Article

Process Analysis of the Conversion of Styrene to Biomass and Medium Chain Length Polyhydroxyalkanoate in a Two-Phase Bioreactor

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Abstract

The improvement and modeling of a process for the supply of the volatile aromatic hydrocarbon, styrene, to a fermentor for increased biomass production of the medium chain length polyhydroxyalkanoate (mcl-PHA) accumulating bacterium Pseudomonas putida CA-3 was investigated. Fed-batch experiments were undertaken using different methods to provide the styrene. Initial experiments where styrene was supplied as a liquid to the bioreactor had detrimental effects on cell growth and inhibited PHA polymer accumulation. By changing the feed of gaseous styrene to liquid styrene through the air sparger a 5.4-fold increase in cell dry-weight was achieved (total of 10.56 g L(-1) ) which corresponds to a fourfold improvement in PHA production (3.36 g L(-1) ) compared to previous studies performed in our laboratory (0.82 g L(-1) ). In addition this final improved feeding strategy reduced the release of styrene from the fermentor 50-fold compared to initial experiments (0.12 mL total styrene released per 48 h run). An unstructured kinetic model was developed to describe cell growth along with substrate and oxygen utilization. The formation of dispersed gas (air) and liquid (styrene) phases in the medium and the transfer of styrene between the aqueous and dispersed liquid droplet phases was also modeled. The model provided a detailed description of these phase transitions and helped explain how the feeding strategy led to improved process performance in terms of final biomass levels. It also highlighted the key factors to be considered during further process improvement. Biotechnol. Bioeng. © 2011 Wiley Periodicals, Inc.

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... In recent years, we have reported on the development of technologies to convert petrochemical plastic waste streams into PHA. The conversion 163 PHA from Carbon-Rich Waste Streams processes have been reported for PS (Goff, Ward, & Oconnor, 2007;Nikodinovic-Runic et al., 2011;Ward, Goff, Donner, Kaminsky, & O'Connor, 2006), PET , PE (Guzik, 2012), and products present in the pyrolysis products of mixed plastic waste . The technologies are characterized by a two-step chemobiotechnological route. ...
... The pyrolysis (520 C) of PS in a fluidized bedreactor (Quartz sand (0.3-0.5 mm)) resulted in the generation of an oil composed of styrene (82.8%, w/w) and low levels of other aromatic compounds. This styrene oil, when supplied as the sole source of carbon and energy, allowed for the growth of P. putida CA-3 and PHA accumulation in shake-flask experiments and in fed-batch fermentations (Goff et al., 2007;Nikodinovic-Runic et al., 2011). The medium chain length PHA (mcl-PHA) accumulated was comprised of 6, 8, and 10 carbon (R)-3hydroxyalkanoic acid monomers in a molar ratio of 0.05:0.44:1.13, ...
... Manipulation of nitrogen concentration during fed-batch fermentations further improved the conversion yield 2.8-fold (Goff et al., 2007). In subsequent developments, the process was further improved through the control of styrene feeding (Nikodinovic-Runic et al., 2011). Initial fed-batch experiments, where styrene was supplied as a liquid to the bioreactor, had detrimental effects on cell growth and inhibited PHA polymer accumulation. ...
Article
Research into the production of biodegradable polymers has been driven by vision for the most part from changes in policy, in Europe and America. These policies have their origins in the Brundtland Report of 1987, which provides a platform for a more sustainable society. Biodegradable polymers are part of the emerging portfolio of renewable raw materials seeking to deliver environmental, social, and economic benefits. Polyhydroxyalkanoates (PHAs) are naturally-occurring biodegradable-polyesters accumulated by bacteria usually in response to inorganic nutrient limitation in the presence of excess carbon. Most of the early research into PHA accumulation and technology development for industrial-scale production was undertaken using virgin starting materials. For example, polyhydroxybutyrate and copolymers such as polyhydroxybutyrate-co-valerate are produced today at industrial scale from corn-derived glucose. However, in recent years, research has been undertaken to convert domestic and industrial wastes to PHA. These wastes in today's context are residuals seen by a growing body of stakeholders as platform resources for a biobased society. In the present review, we consider residuals from food, plastic, forest and lignocellulosic, and biodiesel manufacturing (glycerol). Thus, this review seeks to gain perspective of opportunities from literature reporting the production of PHA from carbon-rich residuals as feedstocks. A discussion on approaches and context for PHA production with reference to pure- and mixed-culture technologies is provided. Literature reports advocate results of the promise of waste conversion to PHA. However, the vast majority of studies on waste to PHA is at laboratory scale. The questions of surmounting the technical and political hurdles to industrialization are generally left unanswered. There are a limited number of studies that have progressed into fermentors and a dearth of pilot-scale demonstration. A number of fermentation studies show that biomass and PHA productivity can be increased, and sometimes dramatically, in a fermentor. The relevant application-specific properties of the polymers from the wastes studied and the effect of altered-waste composition on polymer properties are generally not well reported and would greatly benefit the progress of the research as high productivity is of limited value without the context of requisite case-specific polymer properties. The proposed use of a waste residual is advantageous from a life cycle viewpoint as it removes the direct or indirect effect of PHA production on land usage and food production. However, the question, of how economic drivers will promote or hinder advancements to demonstration scale, when wastes generally become understood as resources for a biobased society, hangs today in the balance due to a lack of shared vision and the legacy of mistakes made with first generation bioproducts.
... The only exception is P. putida CA-3, a patent strain that can store mcl-PHA up to 33% CDM and 31.8% CDM under shake-flask and fermenter conditions, respectively (27,28). Therefore, the pool of bacterial strains with the metabolic capacity to efficiently bioconvert styrene into mcl-PHA needs to be increased. ...
... CDM) and PHA mass fraction (324.90±23.98 mg gCDM −1 ) were also observed and found to be similar to values obtained by P. putida CA-3 (28). The extracted PHA polymer mainly comprised C 6 , C 8 , C 10 , C 12 , C 14 at a ratio of 2:42:1257:17:1, showing an increased dominance of the C 10 monomer and an additional C 14 monomer ( Fig. 2B and Fig. 3), which were previously not observed under shake-flask cultivation ( Table 1). ...
Article
Full-text available
Styrene is a toxic pollutant commonly found in waste effluents from plastic processing industries. We herein identified and characterized microorganisms for bioconversion of the organic eco-pollutant styrene into a valuable biopolymer medium-chain-length poly(hydroxyalkanoate) (mcl-PHA). Twelve newly-isolated styrene-degrading Pseudomonads were obtained and partial phaC genes were detected by PCR in these isolates. These isolates assimilated styrene to produce mcl-PHA, forming PHA contents between 0.05±0.00 and 23.10±3.25% cell dry mass (% CDM). The best-performing isolate was identified as Pseudomonas putida NBUS12. A genetic analysis of 16S rDNA and phaZ genes revealed P.putida NBUS12 as a genetically-distinct strain from existing phenotypically-similar bacterial strains. This bacterium achieved a final biomass of 1.28±0.10 g L−1 and PHA content of 32.49±2.40% CDM. The extracted polymer was mainly comprised of 3-hydroxyhexanoate (C6), 3-hydroxyoctanoate (C8), 3-hydroxydecanoate (C10), 3-hydroxydodecanoate (C12), and 3-hydroxytetradecanoate (C14) monomers at a ratio of 2:42:1257:17:1. These results collectively suggested that P. putida NBUS12 is a promising candidate for the biotechnological conversion of styrene into mcl-PHA.
