Coexistence of specialist and generalist species within mixed plastic derivative-utilizing microbial communities.

BACKGROUND: Plastic-degrading microbial isolates offer great potential to degrade, transform, and upcycle plastic waste. Tandem chemical and biological processing of plastic wastes has been shown to substantially increase the rates of plastic degradation; however, the focus of this work has been almost entirely on microbial isolates (either bioengineered or naturally occurring). We propose that a microbial community has even greater potential for plastic upcycling. A microbial community has greater metabolic diversity to process mixed plastic waste streams and has built-in functional redundancy for optimal resilience. RESULTS: Here, we used two plastic-derivative degrading communities as a model system to investigate the roles of specialist and generalist species within the microbial communities. These communities were grown on five plastic-derived substrates: pyrolysis treated high-density polyethylene, chemically deconstructed polyethylene terephthalate, disodium terephthalate, terephthalamide, and ethylene glycol. Short-read metagenomic and metatranscriptomic sequencing were performed to evaluate activity of microorganisms in each treatment. Long-read metagenomic sequencing was performed to obtain high-quality metagenome assembled genomes and evaluate division of labor. CONCLUSIONS: Data presented here show that the communities are primarily dominated by Rhodococcus generalists and lower abundance specialists for each of the plastic-derived substrates investigated here, supporting previous research that generalist species dominate batch culture. Additionally, division of labor may be present between Hydrogenophaga terephthalate degrading specialists and lower abundance protocatechuate degrading specialists. Video Abstract.

Deconstructed Plastic Substrate Preferences of Microbial Populations from the Natural Environment.

Over half of the world's plastic waste is landfilled, where it is estimated to take hundreds of years to degrade. Given the continued use and disposal of plastic products, it is vital that we develop fast and effective ways to utilize plastic waste. Here, we explore the potential of tandem chemical and biological processing to process various plastics quickly and effectively. Four samples of compost or sediment were used to set up enrichment cultures grown on mixtures of compounds, including disodium terephthalate and terephthalic acid (monomers of polyethylene terephthalate), compounds derived from the chemical deconstruction of polycarbonate, and pyrolysis oil derived from high-density polyethylene plastics. Established enrichment communities were also grown on individual substrates to investigate the substrate preferences of different taxa. Biomass harvested from the cultures was characterized using 16S rRNA gene amplicon sequencing and shotgun metagenomic sequencing. These data reveal low-diversity microbial communities structured by differences in culture inoculum, culture substrate source plastic type, and time. Microbial populations from the classes Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, and Acidobacteriae were significantly enriched when grown on substrates derived from high-density polyethylene and polycarbonate. The metagenomic data contain abundant aromatic and aliphatic hydrocarbon degradation genes relevant to the biodegradation of deconstructed plastic substrates used here. We show that microbial populations from diverse environments are capable of growth on substrates derived from the chemical deconstruction or pyrolysis of multiple plastic types and that paired chemical and biological processing of plastics should be further developed for industrial applications to manage plastic waste. IMPORTANCE The durability and impermeable nature of plastics have made them a popular material for numerous applications, but these same qualities make plastics difficult to dispose of, resulting in massive amounts of accumulated plastic waste in landfills and the natural environment. Since plastic use and disposal are projected to increase in the future, novel methods to effectively break down and dispose of current and future plastic waste are desperately needed. We show that the products of chemical deconstruction or pyrolysis of plastic can successfully sustain the growth of low-diversity microbial communities. These communities were enriched from multiple environmental sources and are capable of degrading complex xenobiotic carbon compounds. This study demonstrates that tandem chemical and biological processing can be used to degrade multiple types of plastics over a relatively short period of time and may be a future avenue for the mitigation of rapidly accumulating plastic waste.

Metagenomic Sequencing of Two Cultures Grown on Chemically Deconstructed Plastic Products.

We report the metagenome sequences of two microbial cultures that were grown with chemically deconstructed plastic products as their sole carbon source. These metagenomes will provide insights into the metabolic capabilities of cultures grown on deconstructed plastics and can serve as a starting point for the identification of novel plastic degradation mechanisms.

Killing two birds with one stone: chemical and biological upcycling of polyethylene terephthalate plastics into food.

