Biodegradation of rubber in cultures of Rhodococcus rhodochrous and by its enzyme latex clearing protein.

The biodegradation of rubber materials is considered as a sustainable recycling alternative, highlighting the use of microorganisms and enzymes in oxidative processes of natural rubber. Currently, the main challenge is the treatment of rubber materials such as waste tyres, where the mixture of rubber polymers with different additives and the cross-linked structure obtained due to the vulcanisation process positions them as highly persistent materials. This study characterises the degradation of different rubber-containing substrates in in vivo and in vitro processes using the bacterium Rhodococcus rhodochrous and the oxygenase latex clearing protein (Lcp) from the same strain. For the first time, the degradation of polyisoprene particles in liquid cultures of R. rhodochrous was analysed, obtaining up to 19.32% mass loss of the polymer when using it as the only carbon source. Scanning electron microscopy analysis demonstrated surface alteration of pure polyisoprene and vulcanised rubber particles after 2 weeks of incubation. The enzyme LcpRR was produced in bioreactors under rhamnose induction and its activity characterised in oxygen consumption assays at different enzyme concentrations. A maximum consumption of 28.38 µmolO2/min was obtained by adding 100 µg/mL LcpRR to a 2% (v/v) latex emulsion as substrate. The bioconversion of natural rubber into reaction degradation products or oligoisoprenoids was calculated to be 32.54%. Furthermore, the mass distribution of the oligoisoprenoids was analysed by liquid chromatography coupled to mass spectrometry (LC-MS) and 17 degradation products, ranging from C20 to C100 oligoisoprenoids, were identified. The multi-enzymatic degradation capacity of R. rhodochrous positions it as a model microorganism in complex degradation processes such as in the case of tyre waste.

Molecular weight and guluronic/mannuronic ratio of alginate produced by Azotobacter vinelandii at two bioreactor scales under diazotrophic conditions.

Alginates can be used to elaborate hydrogels, and their properties depend on the molecular weight (MW) and the guluronic (G) and mannuronic (M) composition. In this study, the MW and G/M ratio were evaluated in cultures of Azotobacter vinelandii to 3 and 30 L scales at different oxygen transfer rates (OTRs) under diazotrophic conditions. An increase in the maximum OTR (OTRmax) improved the alginate production, reaching 3.3 ± 0.2 g L-1. In the cultures conducted to an OTR of 10.4 mmol L-1 h-1 (500 rpm), the G/M increased during the cell growth phase and decreased during the stationary phase; whereas, in the cultures at 19.2 mmol L-1 h-1 was constant throughout the cultivation. A higher alginate MW (520 ± 43 kDa) and G/M ratio (0.86 ± 0.01) were obtained in the cultures conducted at 10.4 mmol L-1 h-1. The OTR as a criterion to scale up alginate production allowed to replicate the concentration and the alginate production rate; however, it was not possible reproduce the MW and G/M ratio. Under a similar specific oxygen uptake rate (qO2) (approximately 65 mmol g-1 h-1) the alginate MW was similar (approximately 365 kDa) in both scales. The evidences revealed that the qO2 can be a parameter adequate to produce alginate MW similar in two bioreactor scales. Overall, the results have shown that the alginate composition could be affected by cellular respiration, and from a technological perspective the evidences contribute to the design process based on oxygen consumption to produce alginates defined.

Impact of additives of commercial rubber compounds on the microbial and enzymatic degradation of poly(cis-1,4-isoprene).