... To develop a nonnatural enzyme cascade to benzoic acid, we rely on the highly efficient oxyfunctionalization of styrene (Gursky, Nikodinovic-Runic, Feenstra, & O'Connor, 2010;Nikodinovic-Runic et al., 2011;Panke, Held, Wubbolts, Witholt, & Schmid, 2002;Paul et al., 2015;Volmer, Lindmeyer, Seipp, Schmid, & Bühler, 2019) and our previously reported four-step biotransformation of styrene to (S)-mandelic acid (Lukito, Sekar, Wu, & Li, 2019;Wu et al., 2016) to establish a seven-step cascade to benzoic acid in this report ( Figure 1a). Moreover, utilizing a deamination-decarboxylation of L-phenylalanine to styrene (McKenna & Nielsen, 2011;, the cascade is extended to achieve efficient cascade biotransformation of L-phenylalanine to benzoic acid (Figure 1b). ...
... 10-25%). On the other hand, the epoxidation of styrene 1a with styrene monooxygenase (SMO) is one of most efficient biocatalytic oxyfunctionalizations with >90% conversion to (S)-styrene oxide 2a in up to 600 mM(Gursky et al., 2010;Nikodinovic-Runic et al., 2011;Panke et al., 2002;Paul et al., 2015;Volmer et al., 2019). Based on SMOcatalyzed epoxidation, we previously developed a four-step biotransformation of styrene 1a to (S)-mandelic acid 5a in 78% conversion and up to 144 mMWu et al., 2016). ...
Article
As an important bulk chemical, benzoic acid is currently manufactured from non‐renewable feedstocks under harsh conditions. Although there are natural pathways for biosynthesis of benzoic acid, they are often inefficient and subjected to complex regulation. Here we develop a non‐natural enzyme cascade to efficiently produce benzoic acid from styrene or biogenic ʟ‐phenylalanine under mild conditions. By using a modular approach, two whole‐cell catalysts E. coli LZ305 and LZ325 are engineered for co‐expressing seven and nine enzymes for production of 133‐146 mM benzoic acid (16.2‐17.8 g/Laq) with 88‐97% conversion via seven‐ and nine‐step cascade biotransformation of styrene and ʟ‐phenylalanine, respectively. The seven‐step cascade represents a formal high‐yielding biocatalytic oxidative cleavage of styrene, and the nine‐step cascade showcases the high efficiency of extended non‐natural enzyme cascades. Moreover, to achieve benzoic acid production directly from low‐cost renewable glycerol, a novel coupled fermentation‐biotransformation process was developed by integration of fermentative production of ʟ‐phenylalanine with in situ biotransformation to give 63‐70 mM benzoic acid (7.6‐8.6 g/Laq), which is around 20 times higher than the reported value via a natural pathway. The coupled fermentation‐biotransformation process could be generally applicable to microbial production of growth‐inhibitory or toxic chemicals in high concentrations. This article is protected by copyright. All rights reserved.
... Employment of such bacteria combines bioremediation with the production of a high value-added material. So far, bacterial strains that belong to the genus of Sphingobacterium, Bacillus, Pseudomonas, and Rhodococcus have been isolated and studied regarding their PHA production potential degrading environmental pollutants, as summarized in Table 2 [3,29,[33][34][35][36][37][38][39]. ...
... However, higher cell density and PHA production, characterized by a conversion yield of 0.28 g/g, were observed when cells were supplied with nitrogen at a feeding rate of 1.5 mg/L/h [37]. Moreover, in a recent study the key challenges of improving transfer and increasing supply of styrene, without inhibiting bacterial growth, were addressed [35]. It was shown that by changing the feed from gaseous to liquid styrene, through the air sparger, release of styrene was reduced 50-fold, biomass concentration was five times higher, while PHA production was four-fold compared to previous experiments, with a PHA content reaching up to 32% in terms of CDW and a conversion yield of 0.17 g/g. ...
Article
Full-text available
Sustainable biofuels, biomaterials, and fine chemicals production is a critical matter that research teams around the globe are focusing on nowadays. Polyhydroxyalkanoates represent one of the biomaterials of the future due to their physicochemical properties, biodegradability, and biocompatibility. Designing efficient and economic bioprocesses, combined with the respective social and environmental benefits, has brought together scientists from different backgrounds highlighting the multidisciplinary character of such a venture. In the current review, challenges and opportunities regarding polyhydroxyalkanoate production are presented and discussed, covering key steps of their overall production process by applying pure and mixed culture biotechnology, from raw bioprocess development to downstream processing.
... work on polystyrene oil conversion by Pseudomonas putida CA-3 produce 32% PHA (Nikodinovic-Runic et al., 2011 ). The mixture of terepthalic acid in the form of solid fraction and supplemented with glycerol waste was used as substrates by bacterial cultures of P. putida F1, P. putida mt-2, P. putida CA-3, and 37% PHA productivity was observed (Nikodinovic et al., 2008). ...
Chapter
Municipal wastewater is one of the wastes which can be used as a substrate for microbial growth. Municipal waste contains sludge, which is considered an end product of wastewater treatment plants, contributing to higher costs for its management. However, the sludge has the highest amount of organic matter, which can produce high-value products, especially polyhydroxyalkanoates (PHAs). Through volarization and fixation of methane by microbial consortium can lower the methane content in the sludge and can be integrated with the production of PHA. PHA has gain far more interest in past few decades due to its biodegradable and biofriendly nature towards the environment. PHA is thermoplastic polyesters produced by various classes of microorganisms such as bacteria, fungi, and algae. These are produced under stress condition like nutrient limitation. The process includes four major steps: i) Treatment of wastewater through enrichment and later, end product directed for PHA sustaining biomass; ii) production of volatile fatty acids (VFA) or VFA rich stream through acidogenic fermentation; iii) production and accumulation of PHA using VFA rich stream; and iv) downstream process for PHA recovery. The following chapter will be detailed summary the effective utilization of sewage sludge to produce PHAs via integrating municipal water treatment and PHA production.
... Recovered styrene from the pyrolysis oil of PS waste can be used as a chemical source in industries for polymerization of PS polymer (Achilias 2007;Frediani et al., 2012). Moreover, polyhydroxyalkanoate a biocompatible and biodegradable plastic can be produced from pyrolysis oil of PS waste (Nikodinovic-Runic et al., 2011). ...
Article
This paper aims to investigate the effect of temperature and reaction time on the yield and quality of liquid oil produced from a pyrolysis process. Polystyrene (PS) type plastic waste was used as a feedstock in a small pilot scale batch pyrolysis reactor. At 400 °C with a reaction time of 75 min, the gas yield was 8% by mass, the char yield was 16% by mass, while the liquid oil yield was 76% by mass. Raising the temperature to 450 °C increased the gas production to 13% by mass, reduced the char production to 6.2% and increased the liquid oil yield to 80.8% by mass. The optimum temperature and reaction time was found to be 450 °C and 75 min. The liquid oil at optimum conditions had a dynamic viscosity of 1.77 mPa s, kinematic viscosity of 1.92 cSt, a density of 0.92 g/cm 3 , a pour point of À60 °C, a freezing point of À64 °C, a flash point of 30.2 °C and a high heating value (HHV) of 41.6 MJ/kg this is similar to conventional diesel. The gas chromatography with mass spectrophotometry (GC–MS) analysis showed that liquid oil contains mainly styrene (48%), toluene (26%) and ethyl-benzene (21%) compounds.
... Recovered styrene from the pyrolysis oil of PS waste can be used as a chemical source in indus- tries for polymerization of PS polymer (Achilias 2007;Frediani et al., 2012). Moreover, polyhydroxyalkanoate a biocompatible and biodegradable plastic can be produced from pyrolysis oil of PS waste (Nikodinovic-Runic et al., 2011). ...