Most polyethylene terephthalate (PET) plastic waste is landfilled or pollutes the environment. Additionally, global food production must increase to support the growing population. This article explores the feasibility of using microorganisms in an industrial system that upcycles PET into edible microbial protein powder to solve both problems simultaneously. Many microorganisms can utilize plastics as feedstock, and the resultant microbial biomass contains fats, nutrients, and proteins similar to those found in human diets. While microbial degradation of PET is promising, biological PET depolymerization is too slow to resolve the global plastic crisis and projected food shortages. Evidence reviewed here suggests that by coupling chemical depolymerization and biological degradation of PET, and using cooperative microbial communities, microbes can efficiently convert PET waste into food.

Study of the Viscosity and Thermal Characteristics of Polyolefins/Solvent Mixtures: Applications for Plastic Pyrolysis.

Plastic pollution is one of the biggest environmental problems that the world is currently facing. Pyrolysis is a frontier technique aimed at converting plastic waste back into virgin-quality resin. However, the transfer of the waste plastic feed into the pyrolysis reactor must be optimized before the process can be upscaled to a continuous process. In this study, a new solvent that reduces the viscosity of molten plastic was introduced and characterized. The results revealed that the polymers are soluble in the ratio of up to 75 wt % plastic and 25 wt % solvent at 240 °C. The viscosity of pure low-density polyethylene (LDPE), high-density polyethylene (HDPE), and polypropylene (PP) in the solvent was measured in different weight percentages of polymer in solvent (30-80 wt %) and at 160, 180, 200, 220, 240, and 260 °C. The viscosity decreased with the decreasing polymer-weight percentage and with increasing temperature. The viscosity of LDPE/solvent and PPs(isotactic)/solvent is much lower than for HDPE/solvent and PPp(polypropylene impact copolymer)/solvent. Differential scanning calorimetry (DSC) and thermal gravimetric analysis (TGA) were applied to characterize the thermal behavior of LDPE, HDPE, and PP in the solvent in three different weight percentages (25, 50, and 75 wt %). The DSC results indicate that in the mixture of PPs/solvent and LDPE/solvent the melting point of PP and LDPE decreases as the amount of solvent increases. Overall, these results indicate that the selected solvent is an effective agent to prepare waste plastics for pyrolysis.

A Review of Environmental Life Cycle Assessments of Liquid Transportation Biofuels in the Pan American Region.

Life-cycle assessment (LCA) has been applied to many biofuel and bioenergy systems to determine potential environmental impacts, but the conclusions have varied. Different methodologies and processes for conducting LCA of biofuels make the results difficult to compare, in-turn making it difficult to make the best possible and informed decision. Of particular importance are the wide variability in country-specific conditions, modeling assumptions, data quality, chosen impact categories and indicators, scale of production, system boundaries, and co-product allocation. This study has a double purpose: conducting a critical evaluation comparing environmental LCA of biofuels from several conversion pathways and in several countries in the Pan American region using both qualitative and quantitative analyses, and making recommendations for harmonization with respect to biofuel LCA study features, such as study assumptions, inventory data, impact indicators, and reporting practices. The environmental management implications are discussed within the context of different national and international regulatory environments using a case study. The results from this study highlight LCA methodology choices that cause high variability in results and limit comparability among different studies, even among the same biofuel pathway, and recommendations are provided for improvement.

Evaluation of hardboard manufacturing process wastewater as a feedstream for ethanol production.

Waste streams from the wood processing industry can serve as feedstream for ethanol production from biomass residues. Hardboard manufacturing process wastewater (HPW) was evaluated on the basis of monomeric sugar recovery and fermentability as a novel feedstream for ethanol production. Dilute acid hydrolysis, coupled with concentration of the wastewater resulted in a hydrolysate with 66 g/l total fermentable sugars. As xylose accounted for 53 % of the total sugars, native xylose-fermenting yeasts were evaluated for their ability to produce ethanol from the hydrolysate. The strains selected were, in decreasing order by ethanol yields from xylose (Y p/s, based on consumed sugars), Scheffersomyces stipitis ATCC 58785 (CBS 6054), Pachysolen tannophilus ATCC 60393, and Kluyveromyces marxianus ATCC 46537. The yeasts were compared on the basis of substrate utilization and ethanol yield during fermentations of the hydrolysate, measured using an HPLC. S. stipitis, P. tannophilus, and K. marxianus produced 0.34, 0.31, and 0.36 g/g, respectively. The yeasts were able to utilize between 58 and 75 % of the available substrate. S. stipitis outperformed the other yeast during the fermentation of the hydrolysate; consuming the highest concentration of available substrate and producing the highest ethanol concentration in 72 h. Due to its high sugar content and low inhibitor levels after hydrolysis, it was concluded that HPW is a suitable feedstream for ethanol production by S. stipitis.