Much fundamental research has already been performed to understand the mechanism of microbial rubber degradation. Due to the increasing amount of rubber waste, biotechnical methods to degrade that particular waste are strongly needed. The present study evaluates whether a microbial or an enzymatic process is more suitable for efficient biodegradation, due to less sensitivity towards rubber additives. Therefore we investigated the impact of 15 different frequently used rubber additives on cells of the potent rubber degrader Gordonia polyisoprenivorans VH2 and the enzyme Lcp1VH2. For this, cells were grown on poly(cis-1,4-isoprene) in presence of these rubber additives. Furthermore, the effect of those additives on the enzymatic cleavage of poly(cis-1,4-isoprene) by Lcp1VH2 was determined by in vitro studies. It was observed that additives, used to accelerate the vulcanization process, like N-cyclohexyl-2-benzothiazolesulfenamide and zinc-bis(N,N-dibenzyl-dithiocarbamate), are diminishing the growth of the microorganism depending on their concentration-higher toxicity with increasing concentration. In contrast, sulfur prevents cell growth, but does not affect Lcp1VH2. Stearic acid and paraffin wax were found to be consumed by G. polyisoprenivorans VH2. Plasticizers mainly prevent growth, but do not interfere with the enzyme activity. This study identified antioxidants as the most interfering group of additives for microbial and enzymatic rubber degradation. It was found that the in vitro degradation by Lcp1VH2 is much more resistant and less sensitive towards the investigated rubber additives, when compared to the in vivo approach. Therefore, an enzymatic process might be a promising method to enhance rubber degradation.

Coenzyme Q production by metabolic engineered Escherichia coli strains in defined medium.

Coenzyme Q (CoQ) plays an important role as an electron transporter in the respiratory chain. It is formed from a benzoquinone ring and an isoprenoid chain of a specific length depending on the organism. We constructed an engineered Escherichia coli strain (menF) unable to produce demethylmenaquinone and menaquinone, compounds that compete for both chorismate, precursor of the benzoquinone ring, and the isoprenoid chain involved in CoQ biosynthesis. In addition, a mutant strain (entC) unable to produce enterobactin, high-affinity siderophore, synthesized from chorismate, and a double mutant (entC-menF) were constructed. The use of glucose or glycerol as carbon sources was also evaluated for the production of CoQ8 in these strains. The double mutant (entC-menF) showed 18% increase in CoQ8-specific content compared to the control strain; however, the single-mutant strains did not show statistically significant differences in CoQ8-specific content respect to the control, in glucose medium in bioreactor experiments. Glycerol was significantly superior compared to glucose for the production of CoQ8 in E. coli, where the CoQ8-specific content increased 126% and 53% in the control and double-mutant strain, respectively. The expression of genes related to CoQ8 biosynthesis is reported, where the entC-menF double-mutant strain showed a significant increase in the expression of CoQ8 biosynthesis-related genes when glycerol was used as sole carbon source. The control strain did not show gene expression difference between both carbon sources, indicating a possible regulation at a different level.

Effects of turning aeration and the initial carbon/nitrogen ratio on the biodegradation of polylactic acid under controlled conditions.

Plastic pollution is primarily caused by the accumulation of petroleum-derived plastics, as they tend to degrade slowly. Sustainable alternatives to these materials are bio-based and biodegradable plastics, such as polylactic acid (PLA). In this study, we assessed how turning aeration and the initial carbon/nitrogen (C/N) ratio impact PLA biodegradation. The study was carried out under controlled composting conditions, over 180 days, with the aim of decreasing the biodegradation time of the PLA. Apple pomace, rice husk, grape pomace compost, and PLA were used as substrates in the composting process. The experiments were conducted using three types of turning aeration: without turning, one turn per week, and two turns per week. Three initial C/N ratios were used: 20, 30, and 40. A stepwise temperature ramp was designed and implemented to simulate industrial composting conditions, which influence microbial activity and thus the rate of decomposition of substrates, including PLA. The data showed behavior; hence, a nonlinear regression model based on the logistic growth equation was used to predict the PLA biodegradation at the end of the composting process. The results showed that two turns per week with an initial C/N ratio of 30 or 40 led to a 90 % biodegradation of the PLA in 130 days. This degradation was verified by Fourier-Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscopy (SEM).

