A Thermostable Lipase Isolated from Brevibacillus thermoruber Strain 7 Degrades Ɛ-Polycaprolactone.

The tremendous problem with plastic waste accumulation has determined an interest in biodegradation by effective degraders and their enzymes, such as thermophilic enzymes, which are characterized by high catalytic rates, thermostability, and optimum temperatures close to the melting points of some plastics. In the present work, we report on the ability of a thermophilic lipase, by Brevibacillus thermoruber strain 7, to degrade Ɛ-polycaprolactone (PCL), as well as the enzyme purification, the characterization of its physicochemical properties, the product degradation, and its disruptive effect on the PCL surface. The pure enzyme showed the highest reported optimum temperature at 55 °C and a pH of 7.5, while its half-life at 60 °C was more than five hours. Its substrate specificity referred the enzyme to the subgroup of lipases in the esterase group. A strong inhibitory effect was observed by detergents, inhibitors, and Fe3+ while Ca2+ enhanced its activity. The monomer Ɛ-caprolactone was a main product of the enzyme degradation. Similar elution profiles of the products received after treatment with ultra-concentrate and pure enzyme were observed. The significant changes in PCL appearance comprising the formation of shallower or deeper in-folds were observed after a week of incubation. The valuable enzyme properties of the lipase from Brevibacillus thermoruber strain 7, which caused a comparatively quick degradation of PCL, suggests further possible exploration of the enzyme for effective and environment-friendly degradation of PCL wastes in the area of thermal basins, or in thermophilic remediation processes.

Degradation of Poly(ε-caprolactone) by a Thermophilic Community and Brevibacillus thermoruber Strain 7 Isolated from Bulgarian Hot Spring.

The continual plastic accumulation in the environment and the hazardous consequences determine the interest in thermophiles as possible effective plastic degraders, due to their unique metabolic mechanisms and change of plastic properties at elevated temperatures. PCL is one of major biodegradable plastics with promising application to replace existing non-biodegradable polymers. Metagenomic analysis of the phylogenetic diversity in plastic contaminated area of Marikostinovo hot spring, Bulgaria revealed a higher number taxonomic groups (11) in the sample enriched without plastic (Marikostinovo community, control sample, MKC-C) than in that enriched in the presence of poly-ε-caprolactone (PCL) (MKC-P), (7). A strong domination of the phylum Proteobacteria was observed for MKC-C, while the dominant phyla in MKC-P were Deinococcus-Thermus and Firmicutes. Among the strains isolated from MKC-P, the highest esterase activity was registered for Brevibacillus thermoruber strain 7 at 55 °C. Its co-cultivation with another isolate resulted in ~10% increase in enzyme activity. During a 28-day biodegradation process, a decrease in PCL molecular weight and weight loss were established resulting in 100% degradation by MKC-P and 63.6% by strain 7. PCL degradation intermediate profiles for MKC-P and pure strain were similar. Broken plastic pieces from PCL surface and formation of a biofilm by MKC-P were observed by SEM, while the pure strain caused significant deformation of PCL probes without biofilm formation.

Plastic Degradation by Extremophilic Bacteria.

Intensive exploitation, poor recycling, low repeatable use, and unusual resistance of plastics to environmental and microbiological action result in accumulation of huge waste amounts in terrestrial and marine environments, causing enormous hazard for human and animal life. In the last decades, much scientific interest has been focused on plastic biodegradation. Due to the comparatively short evolutionary period of their appearance in nature, sufficiently effective enzymes for their biodegradation are not available. Plastics are designed for use in conditions typical for human activity, and their physicochemical properties roughly change at extreme environmental parameters like low temperatures, salt, or low or high pH that are typical for the life of extremophilic microorganisms and the activity of their enzymes. This review represents a first attempt to summarize the extraordinarily limited information on biodegradation of conventional synthetic plastics by thermophilic, alkaliphilic, halophilic, and psychrophilic bacteria in natural environments and laboratory conditions. Most of the available data was reported in the last several years and concerns moderate extremophiles. Two main questions are highlighted in it: which extremophilic bacteria and their enzymes are reported to be involved in the degradation of different synthetic plastics, and what could be the impact of extremophiles in future technologies for resolving of pollution problems.

High bioreactor production and emulsifying activity of an unusual exopolymer by Chromohalobacter canadensis 28.