Article
Full-text available
This paper aims to investigate the effect of temperature and reaction time on the yield and quality of liquid oil produced from a pyrolysis process. Polystyrene (PS) type plastic waste was used as a feedstock in a small pilot scale batch pyrolysis reactor. At 400 �C with a reaction time of 75 min, the gas yield was 8% by mass, the char yield was 16% by mass, while the liquid oil yield was 76% by mass. Raising the temperature to 450 �C increased the gas production to 13% by mass, reduced the char production to 6.2% and increased the liquid oil yield to 80.8% by mass. The optimum temperature and reaction time was found to be 450 �C and 75 min. The liquid oil at optimum conditions had a dynamic viscosity of 1.77 mPa s, kinematic viscosity of 1.92 cSt, a density of 0.92 g/cm3, a pour point of �60 �C, a freezing point of �64 �C, a flash point of 30.2 �C and a high heating value (HHV) of 41.6 MJ/kg this is similar to conventional diesel. The gas chromatography with mass spectrophotometry (GC–MS) analysis showed that liquid oil contains mainly styrene (48%), toluene (26%) and ethyl-benzene (21%) compounds.
... The recovery of these aromatic compounds from produced liquid oils can be a potential source of precursor chemicals in industries for polymerization of plastic monomers (Miandad et al., 2016a,b;Shah and Jan 2014;Sarker and Rashid, 2013). According to Nikodinovic-Runic et al. (2011), a biocompatible and biodegradable plastic can be produced from pyrolysis oil of PS plastic waste. ...
... Recovered styrene can be used as feedstock in various industries for PS polymerization (Achilias, 2007). Biodegradable plastic i.e. polyhydroxyalkanoate can also be produced from pyrolytic liquid oil produced from thermal degradation of PS plastic wastes (Nikodinovic-Runic et al., 2011). ...
Article
The aim of this study was to determine the quality and applications of liquid oil produced by thermal and catalytic pyrolysis of polystyrene (PS) plastic waste by using a small pilot scale pyrolysis reactor. Thermal pyrolysis produced maximum liquid oil (80.8%) with gases (13%) and char (6.2%), while catalytic pyrolysis using synthetic and natural zeolite decreased the liquid oil yield (52%) with an increase in gases (17.7%) and char (30.1%) production. The lower yield but improved quality of liquid oil through catalytic pyrolysis are due to catalytic features of zeolites such as microporous structure and high BET surface area. The liquid oils, both from thermal and catalytic pyrolysis consist of around 99% aromatic hydrocarbons, as further confirmed by GC-MS results. FT-IR analysis further showed chemical bonding and functional groups of mostly aromatic hydrocarbons, which is consistent with GC-MS results. The produced liquid oils can be suitable for energy generation and heating purposes after the removal of acid, solid residues and contaminants. Further upgrading of liquid oil and blending with diesel is required for its potential use as a transport fuel.
... Recovered styrene can be used as feedstock in various industries for PS polymerization (Achilias, 2007). Biodegradable plastic i.e. polyhydroxyalkanoate can also be produced from pyrolytic liquid oil produced from thermal degradation of PS plastic wastes (Nikodinovic-Runic et al., 2011). ...
Poster
Catalytic Pyrolysis is a promising technique to convert the plastic waste into liquid oil and value added products. However use of synthetic catalyst may increase the cost of the project. To overcome the economical problem use of natural zeolite was carried out.
... Bacteria of the genus Rhodococcus, Cupriavidus, Pseudomonas, and Burkholderia, Achromobacter are specialized aromatic degraders along with the ability to form PHA under nutrient imbalance from styrene (Ward et al., 2005), benzene-toluene-ethylbenzene-p-xylene (BTEX) (Nikodinovic et al., 2008), toluene (Hori et al., 2009), furfural (Pan et al., 2012), 4-hydroxybenzoic acid, vanillic acid (Tomizawa et al., 2014), and phenol (Zhang et al., 2018), but to limited amounts -less than 0.5 g L −1 of the polyester. Nevertheless, in a fed-batch process, one of the highest mcl-PHA titre (3.4 g L −1 ) was obtained from styrene in P. putida CA-3 (Nikodinovic-Runic et al., 2011). In the last decade, the focus of research has turned into the use of aromatics derived from lignin for obtaining next-generation biochemicals (Becker & Wittmann, 2019;van Duuren et al., 2020). ...
Article
Full-text available
Microbial production of biopolymers derived from renewable substrates and waste streams reduces our heavy reliance on petrochemical plastics. One of the most important biodegradable polymers is the family of polyhydroxyalkanoates (PHAs), naturally occurring intracellular polyoxoesters produced for decades by bacterial fermentation of sugars and fatty acids at the industrial scale. Despite the advances, PHA production still suffers from heavy costs associated with carbon substrates and downstream processing to recover the intracellular product, thus restricting market positioning. In recent years, model‐aided metabolic engineering and novel synthetic biology approaches have spurred our understanding of carbon flux partitioning through competing pathways and cellular resource allocation during PHA synthesis, enabling the rational design of superior biopolymer producers and programmable cellular lytic systems. This review describes these attempts to rationally engineering the cellular operation of several microbes to elevate PHA production on specific substrates and waste products. We also delve into genome reduction, morphology, and redox cofactor engineering to boost PHA biosynthesis. Besides, we critically evaluate engineered bacterial strains in various fermentation modes in terms of PHA productivity and the period required for product recovery. This review describes the attempts to rationally engineering the cellular operation of several microbes to elevate PHA production on specific substrates and waste products. We also delve into genome reduction, morphology, and redox cofactor engineering to boost PHA biosynthesis. Besides, we critically evaluate engineered bacterial strains in various fermentation modes in terms of PHA productivity and the period required for product recovery.
... In 2005, Ward et al. first found that Pseudomonas putida CA-3 could convert the metabolite of styrene, PAA, into polyhydroxyalkanoate (PHA) when a limiting concentration of nitrogen was added to the growth medium. Their finding built the metabolic link between styrene degradation and PHA accumulation in P. putida CA-3 and found a trail for the microbial valorization of PS waste into valuable chemicals Nikodinovic-Runic et al., 2011). ...
Article
Full-text available
A growing accumulation of plastic wastes has become a severe environmental and social issue. It is urgent to develop innovative approaches for the disposal of plastic wastes. In recent years, reports on biodegradation of synthetic plastics by microorganisms or enzymes have sprung up, and these offer a possibility to develop biological treatment technology for plastic wastes. In this review, we have comprehensively summarized the microorganisms and enzymes that are able to degrade a variety of generally used synthetic plastics, such as polyethylene (PE), polystyrene (PS), polypropylene (PP), polyvinyl chloride (PVC), polyurethane (PUR), and polyethylene terephthalate (PET). In addition, we have highlighted the microbial metabolic pathways for plastic depolymerization products and the current attempts toward utilization of such products as feedstocks for microbial production of chemicals with high value. Taken together, these findings will contribute to building a conception of bio-upcycling plastic wastes by connecting the biodegradation of plastic wastes to the biosynthesis of valuable chemicals in microorganisms. Last, but not least, we have discussed the challenges toward microbial degradation and valorization of plastic wastes.
... PHAs can be produced by Pseudomonas species from TPA, as well as EG (Kenny et al. 2008(Kenny et al. , 2012Franden et al. 2018). Pseudomonas putida has often been suggested as a candidate to serve PHA production, not only from TPA and EG but also from the styrene degradation product phenylacetic acid (PAA) (Ward et al. 2006;Nikodinovic-Runic et al. 2011). The degradation products, TPA and EG, could serve as substrates for the bioplastic industry, where PHAs, such as polyhydroxybutyrate, are produced. ...