Characterization of the conversion system of natural rubber to poly(3-Hydroxyalkanoate) in Piscinibacter gummiphilus strain NS21T.

Poly(3-hydroxyalkanoate) (PHA), a bacteria-synthesized biodegradable polyester, is a useful alternative to fossil resources, and current systems for its production rely predominantly on edible resources, raising concerns about microbial competition for nutrients. Therefore, we investigated mechanisms underlying PHA production from non-edible resources by Piscinibacter gummiphilus strain NS21T. Strain NS21T can utilize natural rubber as a carbon source on solid media and potentially produces PHA. Gas chromatography and nuclear magnetic resonance analyses of NS21T cell extracts revealed the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(3-hydroxybutyrate) from natural rubber and glucose, respectively. Transcriptional analysis suggested that phaC is involved in PHA production. An increased PHBV accumulation rate under nitrogen-limiting conditions indicates the potential of this strain to be used as a PHBV production enhancement strategy. Furthermore, the disruption of PHA depolymerase genes resulted in enhanced PHA production, indicating the involvement of these genes in PHA degradation. These findings highlight the potential of NS21T for PHBV production from natural rubber, a non-edible resource.

MnO2/TiO2-Catalyzed ozonolysis: enhancing Pentachlorophenol degradation and understanding intermediates.

Pentachlorophenol is a pesticide widely known for its harmful effects on sewage, causing harm to the environment. In previous studies, our group identified adsorption as a crucial factor in catalytic ozonation processes, and subsequent observations revealed the catalyst's role in reducing toxicity during degradation. In this research, we quantified organochlorine intermediates and low molecular weight organic acids generated under optimal pH conditions (pH 9), with and without the catalyst. Additionally, we assessed the reactivity of these intermediates through theoretical calculations. Our findings indicate that the catalyst reduces the duration of intermediates. Additionally, the presence of CO2 suggests enhanced mineralization of pentachlorophenol, a process notably facilitated by the catalyst. Theoretical calculations, such as Fukui analysis, offer insights into potential pathways for the dechlorination of aromatic molecules by radicals like OH, indicating the significance of this pathway.

Relation of 3HV fraction and thermomechanical properties of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) produced by Azotobacter vinelandii OP.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) has attracted substantial attention as a promising material for industrial applications. In this study, different PHBV films with distinct 3-hydroxyvalerate (3HV) contents produced by Azotobacter vinelandii OP were evaluated. The 3HV fraction ranged from 18.6 to 36.7 mol%, and the number-average molecular weight (Mn) was between 238 and 434 kDa. In the bioreactor, a 3HV fraction (36.7 mol%) and an Mn value of 409 kDa were obtained with an oxygen transfer rate (OTR) of 12.5 mmol L-1 h-1. Thermal analysis measurements showed decreased melting (Tm) and glass transition (Tg) temperatures, and values with relatively high 3HV fractions indicated improved thermomechanical properties. The incorporation of the 3HV fraction in the PHBV chain improved the thermal stability of the films, reduced the polymer Tm, and affected the tensile strength. PHBV film with 36.7 mol% 3HV showed an increase in its tensile strength (51.8 MPa) and a decrease in its Tm (170.61 °C) compared with PHB. Finally, scanning electron microscopy (SEM) results revealed that the PHBV film with 32.8 mol% 3HV showed a degradation upon contact with soil, water, or soil bacteria, showing more porous surfaces after degradation. The latter phenomenon indicated that thermomechanical properties played an important role in biodegradation.

Efficient production of a polyhydroxyalkanoate by Azotobacter vinelandii OP using apple residues as promising feedstock.

