilized into polyurethane foam in a 30 L laboratory fermenter. The process of immobilization was investigated in terms of the carrier porosity, its type, amount, and shape, location inside the fermenter, mixing, and aeration parameters during the culture, as well as downstream processing operations. The selected conditions allowed for immobilization of approximately 7 g of defatted and dried mycelium in 1 g of carrier, i.e., seven times more than achievable in 1 L shake-flasks. Enzymatic preparation obtained by this method exhibited both the chitosanolytic (ACH 432.5 ± 6.8 unit/g) and lipolytic (AL 150.0 ± 9.3 U/g) activities. The immobilized preparation was successfully used in chitosan hydrolysis to produce chitooligosaccharides and low molecular weight chitosan, as well as in waste fats degradation and in esters synthesis in nonaqueous media. It was found that the half-life of immobilized preparations stored at room temperature is on average of 200 days.