Chapter
Millions of tons of plastics entering the sea each year are a substantial environmental problem. It is expected that ocean plastic pollution will increase when considering the rapidly rising rates in global plastic production, in contrast to the relatively slow growth in plastic recycling rates, and future projections of increasing population densities in coastal areas. However, a significant discrepancy exists between the vast quantities of plastic entering the ocean and the orders of magnitude lower amounts afloat at the sea surface, indicating a substantial sink for ocean plastics. Plastics are probably degraded in a multi-step process facilitated by abiotic and biotic factors. Abiotic factors, such as shear stress induced by wave action, solar ultraviolet radiation, and heat embrittle and fragment plastics. Fragmentation of macroplastics results in micro and nanoscale particles. Photooxidation causes the release of chain scission products from the polymer matrix, e.g., nanoplastics, low-molecular-weight polymer fragments, and hydrocarbon gases. Biodegradation of plastics is mediated by microbes that have enzymes capable of inducing (1) chain scission and depolymerization, and (2) assimilate and terminally oxidize the intermediate products of initial degradation. Plastic degradation products from UV radiation could be a useful carbon source for microbes, while the role of marine microbes as initial degraders is not well understood. Several terrestrial microorganisms (bacteria, fungi) are known to degrade specific plastic polymers. For example, the bacterium Ideonella sakaiensis hydrolyses polyethylene terephthalate (PET) with a novel cutinase (termed PETase) and utilizes the degradation products as energy and carbon source. In the marine environment, complex hydrocarbon-degrading bacteria have repetitively been found in association with plastics. These bacteria have genes encoding for monooxygenases, peroxidases, and dehydrogenases, enzymes which can, in principle, facilitate the initial breakdown of plastics. Most commonly applied methods to investigate plastic biodegradation are based on monitoring weight loss of plastic over time, determining chemical changes of the polymer, investigating colonization of plastics by microbes, and measuring CO2 production rates. However, these evaluation methods often lack rigor in confirming initial depolymerization, assimilation, and mineralization. This chapter provides an overview of plastic biodegradation in the marine realm. Identified and potential microbial plastic degraders will be covered. Their metabolic and enzymatic capabilities will be highlighted with respect to valorization their potential in the future.
... The pyrolysis products from the PS and PET were directly used as carbon source for the production of mclPHAs by variety of wild type Pseudomonas spp. and moderate to good yields were obtained using optimization of fermentation strategies (Goff et al., 2007;Kenny et al., 2008;Nikodinovic-Runic et al., 2011). PE pyrolysis wax consisting of aliphatic hydrocarbons ranging from C8 to C32 was used by Pesudomonas areuginosa GL-1 and P. oleovorans B-14682, in the presence of a biosurfactant, for the production of mclPHA (Guzik et al., 2014). ...
Article
Full-text available
Inspirational concepts, and the transfer of analogs from natural biology to science and engineering, has produced many excellent technologies to date, spanning vaccines to modern architectural feats. This review highlights that answers to the pressing global petroleum-based plastic waste challenges, can be found within the mechanics and mechanisms natural ecosystems. Here, a suite of technological and engineering approaches, which can be implemented to operate in tandem with nature's prescription for regenerative material circularity, is presented as a route to plastics sustainability. A number of mechanical/green chemical (pre)treatment methodologies, which simulate natural weathering and arthropodal dismantling activities are reviewed, including: mechanical milling, reactive extrusion, ultrasonic-, UV- and degradation using supercritical CO2. Akin to natural mechanical degradation, the purpose of the pretreatments is to render the plastic materials more amenable to microbial and biocatalytic activities, to yield effective depolymerization and (re)valorization. While biotechnological based degradation and depolymerization of both recalcitrant and bioplastics are at a relatively early stage of development, the potential for acceleration and expedition of valuable output monomers and oligomers yields is considerable. To date a limited number of independent mechano-green chemical approaches and a considerable and growing number of standalone enzymatic and microbial degradation studies have been reported. A convergent strategy, one which forges mechano-green chemical treatments together with the enzymatic and microbial actions, is largely lacking at this time. An overview of the reported microbial and enzymatic degradations of petroleum-based synthetic polymer plastics, specifically: low-density polyethylene (LDPE), high-density polyethylene (HDPE), polystyrene (PS), polyethylene terephthalate (PET), polyurethanes (PU) and polycaprolactone (PCL) and selected prevalent bio-based or bio-polymers [polylactic acid (PLA), polyhydroxyalkanoates (PHAs) and polybutylene succinate (PBS)], is detailed. The harvesting of depolymerization products to produce new materials and higher-value products is also a key endeavor in effectively completing the circle for plastics. Our challenge is now to effectively combine and conjugate the requisite cross disciplinary approaches and progress the essential science and engineering technologies to categorically complete the life-cycle for plastics.
... Styrene or phenylacetate as substrate for PHA production was presented by the group of O'Connor (Ward et al., 2005). They went on and used PS pyrolysis oil to convert 64 g of plastic waste into 6.4 g of PHA (Ward et al., 2006), which could be enhanced even further (Goff et al., 2007;Nikodinovic-Runic et al., 2011). This is remarkable as most microbes could not even grow or survive in the presence of a second phase of styrene (i.e., at styrene concentrations above 2 mM). ...
Article
Full-text available
The plastic crisis requires drastic measures, especially for the plastics’ end-of-life. Mixed plastic fractions are currently difficult to recycle, but microbial metabolism might open new pathways. With new technologies for degradation of plastics to oligo- and monomers, these carbon sources can be used in biotechnology for the upcycling of plastic waste to valuable products, such as bioplastics and biosurfactants. We briefly summarize well-known monomer degradation pathways and computed their theoretical yields for industrially interesting products. With this information in hand, we calculated replacement scenarios of existing fossil-based synthesis routes for the same products. Thereby, we highlight fossil-based products for which plastic monomers might be attractive alternative carbon sources. Notably, not the highest yield of product on substrate of the biochemical route, but rather the (in-)efficiency of the petrochemical routes (i.e., carbon, energy use) determines the potential of biochemical plastic upcycling. Our results might serve as a guide for future metabolic engineering efforts towards a sustainable plastic economy.
... The recovered aromatic compounds present in liquid oil can also be utilized as raw materials in the chemical industries for plastic monomer polymerization (Shah and Jan 2014). The produced liquid oil from PS can also be used for the synthesis of a biodegradable and biocompatible plastic (Nikodinovic-Runic et al., 2011). ...
Article
This study aims to examine the catalytic pyrolysis of various plastic wastes in the presence of natural and synthetic zeolite catalysts. A small pilot scale reactor was commissioned to carry out the catalytic pyrolysis of polystyrene (PS), polypropylene (PP), polyethylene (PE) and their mixtures in different ratios at 450 °C and 75 min. PS plastic waste resulted in the highest liquid oil yield of 54% using natural zeolite and 50% using synthetic zeolite catalysts. Mixing of PS with other plastic wastes lowered the liquid oil yield whereas all mixtures of PP and PE resulted in higher liquid oil yield than the individual plastic feedstocks using both catalysts. The GC–MS analysis revealed that the pyrolysis liquid oils from all samples mainly consisted of aromatic hydrocarbons with a few aliphatic hydrocarbon compounds. The types and amounts of different compounds present in liquid oils vary with some common compounds such as styrene, ethylbenzene, benzene, azulene, naphthalene, and toluene. The FT-IR data also confirmed that liquid oil contained mostly aromatic compounds with some alkanes, alkenes and small amounts of phenol group. The produced liquid oils have high heating values (HHV) of 40.2–45 MJ/kg, which are similar to conventional diesel. The liquid oil has potential to be used as an alternative source of energy or fuel production.