Feedstock mixture effects on sugar monomer recovery following dilute acid pretreatment and enzymatic hydrolysis.

This study seeks to investigate the effects of biomass mixtures on overall sugar recovery from the combined processes of dilute acid pretreatment and enzymatic hydrolysis. Aspen, a hardwood species well suited to biochemical processing, was chosen as the model species for this study. Balsam, a high-lignin softwood species, and switchgrass, an herbaceous energy crop with high ash content, were chosen as adjuncts. A matrix of three different dilute acid pretreatment severities and three different enzyme loading levels was used to characterize interactions between pretreatment and enzymatic hydrolysis. No synergism or antagonism was observed for any of the feedstock mixtures. Maximum glucose yield was 70% of theoretical for switchgrass and maximum xylose yield was 99.7% of theoretical for aspen. Supplemental ß-glucosidase increased glucose yield from enzymatic hydrolysis by an average of 15%. Total sugar recoveries for mixtures could be predicted to within 4% by linear interpolation of the pure species results.

Effects of dilute acid pretreatment conditions on enzymatic hydrolysis monomer and oligomer sugar yields for aspen, balsam, and switchgrass.

The effects of dilute acid hydrolysis conditions were investigated on total sugar (glucose and xylose) yields after enzymatic hydrolysis with additional analyses on glucose and xylose monomer and oligomer yields from the individual hydrolysis steps for aspen (a hardwood), balsam (a softwood), and switchgrass (a herbaceous energy crop). The results of this study, in the form of measured versus theoretical yields and a severity analysis, show that for aspen and balsam, high dilute acid hydrolysis xylose yields were obtainable at all acid concentrations (0.25-0.75 wt.%) and temperatures (150-175 degrees C) studied as long as reaction time was optimized. Switchgrass shows a relatively stronger dependence on dilute acid hydrolysis acid concentration due to its higher neutralizing mineral content. Maximum total sugar (xylose and glucose; monomer plus oligomer) yields post-enzymatic hydrolysis for aspen, balsam, and switchgrass, were 88.3%, 21.2%, and 97.6%, respectively. In general, highest yields of total sugars (xylose and glucose; monomer plus oligomer) were achieved at combined severity parameter values (log CS) between 2.20 and 2.40 for the biomass species studied.

Kinetic characterization for dilute sulfuric acid hydrolysis of timber varieties and switchgrass.

Hydrolysis of four timber species (aspen, balsam fir, basswood, and red maple) and switchgrass was studied using dilute sulfuric acid at 50 g dry biomass/L under similar conditions previously described as acid pretreatment. The primary goal was to obtain detailed kinetic data of xylose formation and degradation from a match between a first order reaction model and the experimental data at various final reactor temperatures (160-190 degrees C), sulfuric acid concentrations (0.25-1.0% w/v), and particle sizes (28-10/20 mesh) in a glass-lined 1L well-mixed batch reactor. Reaction rates for the generation of xylose from hemicellulose and the generation of furfural from xylose were strongly dependent on both temperature and acid concentration. However, no effect was observed for the particle sizes studied. Oligomer sugars, representing incomplete products of hydrolysis, were observed early in the reaction period for all sugars (xylose, glucose, arabinose, mannose, and galactose), but were reduced to low concentrations at later times (higher hemicellulose conversions). Maximum yields for xylose ranged from 70% (balsam) to 94% (switchgrass), for glucose from 10.6% to 13.6%, and for other minor sugars from 8.6% to 58.9%. Xylose formation activation energies and the pre-exponential factors for the timber species and switchgrass were in a range of 49-180 kJ/mol and from 7.5 x 10(4) to 2.6 x 10(20)min(-1), respectively. In addition, for xylose degradation, the activation energies and the pre-exponential factors ranged from 130 to 170 kJ/mol and from 6.8 x 10(13) to 3.7 x 10(17)min(-1), respectively. There was a near linear dependence on acid concentration observed for xylose degradation. Our results suggest that mixtures of biomass species may be processed together and still achieve high yields for all species.

Characterization of free and immobilized (S)-aminotransferase for acetophenone production.