Fruit residues are attractive substrates for the production of bacterial polyhydroxyalkanoates due to the high contents of fermentable sugars and the fast, simple, and efficient pretreatment methods required. In this study, apple residues, mainly apple peel, were used as the sole carbon source in cultures of the bacterium Azotobacter vinelandii OP to produce poly-3-hydroxybutyrate (P3HB). Conversion from the residue to total sugars was highly effective, achieving conversions of up to 65.4 % w w-1 when using 1 % v v-1 sulfuric acid and 58.3 % w w-1 in the absence of acid (only water). The cultures were evaluated at the shake-flask scale and in 3-L bioreactors using a defined medium under nitrogen starvation conditions. The results showed the production of up to 3.94 g L-1 P3HB in a bioreactor, reaching an accumulation of 67.3 % w w-1 when using apple residues. For the PHB obtained from the cultures with apple residues, a melting point of 179.99 °C and a maximum degradation temperature of 274.64 °C were calculated. A P3HB production strategy is shown using easily hydrolysable fruit residues to achieve production yields comparable to those obtained with pure sugars under similar cultivation conditions.

Bioengineering of biowaste to recover bioproducts and bioenergy: A circular economy approach towards sustainable zero-waste environment.

The inevitable need for waste valorisation and management has revolutionized the way in which the waste is visualised as a potential biorefinery for various product development rather than offensive trash. Biowaste has emerged as a potential feedstock to produce several value-added products. Bioenergy generation is one of the potential applications originating from the valorisation of biowaste. Bioenergy production requires analysis and optimization of various parameters such as biowaste composition and conversion potential to develop innovative and sustainable technologies for most effective utilization of biowaste with enhanced bioenergy production. In this context, feedstocks, such as food, agriculture, beverage, and municipal solid waste act as promising resources to produce renewable energy. Similarly, the concept of microbial fuel cells employing biowaste has clearly gained research focus in the past few decades. Despite of these potential benefits, the area of bioenergy generation still is in infancy and requires more interdisciplinary research to be sustainable alternatives. This review is aimed at analysing the bioconversion potential of biowaste to renewable energy. The possibility of valorising underutilized biowaste substrates is elaborately presented. In addition, the application and efficiency of microbial fuel cells in utilizing biowaste are described in detail taking into consideration of its great scope. Furthermore, the review addresses the significance bioreactor development for energy production along with major challenges and future prospects in bioenergy production. Based on this review it can be concluded that bioenergy production utilizing biowaste can clearly open new avenues in the field of waste valorisation and energy research. Systematic and strategic developments considering the techno economic feasibilities of this excellent energy generation process will make them a true sustainable alternative for conventional energy sources.

Microbial engineering strategies for synthetic microplastics clean up: A review on recent approaches.

Microplastics are the small fragments of the plastic molecules which find their applications in various routine products such as beauty products. Later, it was realized that it has several toxic effects on marine and terrestrial organisms. This review is an approach in understanding the microplastics, their origin, dispersal in the aquatic system, their biodegradation and factors affecting biodegradation. In addition, the paper discusses the major engineering approaches applied in microbial biotechnology. Specifically, it reviews microbial genetic engineering, such as PET-ase engineering, MHET-ase engineering, and immobilization approaches. Moreover, the major challenges associated with the plastic removal are presented by evaluating the recent reports available.

Commercialization potential of agro-based polyhydroxyalkanoates biorefinery: A technical perspective on advances and critical barriers.

The exponential increase in the use and careless discard of synthetic plastics has created an alarming concern over the environmental health due to the detrimental effects of petroleum based synthetic polymeric compounds. Piling up of these plastic commodities on various ecological niches and entry of their fragmented parts into soil and water has clearly affected the quality of these ecosystems in the past few decades. Among the many constructive strategies developed to tackle this global issue, use of biopolymers like polyhydroxyalkanoates as sustainable alternatives for synthetic plastics has gained momentum. Despite their excellent material properties and significant biodegradability, polyhydroxyalkanoates still fails to compete with their synthetic counterparts majorly due to the high cost associated with their production and purification thereby limiting their commercialization. Usage of renewable feedstocks as substrates for polyhydroxyalkanoates production has been the thrust area of research to attain the sustainability tag. This review work attempts to provide insights about the recent developments in the production of polyhydroxyalkanoates using renewable feedstock along with various pretreatment methods used for substrate preparation for polyhydroxyalkanoates production. Further, the application of blends based on polyhydroxyalkanoates, and the challenges associated with the waste valorization based polyhydroxyalkanoates production strategy is elaborated in this review work.