Chapter
This chapter discusses a plethora of microbial biopolymers. The production strategy for a microbialpolyhydroxyalkanoatebiopolymerisdiscussedindetail
Chapter
The glory of synthetic polymer and the production and application can be contributed to their prize, permanence, and endowment for the proficiency in daily living. Although, one time utilization of synthetic plastic, longevity and rebellious characteristics is opened a continuous enchancement in synthetic polymer as a part of garbage. Requirement for the replacement of one-time utilized plastics which is hard to detect has motivated many researchers to work regarding the permanent solution to replace synthetic polymers that are oil-based plastics. Poly-β-hydroxybutyrate is a naturally formed biopolymer that can be used as plastic produced by various microorganisms. It has aquired importance because of its structural variations and similar analogy to synthetic polymer. Additionally, specific physico-chemical, bio-logical, and degradation characterisitics of biodegradable plastics is key factor which make bioplastic important for various uses in different field. This chapter contains classification, biosynthesis, detection techniques, and recovery methods of microbially produced biodegradable polymer. We then discuss the application of biodegradable polymers in various fields to reduce the dependence on synthetic plastic.
Article
Benzene, toluene, ethylbenzene, xylenes and styrene (BTEXS) are toxic pollutants that co-occur in wastewater effluents from petrochemical and chemical industries. Seeking effective and efficient treatments for these effluents is crucial in eliminating serious health and environmental issues that would otherwise arise from the anthropogenic release of BTEXS. This work examined the evolution in bacterial profiles under prolonged BTEXS enrichment and identified biomarkers associated with the enriched microbial community. The volatile suspended solids (VSS) increased by 24% within 15 months, indicating that the microbial community had evolved to assimilate BTEXS as an energy and carbon source. Six key biomarkers and three indicative biomarkers were identified at the bacterial order level. Rhizobales, Burkholderiales and Actinomycetales were identified as key biomarkers of the core BTEXS-degrading population while Sphingobacteriales, Flavobacteriales and Bacteroidales were identified as key biomarkers of the secondary BTEXS-degrading population. Xanthomonadales, Pseudomonadales and Clostridales may serve as indicative biomarkers of the operating conditions (i.e. BTEXS loading and oxygen levels). This new knowledge is beneficial to engineers in selecting seed inoculum and monitoring reactor stability. Furthermore, it potentially enables engineers to leverage on innate microbial characteristics to develop biological treatments for an effective and efficient remediation of BTEXS-laden wastewater effluents.
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During the past fifty years, the environmental pollution from non‐biodegradable petroleum‐based plastics has aggravated and become a real threat to marine life and human health. Thus, urgent solutions to plastic pollution are needed for reducing the contamination of soil and water resources. Developing alternative biodegradable plastics derived from renewable resources is an emerging research focus to alleviate the accumulation of plastic waste in the environment. Polyhydroxyalkanoates (PHAs) are one of the promising biodegradable biopolymers for replacing plastics derived from fossil fuel resources. The large scale commercialization of PHAs is not yet feasible due to their high production cost largely associated with the feedstock cost. Use of readily available carbon sources from underutilized lignocellulosic biomass and non‐recyclable plastic wastes allows for the feedstock cost reduction, production of value‐added PHA bioplastics, and significantly contributes to the solution of plastic pollution in a circular economy approach. This review highlights the recent efforts for valorizing plastic and lignocellulosic wastes to produce PHAs through a biotechnological approach using a two‐step methodology. In the first step, plastic (PE, PP, PS, PET) and lignocellulosic (cellulose, hemicellulose, lignin) macromolecules are depolymerized and converted to smaller fragments/ monomers, which will be utilized for the subsequent bio‐upcycling step via fermentation process to produce PHAs. Pyrolyzed plastic wastes and hydrolysates from lignocellulosic waste biomass will facilitate the transition from linear to circular economy, lower the production cost of PHAs, and contribute the solution of plastic pollution in a practical, economical, and sustainable approach. This article is protected by copyright. All rights reserved.
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Petroleum-based synthetic plastics are difficult to be biodegraded due to their large molecular weight, high hydrophobicity, and high chemical bond energy. The increasing accumulation of waste plastic in the environment has resulted in severe “white pollution.” Currently, landfilling and incineration are commonly used for waste plastic disposal. However, life-cycle assessment (LCA) studies indicate that landfilling is the worst end-of-life management option, while incineration is also not a viable solution for plastic pollution due to the considerable release of greenhouse gas and dangerous substances. Therefore, it is urgent to develop a green and efficient recycling technology for waste plastic management. In recent years, studies on plastics biodegradation using microorganisms or enzymes have achieved a breakthrough progress, exhibiting a promising prospect for developing biodegradation and biotransformation technologies toward waste plastics disposal. In this chapter, we comprehensively summarized the microorganisms and enzymes involved in plastics depolymerization, with emphasis on the analysis of the biodepolymerization pathways and degradants derived from specific plastics. In addition, application of these depolymerization products as substrates for high-value added chemicals production is highlighted. Moreover, the obstacles lying on the way for waste plastics biodegradation, including the lack of depolymerases, low depolymerization efficiency, and difficulties against plastic degradants utilization are deeply discussed.
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With the impending fossil fuel crisis, the search for and development of alternative chemical/material substitutes is pivotal in reducing mankind’s dependency on fossil resources. One of the potential substitute candidates is polyhydroxyalkanoate (PHA). PHA is a carbon-neutral and valuable polymer that could be produced from many renewable carbon sources by microorganisms, making it a sustainable and environmental-friendly material. At present, PHA is not cost competitive compared to fossil-derived products. Encouraging and intensifying research work on PHA is anticipated to enhance its economic viability in the future. The development of various biomolecular and chemical techniques for PHA analysis has led to the identification of many PHA-producing microbial strains, some of which are deposited in culture collections. Research work on PHA could be rapidly initiated with these ready-to-use techniques and microbial strains. This review aims to facilitate the start-up of PHA research by providing a summary of commercially available PHA-accumulating microbial cultures, PHA biosynthetic pathways, and methods for PHA detection, extraction and analysis.
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A work applied response surface methodology coupled with Box–Behnken design (RSM-BBD) has been developed to enhance styrene recovery from waste polystyrene (WPS) through pyrolysis. The relationship between styrene yield and three selected operating parameters (i.e., temperature, heating rate, and carrier gas flow rate) was investigated. A second order polynomial equation was successfully built to describe the process and predict styrene yield under the study conditions. The factors identified as statistically significant to styrene production were: temperature, with a quadratic effect; heating rate, with a linear effect; carrier gas flow rate, with a quadratic effect; interaction between temperature and carrier gas flow rate; and interaction between heating rate and carrier gas flow rate. The optimum conditions for the current system were determined to be at a temperature range of 470–505 °C, a heating rate of 40 °C/min, and a carrier gas flow rate range of 115–140 mL/min. Under such conditions, 64.52% WPS was recovered as styrene, which was 12% more than the highest reported yield for reactors of similar size. It is concluded that RSM-BBD is an effective approach for yield optimization of styrene recovery from WPS pyrolysis.