Unusual composition of an exopolymer (EP) from an obligate halophilic bacterium Chromohalobacter canadensis 28 has triggered an interest in development of an effective bioreactor process for its production. Its synthesis was investigated in 2-L bioreactor at agitation speeds at interval 600-1000 rpm, at a constant air flow rate of 0.5 vvm; aeration rates of 0.5, 1.0, and 1.5 vvm were tested at constant agitation rate of 900 rpm. EP production was affected by both, agitation and aeration. As a result twofold increase of EP yield was observed and additionally increased up to 3.08 mg/mL in a presence of surfactants. For effective scale-up of bioreactors mass transfer parameters were estimated and lowest values of KLa obtained for the highest productivity fermentation was established. Emulsification activity of EP exceeded that of trade hydrocolloids xanthan, guar gum, and cellulose. A good synergism between EP and commercial cellulose proved its potential exploration as an enhancer of emulsifying properties of trade emulsions. A pronounced lipophilic effect of EP was established toward olive oil and liquid paraffin. Cultivation of human keratinocyte cells (HaCaT) with crude EP and purified γ-polyglutamic acid (PGA) showed higher viability than control group.

Extracellular polymer substance synthesized by a halophilic bacterium Chromohalobacter canadensis 28.

Halophilic microorganisms are producers of a lot of new compounds whose properties suggest promising perspectives for their biotechnological exploration. Moderate halophilic bacterium Chromohalobacter canadensis 28 was isolated from Pomorie salterns as an extracellular polymer substance (EP) producer. The best carbon source for extracellular polymer production was found to be lactose, a sugar received as a by-product from the dairy industry. After optimization of the culture medium and physicochemical conditions for cultivation, polymer biosynthesis increased more than 2-fold. The highest level of extracellular polymer synthesis by C. canadensis 28 was observed in an unusually high NaCl concentration (15% w/v). Chemical analysis of the purified polymer revealed the presence of an exopolysaccharide (EPS) fraction (14.3% w/w) and protein fraction (72% w/w). HPLC analysis of the protein fraction showed the main presence of polyglutamic acid (PGA) (75.7% w/w). EPS fraction analysis revealed the following sugar composition (% w/w): glucosamine 36.7, glucose 32.3, rhamnose 25.4, xylose 1.7, and not identified sugar 3.9. The hydrogel formed by PGA and EPS fractions showed high swelling behavior, very good emulsifying and stabilizing properties, and good foaming ability. This is the first report for halophilic bacterium able to synthesize a polymer containing PGA fraction. The synthesized biopolymer shows an extremely high hydrophilicity, due to the simultaneous presence of PGA and EPS. The analysis of its functional properties and the presence of glucosamine in the highest proportion in EPS fraction clearly determine the potential of EP synthesized by C. canadensis 28 for application in the cosmetics industry.

Isolation of novel alkaliphilic bacillus strains for cyclodextrin glucanotransferase production.

New alkaliphilic Bacillus producers of cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) were isolated from 17 Bulgarian alkaline and normal habitats (springs and soils) by three steps of a selection. None of the isolates obtained, producing CGTase, appeared to be thermophilic in character. One hundred and thirty-seven strains were estimated for CGTase activity by batch cultivation in a liquid alkaline medium. Twenty-seven of them had a detectable CGTase activity in their culture supernatants under the enzyme assay conditions, despite of the significant growth of all isolates. The phenotypic properties of three selected strains (20RF, 8SB and 24WE) were determined. They were aerobic endospore-forming Bacillus strains: two of them were obligated alkaliphiles (20RF and 8SB) and one, alkalitolerant (24WE). Both obligated alkaliphiles were further characterised by 16S rRNA analysis. According to the full 16S rRNA gene sequences obtained and deposited to the NCBI GenBank database, both isolated obligated alkaliphiles 20RF and 8SB were clustered into the group of alkaliphilic Bacillus species. The exhibited CGTase production by them (230-250 U ml(-1) for 20RF and 130-160 U ml(-1) for 8SB) defined these new isolates as promising producers of the enzyme, especially Bacillus sp. 8SB synthesising thermostable alkaline beta-CGTase. Both new enzymes from 20RF and 8SB Bacillus strains formed only two types of cyclodextrins, beta and gamma, which could be of interest for their easy separation and industrial production.