Enzyme immobilization often improves process economics, but changes in kinetic properties may also occur. The immobilization of a recombinant thermostable (S)-aminotransferase was made by entrapment on calcium alginate-3% (w/v)-and tested with (S)-(-)-(alpha)-methylbenzylamine for acetophenone production. The best immobilization results were obtained for beads of concentration of 10 mg of spray-dried cells (containing recombinant (S)-aminotransferase) per milliliter of sodium alginate bead. As a result of immobilization, the properties of immobilized spray-dried cells differed from the properties of free spray-dried cells. V (m) for the immobilized enzyme was between 0.08 and 0.09 mM/min, while the V (m) for free enzyme was 0.06-0.07 mM/min. K (m) values differed for immobilized and free spray-dried cells by a factor of between 3 and 5 for (S)-(-)-(alpha)-methylbenzylamine (6.05 mM for immobilized, 1.78 mM for free) and pyruvate (5.0 mM for immobilized, 1.01 mM for free) at 55 degrees C. Optimum pH values were 7.7 and 8.1 for the free spray-dried cells and the immobilized formulation, respectively. The maximum activity for free spray-dried cells was measured at 55 degrees C, whereas for immobilized ones, it was at 60 degrees C. Activation and deactivation energy values for free spray-dried cells were 15.13 and 41.73 kcal/mol, while those for immobilized spray-dried cells were 8.86 and 48.88 kcal/mol, respectively. Overall, as a result of immobilization, an increase in V (m) was measured for the (S)-aminotransferase by 28 to 33% with respect to free enzyme; K (m) increased by a factor of three- to fivefold and had a shift of 5 degrees C in optimum temperature, and the activation energy was 41% lower than the activation energy of free (S)-aminotransferase.

Kinetic characterization and in vitro toxicity evaluation of a luciferase less susceptible to HPV chemical inhibition.

Enzyme-based in vitro toxicity assays are often susceptible to inhibition by test compounds. A mutant luciferase selected to be less susceptible to inhibition by chloroform (CNBLuc03-06) and other high production volume (HPV) chemicals, consisting of three point mutations was created and characterized. The mutant luciferase was less inhibited by chloroform, other HPV chemicals and common surfactant release reagents (Triton-X and SDS) compared to the wild-type. Inhibition was shown to be competitive. CNBLuc03-06 was a factor of 1.5-3.2 more active than wild type and exhibited a much higher affinity for ATP. CNBLuc03-06 was more thermostable than wild-type and also more active at pH values higher than 10. Both luciferases exhibited a significant tradeoff between activation and susceptibility to chemical inhibition in the presences of the reducing agent DTT. Inhibition to HPV chemicals was eliminated using an "optimum" formulation of DTT and co-solvent ethanol. The performance of CNBLuc03-06 in cell-based in vitro toxicity assays was shown to be superior to the current commercial formulation.

Creating a mutant luciferase resistant to HPV chemical inhibition by random mutagenesis and colony-level screening.

Firefly luciferase covers a wide range of applications. One common usage of the bioluminescence assay is the measurement of intracellular concentration of adenosine triphosphate (ATP) for cell viability. However, inhibition of the enzyme reaction by chemicals in the assay has so far limited the application of luciferase for high production volume (HPV) chemical testing. The objective of this research was to obtain a mutant luciferase with increased stability to inhibition by HPV chemicals, yet retaining specific activity comparable to, or better than, wild-type luciferase. The enzymatic properties of the wild-type luciferase were improved by random mutagenesis and colony-level screening. In this paper, the detailed process of creating mutant luciferases for testing the toxicity of HPV chemicals is described. As a result, two mutant luciferases were created, with different degrees of improved tolerance to inhibition by chloroform and other HPV chemicals.

Comparative life-cycle assessments for biomass-to-ethanol production from different regional feedstocks.

This study compares life-cycle (cradle-to-gate) energy consumption and environmental impacts for producing ethanol via fermentation-based processes starting with two lignocellulosic feedstocks: virgin timber resources or recycled newsprint from an urban area. The life-cycle assessment in this study employed a novel combination of computer-aided tools. These tools include fermentation process simulation coupled with an impact assessment software tool for the manufacturing process life-cycle stage impacts. The process simulation file was provided by the National Renewable Energy Laboratory (NREL) and was modified slightly to accommodate these different feedstocks. For the premanufacturing process life-cycle stage impacts, such as the fuels and process chemicals used, transportation, and some preparatory steps (wood chipping, etc.), a life-cycle inventory database (the Boustead Model) coupled with an impact assessment software tool were used (the Environmental Fate and Risk Assessment Tool). The Newsprint process has a slightly lower overall composite environmental index (created from eight impact categories) compared to the Timber process. However, the Timber process consumes less electricity, produces fewer emissions in total, and has less of a human health impact. The amount of life-cycle fossil energy required to produce ethanol is 14% of the energy content of the product, making the overall efficiency 86%. Process improvement strategies were evaluated for both feedstock processes, including recycle of reactor vent air and heat integration. Heat integration has the greatest potential to reduce fossil-derived energy consumption, to an extent that fossil-derived energy over the life cycle is actually saved per unit of ethanol produced. These energy efficiency values are superior to those observed in conventional fossil-based transportation fuels.