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The present rate of economic growth is unsustainable without saving of fossil energy like crude oil, natural gas or coal. Thus mankind has to rely on the alternate/renewable energy sources like biomass, hydropower, geothermal energy, wind energy, solar energy, nuclear energy, etc. On the other hand, suitable waste management strategy is another important aspect of sustainable development. The growth of welfare levels in modern society during the past decades has brought about a huge increase in the production of all kinds of commodities, which indirectly generate waste. Plastics have been one of the materials with the fastest growth because of their wide range of applications due to versatility and relatively low cost. Since the duration of life of plastic products is relatively small, there is a vast plastics waste stream that reaches each year to the final recipients creating a serious environmental problem. Again, because disposal of post consumer plastics is increasingly being constrained by legislation and escalating costs, there is considerable demand for alternatives to disposal or land filling. Advanced research in the field of green chemistry could yield biodegradable/green polymers but is too limited at this point of time to substitute the non-biodegradable plastics in different applications. Once standards are developed for degradable plastics they can be used to evaluate the specific formulations of materials which will find best application in this state as regards their performance and use characteristics. Among the alternatives available are source reduction, reuse, recycling, and recovery of the inherent energy value through waste-to-energy incineration and processed fuel applications. Production of liquid fuel would be a better alternative as the calorific value of the plastics is comparable to that of fuels, around 40Â MJ/kg. Each of these options potentially reduces waste and conserves natural resources. Plastics recycling, continues to progress with a wide range of old and new technologies. Many research projects have been undertaken on chemical recycling of waste plastics to fuel and monomer. This is also reflected by a number of pilot, demonstration, and commercial plants processing various types of plastic wastes in Germany, Japan, USA, India, and elsewhere. Further investigations are required to enhance the generation of value added products (fuel) with low investments without affecting the environment. The paper reviews the available literature in this field of active research and identifies the gaps that need further attention.
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Twelve styrene-utilizing bacteria were isolated from a biofilter used for treating gaseous styrene. A gramnegative strain had a high styrene-degrading activity and was identified as Pseudomonas putida SN1 by 16S rDNA analysis. The styrene degradation in SN1 was regarded to start with a monooxygenase enzyme which converted styrene to styrene oxide, a potentially important chiral building block in organic synthesis. SN1 could grow on styrene and styrene oxide, but not on benzene and toluene. The styrene degradation activity in SN1 was induced when incubated with styrene, and the induction was not inhibited by the presence of readily usable carbon sources such as glucose and citrate. The optimal activity was shown at pH 7.0 and 30 °C and estimated as 170 unit/g cell.
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Here, we report the use of petrochemical aromatic hydrocarbons as a feedstock for the biotechnological conversion into valuable biodegradable plastic polymers--polyhydroxyalkanoates (PHAs). We assessed the ability of the known Pseudomonas putida species that are able to utilize benzene, toluene, ethylbenzene, p-xylene (BTEX) compounds as a sole carbon and energy source for their ability to produce PHA from the single substrates. P. putida F1 is able to accumulate medium-chain-length (mcl) PHA when supplied with toluene, benzene, or ethylbenzene. P. putida mt-2 accumulates mcl-PHA when supplied with toluene or p-xylene. The highest level of PHA accumulated by cultures in shake flask was 26% cell dry weight for P. putida mt-2 supplied with p-xylene. A synthetic mixture of benzene, toluene, ethylbenzene, p-xylene, and styrene (BTEXS) which mimics the aromatic fraction of mixed plastic pyrolysis oil was supplied to a defined mixed culture of P. putida F1, mt-2, and CA-3 in the shake flasks and fermentation experiments. PHA was accumulated to 24% and to 36% of the cell dry weight of the shake flask and fermentation grown cultures respectively. In addition a three-fold higher cell density was achieved with the mixed culture grown in the bioreactor compared to shake flask experiments. A run in the 5-l fermentor resulted in the utilization of 59.6 g (67.5 ml) of the BTEXS mixture and the production of 6 g of mcl-PHA. The monomer composition of PHA accumulated by the mixed culture was the same as that accumulated by single strains supplied with single substrates with 3-hydroxydecanoic acid occurring as the predominant monomer. The purified polymer was partially crystalline with an average molecular weight of 86.9 kDa. It has a thermal degradation temperature of 350 degrees C and a glass transition temperature of -48.5 degrees C.
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Growth of Pseudomonas oleovorans GPol in continuous culture containing a bulk n-octane phase resulted in changes of the fatty acid composition of the membrane lipids. Compared to citrate-grown cells, the ratio of C18 to C16 fatty acids and the ratio of unsaturated to saturated fatty acids increased as a result of growth on octane. Trans-unsaturated fatty acids, which are rarely found in bacteria, were formed during continuous growth of P. oleovorans on octane. Moreover, the mean acyl chain length and unsaturated fatty acids also increased as the growth rates increased both in octane-grown and citrate-grown cells. Differential scanning calorimetry measurements of extracted lipids showed the transition temperature of membrane lipids from octane-grown cells increased from about 24°C to 32°C as the growth rate increased, whereas cells grown on citrate showed a constant transition temperature of about 6°C at all growth rates tested, indicating a decrease of membrane lipid fluidity in octane-grown cells. Because alkanes are known to increase bilayer fluidity by intercalating between lipid fatty acyl chains, the increased transition temperature of the lipids of cells grown on octane may be a physiological response of P. oleovorans to compensate for the direct effects of octane on its cellular membranes.
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Pseudomonas putida CA-3 is a styrene-degrading bacterium capable of accumulating medium-chain-length polyhydroxyalkanoate (mclPHA) when exposed to limiting concentrations of a nitrogen source in the growth medium. Using shotgun proteomics we analysed global proteome expression in P. putida CA-3 supplied with styrene as the sole carbon and energy source under N-limiting (condition permissive for mclPHA synthesis) and non-limiting (condition non-permissive for mclPHA accumulation) growth conditions in order to provide insight into the molecular response of P. putida CA-3 to limitation of nitrogen when grown on styrene. A total of 1761 proteins were identified with high confidence and the detected proteins could be assigned to functional groups including styrene degradation, energy, nucleotide metabolism, protein synthesis, transport, stress response and motility. Proteins involved in the upper and lower styrene degradation pathway were expressed throughout the 48 h growth period under both nitrogen limitation and excess. Proteins involved in polyhydroxyalkanoate (PHA) biosynthesis, nitrogen assimilation and amino acid transport, and outer membrane proteins were upregulated under nitrogen limitation. PHA accumulation and biosynthesis were only expressed under nitrogen limitation. Nitrogen assimilation proteins were detected on average at twofold higher amounts under nitrogen limitation. Expression of the branched-chain amino acid ABC transporter was up to 16-fold higher under nitrogen-limiting conditions. Branched chain amino acid uptake by nitrogen-limited cultures was also higher than that by non-limited cultures. Outer membrane lipoproteins were expressed at twofold higher levels under nitrogen limitation. This was confirmed by Western blotting (immunochemical detection) of cells grown under nitrogen limitation. Our study provides the first global description of protein expression changes during growth of any organism on styrene and accumulating mclPHA (nitrogen-limited growth).
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Styrene metabolism in styrene-degrading Pseudomonas putida CA-3 cells has been shown to proceed via styrene oxide, phenylacetaldehyde, and phenylacetic acid. The initial step in styrene degradation by strain CA-3 is oxygen-dependent epoxidation of styrene to styrene oxide, which is subsequently isomerized to phenylacetaldehyde. Phenylacetaldehyde is then oxidized to phenylacetic acid. Styrene, styrene oxide, and phenylacetaldehyde induce the enzymes involved in the degradation of styrene to phenylacetic acid by P. putida CA-3. Phenylacetic acid-induced cells do not oxidize styrene or styrene oxide. Thus, styrene degradation by P. putida CA-3 can be subdivided further into an upper pathway which consists of styrene, styrene oxide, and phenylacetaldehyde and a lower pathway which begins with phenylacetic acid. Studies of the repression of styrene degradation by P. putida CA-3 show that glucose has no effect on the activity of styrene-degrading enzymes. However, both glutamate and citrate repress styrene degradation and phenylacetic acid degradation, showing a common control mechanism on upper pathway and lower pathway intermediates.