Extended batch cultures for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production by Azotobacter vinelandii OP growing at different aeration rates.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a polymer produced by Azotobacter vinelandii OP. In the bioreactor, PHBV production and its molar composition are affected by aeration rate. PHBV production by A. vinelandii OP was evaluated using extended batch cultures at different aeration rates, which determined different oxygen transfer rates (OTR) in the cultures. Under the conditions evaluated, PHBV with different 3-hydroxyvalerate (3HV) fractions were obtained. In the cultures with a low OTR (6.7 mmol L-1 h-1, at 0.3 vvm), a PHBV content of 38% w w-1 with 9.1 mol % 3HV was achieved. The maximum PHBV production (72% w w-1) was obtained at a high OTR (18.2 mmol L-1 h-1, at 1.0 vvm), both at 48 h. Thus, PHBV production increased in the bioreactor with an increased aeration rate, but not the 3HV fraction in the polymer chain. An OTR of 24.9 mmol L-1 h-1 (at 2.1 vvm) was the most suitable for improving the PHBV content (61% w w-1) and a high 3HV fraction of 20.8 mol % (at 48 h); and volumetric productivity (0.15 g L-1 h-1). The findings indicate that the extended batch culture at 2.1 vvm is the most adequate mode of cultivation to produce higher biomass and PHBV with a high 3HV fraction. Overall, the results have shown that the PHBV production and 3HV fraction could be affected by the aeration rate and the proposed approach could be applied to implement cultivation strategies to optimize PHBV production for different biotechnological applications.

Biotechnological Aspects and Mathematical Modeling of the Biodegradation of Plastics under Controlled Conditions.

The strong environmental impact caused by plastic pollution has led research to address studies from different perspectives. The mathematical modeling of the biodegradation kinetics of solid materials is a major challenge since there are many influential variables in the process and there is interdependence of microorganisms with internal and external factors. In addition, as solid substrates that are highly hydrophobic, mass transfer limitations condition degradation rates. Some mathematical models have been postulated in order to understand the biodegradation of plastics in natural environments such as oceans. However, if tangible and optimizable solutions are to be found, it is necessary to study the biodegradation process under controlled conditions, such as using bioreactors and composting systems. This review summarizes the biochemical fundamentals of the main plastics (both petrochemical and biological origins) involved in biodegradation processes and combines them with the main mathematical equations and models proposed to date. The different biodegradation studies of plastics under controlled conditions are addressed, analyzing the influencing factors, assumptions, model developments, and correlations with laboratory-scale results. It is hoped that this review will provide a comprehensive overview of the process and will serve as a reference for future studies, combining practical experimental work and bioprocess modeling systems.

Emerging challenges for the agro-industrial food waste utilization: A review on food waste biorefinery.

Modernization and industrialization has undoubtedly revolutionized the food and agro-industrial sector leading to the drastic increase in their productivity and marketing thereby accelerating the amount of agro-industrial food waste generated. In the past few decades the potential of these agro-industrial food waste to serve as bio refineries for the extraction of commercially viable products like organic acids, biochemical and biofuels was largely discussed and explored over the conventional method of disposing in landfills. The sustainable development of such strategies largely depends on understanding the techno economic challenges and planning for future strategies to overcome these hurdles. This review work presents a comprehensive outlook on the complex nature of agro-industrial food waste and pretreatment methods for their valorization into commercially viable products along with the challenges in the commercialization of food waste bio refineries that need critical attention to popularize the concept of circular bio economy.