Development and evaluation of an environmental multimedia fate model CHEMGL for the Great Lakes region.

This paper describes the development of a multimedia compartmental model--CHEMGL--which predicts the fate and transport of chemicals in the Great Lakes region and can be used for risk assessment. CHEMGL includes 10 compartments that describe a given region: air boundary layer, free troposphere, lower stratosphere, surface water, sediment, surface soil, vadose soil, groundwater zone, plant foliage and plant root. The model assumes that the compartments are completely mixed and chemical equilibrium between the phases within each compartment is assumed (e.g., suspended solids and biota in water). The attenuation mechanisms include advection, transformation reactions, and diffusive and nondiffusive intermedia transport between compartments. Input parameters include a description of each environmental media, emission rates, and chemical-specific properties and reaction rates. The numerical model results are in good agreement with the analytical solution for an example that examines the fate of benzene. Accordingly, the mathematical and computational components of the model were verified. CHEMGL predicted the concentration of four representative chemicals (atrazine, benzo[a]pyrene, benzene and hexachlorobenzene) in all five basins: Superior, Michigan, Huron, Erie and Ontario. The predicted concentrations fell within one to two orders of magnitude of data reported in the literature. These results suggest that the model is appropriate for estimating the fate and exposure of chemicals for a screening level risk assessment.

Sustainability science and engineering: the emergence of a new metadiscipline.

A case is made for growth of a new metadiscipline of sustainability science and engineering. This new field integrates industrial, social, and environmental processes in a global context. The skills required for this higher level discipline represent a metadisciplinary endeavor, combining information and insights across multiple disciplines and perspectives with the common goal of achieving a desired balance among economic, environmental, and societal objectives. Skills and capabilities that are required to support the new metadiscipline are summarized. Examples of integrative projects are discussed in the areas of sustainability metrics and integration of industrial, societal, and environmental impacts. It is clear that a focus on green engineering that employs pollution prevention and industrial ecology alone are not sufficient to achieve sustainability, because even systems with efficient material and energy use can overwhelm the carrying capacity of a region or lead to other socially unacceptable outcomes. To meet the educational and human resource needs required for this new discipline, the technological and environmental awareness of society must be elevated and a sufficient and diverse pool of human talent must be attracted to this discipline.

Industrial applications using BASF eco-efficiency analysis: perspectives on green engineering principles.

Life without chemicals would be inconceivable, but the potential risks and impacts to the environment associated with chemical production and chemical products are viewed critically. Eco-efficiency analysis considers the economic and life cycle environmental effects of a product or process, giving these equal weighting. The major elements of the environmental assessment include primary energy use, raw materials utilization, emissions to all media, toxicity, safety risk, and land use. The relevance of each environmental category and also for the economic versus the environmental impacts is evaluated using national emissions and economic data. The eco-efficiency analysis method of BASF is briefly presented, and results from three applications to chemical processes and products are summarized. Through these applications, the eco-efficiency analyses mostly confirm the 12 Principles listed in Anastas and Zimmerman (Environ. Sci. Technol. 2003, 37(5), 94A), with the exception that, in one application, production systems based on bio-based feedstocks were not the most eco-efficient as compared to those based on fossil resources. Over 180 eco-efficiency analyses have been conducted at BASF, and their results have been used to support strategic decision-making, marketing, research and development, and communication with external parties. Eco-efficiency analysis, as one important strategy and success factor in sustainable development, will continue to be a very strong operational tool at BASF.

Green engineering education through a U.S. EPA/academia collaboration.

The need to use resources efficiently and reduce environmental impacts of industrial products and processes is becoming increasingly important in engineering design; therefore, green engineering principles are gaining prominence within engineering education. This paper describes a general framework for incorporating green engineering design principles into engineering curricula, with specific examples for chemical engineering. The framework for teaching green engineering discussed in this paper mirrors the 12 Principles of Green Engineering proposed by Anastas and Zimmerman (Environ. Sci. Technol. 2003, 37, 94A-101A), especially in methods for estimating the hazardous nature of chemicals, strategies for pollution prevention, and approaches leading to efficient energy and material utilization. The key elements in green engineering education, which enlarge the "box" for engineering design, are environmental literacy, environmentally conscious design, and beyond-the-plant boundary considerations.