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Genomic analysis of Pseudomonas putida sheds light on metabolic pathways that may be exploited for a variety of biotechnological applications.
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Pseudomonas putida CA-3 is capable of converting the aromatic hydrocarbon styrene, its metabolite phenylacetic acid, and glucose into polyhydroxyalkanoate (PHA) when a limiting concentration of nitrogen (as sodium ammonium phosphate) is supplied to the growth medium. PHA accumulation occurs to a low level when the nitrogen concentration drops below 26.8 mg/liter and increases rapidly once the nitrogen is no longer detectable in the growth medium. The depletion of nitrogen and the onset of PHA accumulation coincided with a decrease in the rate of substrate utilization and biochemical activity of whole cells grown on styrene, phenylacetic acid, and glucose. However, the efficiency of carbon conversion to PHA dramatically increased once the nitrogen concentration dropped below 26.8 mg/liter in the growth medium. When supplied with 67 mg of nitrogen/liter, the carbon-to-nitrogen (C:N) ratios that result in a maximum yield of PHA (grams of PHA per gram of carbon) for styrene, phenylacetic acid, and glucose are 28:1, 21:1, and 18:1, respectively. In cells grown on styrene and phenylacetic acid, decreasing the carbon-to-nitrogen ratio below 28:1 and 21:1, respectively, by increasing the nitrogen concentration and using a fixed carbon concentration leads to lower levels of PHA per cell and lower levels of PHA per batch of cells. Increasing the carbon-to-nitrogen ratio above 28:1 and 21:1 for cells grown on styrene and phenylacetic acid, respectively, by decreasing the nitrogen concentration and using a fixed carbon concentration increases the level of PHA per cell but results in a lower level of PHA per batch of cells. Increasing the carbon and nitrogen concentrations but maintaining the carbon-to-nitrogen ratio of 28:1 and 21:1 for cells grown on styrene and phenylacetic acid, respectively, results in an increase in the total PHA per batch of cells. The maximum yields for PHA from styrene, phenylacetic acid, and glucose are 0.11, 0.17, and 0.22 g of PHA per g of carbon, respectively.
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A modeling study was conducted on growth kinetics of three different strains of Pseudomonas spp. (Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida) during benzene degradation to determine optimum substrate concentrations for most efficient biodegradation. Batch tests were performed for eight different initial substrate concentrations to observe cell growth and associated substrate degradation using benzene-adapted cells. Kinetic parameters of both inhibitory (Haldane-Andrews, Aiba-Edwards) and noninhibitory (Monod) models were fitted to the relationship between specific growth rate and substrate concentration obtained from the growth curves. Results showed that half-saturation constant of P. fluorescens was the highest among the three strains, indicating that this strain could grow well at high concentration, while P. putida could grow best at low concentration. The inhibition constant of P. aeruginosa was the highest, implying that it could tolerate high benzene concentration and therefore could grow at a wider concentration range. Estimated specific growth rate of P. putida was lower, but half-saturation constant was higher than those from literature study due to high substrate concentration range used in this study. These two kinetic parameters resulted in substantial difference between Monod- and Haldane-type models, indicating that distinction should be made in applying those models.
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Pseudomonas oleovorans grows on C(6) to C(12)n-alkanes and 1-alkenes. These substrates are oxidized to the corresponding fatty acids, which are oxidized further via the beta-oxidation pathway, yielding shorter fatty acids which have lost one or more C(2) units. P. oleovorans normally utilizes beta-oxidation pathway intermediates for growth, but in this paper we show that the intermediate 3-hydroxy fatty acids can also be polymerized to intracellular poly-(R)-3-hydroxyalkanoates (PHAs) when the medium contains limiting amounts of essential elements, such as nitrogen. The monomer composition of these polyesters is a reflection of the substrates used for growth of P. oleovorans. The largest monomer found in PHAs always contained as many C atoms as did the n-alkane used as a substrate. Monomers which were shorter by one or more C(2) units were also observed. Thus, for C-even substrates, only C-even monomers were found, the smallest being (R)-3-hydroxyhexanoate. For C-odd substrates, only C-odd monomers were found, with (R)-3-hydroxyheptanoate as the smallest monomer. 1-Alkenes were also incorporated into PHAs, albeit less efficiently and with lower yields than n-alkanes. These PHAs contained both saturated and unsaturated monomers, apparently because the 1-alkene substrates could be oxidized to carboxylic acids at either the saturated or the unsaturated ends. Up to 55% of the PHA monomers contained terminal double bonds when P. oleovorans was grown on 1-alkenes. The degree of unsaturation of PHAs could be modulated by varying the ratio of alkenes to alkanes in the growth medium. Since 1-alkenes were also shortened before being polymerized, as was the case for n-alkanes, copolymers which varied with respect to both monomer chain length and the percentage of terminal double bonds were formed during nitrogen-limited growth of P. oleovorans on 1-alkenes. Such polymers are expected to be useful for future chemical modifications.
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Pseudomonas oleovorans was grown in homogeneous media containing n-alkanoic acids, from formate to decanoate, as the sole carbon sources. Formation of intracellular poly(beta-hydroxyalkanoates) was observed only for hexanoate and the higher n-alkanoic acids. The maximum isolated polymer yields were approximately 30% of the cellular dry weight with growth on either octanoate or nonanoate. In most cases, the major repeating unit in the polymer had the same chain length as the n-alkanoic acid used for growth, but units with two carbon atoms less or more than the acid used as a carbon source were also generally present in the polyesters formed. Indeed, copolymers containing as many as six different types of beta-hydroxyalkanoate units were formed. The weight average molecular weights of the poly(beta-hydroxyalkanoate) copolymers produced by P. oleovorans ranged from 90,000 to 370,000. In spite of the higher cell yields obtained with octanoate and nonanoate, the use of hexanoate and heptanoate yielded higher-molecular-weight polymers. These copolyesters represent an entirely new class of biodegradable thermoplastics.
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Polyhydroxyalkanoates (PHAs) have been established as biodegradable polymers since the second half of the twentieth century. Altering monomer composition of PHAs allows the development of polymers with favorable mechanical properties, biocompatibility and desirable degradation rates, under specific physiological conditions. Hence, the medical applications of PHAs have been explored extensively in recent years. PHAs have been used to develop devices, including sutures, nerve repair devices, repair patches, slings, cardiovascular patches, orthopedic pins, adhesion barriers, stents, guided tissue repair/regeneration devices, articular cartilage repair devices, nerve guides, tendon repair devices, bone-marrow scaffolds, tissue engineered cardiovascular devices and wound dressings. So far, various tests on animal models have shown polymers, from the PHA family, to be compatible with a range of tissues. Often, pyrogenic contaminants copurified with PHAs limit their pharmacological application rather than the monomeric composition of the PHAs and thus the purity of the PHA material is critical. This review summarizes the animal testing, tissue response, in vivo molecular stability and challenges of using PHAs for medical applications. In future, PHAs may become the materials of choice for various medical applications.
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This welcome new edition discusses bioprocess engineering from the perspective of biology students. It includes a great deal of new material and has been extensively revised and expanded. These updates strengthen the book and maintain its position as the book of choice for senior undergraduates and graduates seeking to move from biochemistry/microbiology/molecular biology to bioprocess engineering.