Biotransformation of poly(cis-1,4-isoprene) in a multiphase enzymatic reactor for continuous extraction of oligo-isoprenoid molecules.

Biotechnological processes for the partial degradation or transformation of poly(cis-1,4-isoprene) rubber have been investigated during recent decades with promising results. The use of the enzyme 'latex clearing protein' (Lcp) to transform the polymer into more hydrophilic oligo-isoprenoids results in modifications of the rubber structure and the synthesis of new material. In order to find an alternative process to recover the degradation products, a continuous extraction method using a biphasic system is described. The enzymatic activity of Lcp1VH2 was studied in the presence of ethyl acetate and pentane as extraction solvents. Oligo(cis-1,4-isoprene) molecular species were isolated from the organic phase and analyzed by Electrospray Ionization Mass Spectrometry. The enzymatic reaction process was evaluated in terms of the biotransformation yield of poly(cis-1,4-isoprene) rubber into the corresponding degradation products. Biotransformation yields of between 42-52 % were achieved depending on the enzymatic reactor design and the extraction solvent. The results also showed that the mass distribution of the oligo(cis-1,4-isoprene) depended on the organic solvent applied. A novel, simple and effective process is demonstrated for biotransformation of poly(cis-1,4-isoprene) rubber with high oligo-isoprenoid molecules recovery yields.

Characterization of the latex clearing protein of the poly(cis-1,4-isoprene) and poly(trans-1,4-isoprene) degrading bacterium Nocardia nova SH22a.

Nocardia nova SH22a is an actinobacterium capable of degrading the polyisoprenes poly(cis-1,4-isoprene) and poly(trans-1,4-isoprene). Sequencing and annotating the genome of this strain led to the identification of a single gene coding for the key enzyme for the degradation of rubber: the latex clearing protein (Lcp). In this study, we showed that LcpSH22a-contrary to other already characterized rubber cleaving enzymes-is responsible for the initial cleavage of both polyisoprene isomers. For this purpose, lcpSH22a was heterologously expressed in an Escherichia coli strain and purified with a functional His6- or Strep-tag. Applying liquid chromatography electrospray ionization time-of-flight mass spectrometry (LC/ESI-ToF-MS) and a spectrophotometric pyridine hemochrome assay, heme b was identified as a cofactor. Furthermore, heme-associated iron was identified using total reflection X-ray fluorescence (TXRF) analysis and inhibition tests. The enzyme's temperature and pH optima at 30°C and 7, respectively, were determined using an oxygen consumption assay. Cleavage of poly(cis-1,4-isoprene) and poly(trans-1,4-isoprene) by the oxygenase was confirmed via detection of carbonyl functional groups containing cleavage products, using Schiff's reagent and electrospray ionization mass spectrometry (ESI-MS).

Synthesis of polyhydroxyalkanoates through the biodegradation of poly(cis-1,4-isoprene) rubber.

The search of alternative substrates for the synthesis of polyhydroxyalkanoates (PHA) has become an important factor in order to decrease the production costs. Therefore, the use of industrial by-products or waste materials as carbon and energy sources for different PHA-producing microorganisms has been evaluated during the last decades. Recombinant strains of Gordonia polyisoprenivorans VH2 harboring plasmid pAK68, which contains phaCAB from Ralstonia eutropha and plasmid pAK71 comprising phaC1 from Pseudomonas aeruginosa were evaluated for PHA production. Cultivations were performed in shake flasks, using different carbon sources under an N-starvation condition. Having in consideration the rubber degrading capability of the actinomycete, poly(cis-1,4-isoprene) was utilized as sole carbon source. After twenty days of cultivation the PHA content was analyzed using GC-MS. In cultures of G. polyisoprenivorans harboring pAK68, the detection of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) monomer units indicated the accumulation of the copolyester poly(3HB-co-3HV). This study proposes a recycling method for rubber waste through its biotransformation into bioplastic.