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Pseudomonas oleovorans is able to produce medium-chain length poly(3-hydroxyalkanoates) (mcl-PHA) in continuous and fed-batch two-liquid phase fermentations using n-octane as a sole carbon and energy source. We have previously shown that it is possible to increase the volumetric productivity of such a system by increasing the concentration of cells and PHA in the fermentor with maximal production limited by the oxygen transfer rate to the cultures in our bioreactor systems and by complex effects of metal ions on biomass yields, leading to a maximal biomass concentration of 37 g l−1. This paper describes further improvements in the cultivation process of P. oleovorans for the production of mcl-PHA in two-liquid phase fermentations that have led to a threefold higher final cell density. In order to further increase cell densities, we determined the growth yields for each of the metal ions and developed an optimized feed of metals. Using a bioreactor with better oxygen transfer capabilities, we were able to increase the final cell density in fed-batch cultivations up to 90 g biomass l−1. By applying a computer-controlled exponential nitrogen feed in combination with the feeding of various metal ions, a cell density of 112 g l−1 was obtained. The PHA content of these cells decreased as the cell density increased above 40–50 g l−1, thus negatively affecting overall PHA yields and productivities. Possible approaches to reducing these PHA losses are discussed.
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The absorption of oxygen and styrene in water–silicone oil emulsions was independently studied in laboratory-scale bubble reactors at a constant gas flow rate for the whole range of emulsion compositions (0–10% v/v). The volumetric mass transfer coefficients to the emulsions were experimentally measured using a dynamic absorption method. It was assumed that the gas phase contacts preferentially the water phase. In the case of oxygen absorption, it was found that the addition of silicone oil hinders oxygen mass transfer compared to an air–water system. Decreases in kLaoxygen of up to 25% were noted. Such decreases in the oxygen mass transfer coefficient, which imply longer aeration times to transfer oxygen, could represent a limiting step in biotechnological processes strongly dependent on oxygen concentration. Nevertheless, as the large affinity of silicone oil for oxygen enables greater amounts of oxygen to be transferred from the gas phase, it appears that the addition of more than 5% silicone oil should be beneficial to increase the oxygen transfer rate. In the case of styrene absorption, it was established that the volumetric mass transfer coefficient based on the emulsion volume is roughly constant with the increase in the emulsion composition. In spite of the relatively high cost of silicone oil, water–silicone oil emulsions remain relevant to treat low-solubility volatile organic compounds, such as styrene, in low-concentration gas streams.
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Fully expanded and revised, this handbook lists over 2,000 organic chemicals while incorporating the extensive new information available on the environmental impact of these substances. New coverage is given on mixtures and preparations, individual chemicals, pesticides, detergents, pthalates, polynuclear aromatics, and PCBs. Special attention is given to pollutants of the abiotic and biotic environment, the correlation of bioaccumulation of chemicals to molecular structure, and the use of water solubility data to estimate the fate of chemicals in the environment. Data for each organic chemical includes synonyms, formula, properties, air and water pollution factors, and biological effects.
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Feedstock recycling of plastic waste by thermal and catalytic processes is a promising route to eliminate this refuse (which is harmful to the environment) by obtaining, at the same time, products that are useful as fuels or chemicals. During the past decade, this option has undergone an important evolution from a promising scientific idea to an alternative that is very close to reality with commercial opportunities. Thus, several commercial processes have been developed worldwide, most of them especially addressed toward the preparation of diesel fuel. The present review highlights the most remarkable achievements of the field, providing a fundamental insight into this fascinating area and highlighting the main milestones that should be achieved in the next future for this alternative to become applied commercially on a large scale.
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A search has been made for relations between published results about gas-liquid mass transfer in stirred vessels filled with water. The dynamic gassing-out method, the sulfite method, the separate determinations of A and kL as well as a number of other experimental methods were judged on their own merits. Most methods appear to be of limited applicability, and the sulfite method is even more restricted in this respect. Correlation of the various reports on mass transfer is possible with power per unit volume and gas superficial velocity. This requires a rigid differentiation between water without and water with ions in solution. Among the equipment variables it is in particular the position of the sparger which appears to be of influence.
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Es wird ein Verfahren zur Submerskultur von Knallgasbakterien beschrieben. Es beruht auf der kräftigen Magnetrührung der Nährlösung unter einem Gemisch von H2, O2 und CO2. Der hohen O2-Empfindlichkeit der Zellen wird durch „Gradientenbegasung” Rechnung getragen. Der fakultativ chemolithotrophe Hydrogenomonas-Stamm 20 wurde bakteriologisch charakterisiert und wachstumsphysiologisch untersucht. Die Generationszeit beträgt während der log-Phase 21/6 Std, die scheinbare Verdoppelungszeit 31/5 Std (28° C).
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The Berthelot reaction, based on development of a deep blue colour when ammonia reacts with phenol and alkaline hypochlorite, was investigated and modified. The reaction variables were studied, and a convenient and reliable analytical procedure was developed for ammonia and Kjeldahl nitrogen determination, well suited for routine analysis of fresh and waste water. The buffering system introduced into the reagents permits examination of domestic wastewater, without pH correction, in the 3–11·5 range. The method gives highly reproducible results in the range of 0.02–1 mg l−1 ammonia nitrogen (1 cm light path). The solutions are ready for photometric measurement after 45 min of colour development at room temperature, and remain stable at least 48 h.Interfering substances likely to be present in domestic wastewater are hardness above 400 mg l−1 and nitrite ions above 5 mg l−1n. In high-ammonia wastewater samples, this interference is eliminated by the dilution required for photometric measurements.
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During the last decade, a new generation of high-performance, computerized bioreactors, on-line monitoring devices for medium components, and techniques for continuous analysis of cell composition have revolutionized experimental fermentation research. In view of this development, it is timely to review the corresponding mathematical models for growth and product of the most important microorganisms: bacteria and fungi. The review is focused on structured models which describe microbial kinetics by means of selected cell components rather than by the undifferential biomass. These models have sufficient details to make them compatible with the new experimental techniques, and together experimental and modelling work may advance our knowledge of microbial physiology or suggest new developments in fermentation technology. Not unnaturally, different authors mean different things by structured microbial models, and their nomenclature may vary considerably. To make it easier to classify different models and to recognize a near identity between several models, our review of bacterial and fungal models is preceded by a discussion of a general framework for the study of microbial kinetics. The use of a two-dimensional discretization of the kinetic model wherein both the individual cell and the cell mass is structured is shown to be helpful in dealing with comlex fermentation systems.
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Medium chain length (mcl) poly(hydroxyalkanoic acids) (PHAs) are polyesters accumulated by fluorescent Pseudomonads and other bacteria. Work on the genetics of mcl-PHA formation has led to polymer synthesis in recombinant bacteria and plants. Several high and medium cost applications are now emerging. With optimized bacterial mcl-PHA synthesis on inexpensive agro-substrates and the development of plant-based mcl-PHAs in the next decade, the production economics of these bioplastics will ultimately permit their sustainable production for bulk applications.
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Pseudomonas putida CA-3 has been shown to accumulate the biodegradable plastic polyhydroxyalkanoate (PHA) when fed styrene or polystyrene pyrolysis oil as the sole carbon and energy source under nitrogen limiting growth conditions (67 mg nitrogen per litre at time 0). Batch fermentation of P. putida CA-3 grown on styrene or polystyrene pyrolysis oil in a stirred tank reactor yields PHA at 30% of the cell dry weight (CDW). The feeding of nitrogen at a rate of 1 mg N/l/h resulted in a 1.1-fold increase in the percentage of CDW accumulated as PHA. An increase in the rate of nitrogen feeding up to 1.5 mg N/l/h resulted in further increases in the percentage of the cell dry weight composed of PHA. However, feeding rates of 1.75 and 2 mg N/l/h resulted in dramatic decreases in the percentage of cell dry weight composed of PHA.Interestingly nitrogen was not detectable in the growth medium after 16 h, in any of the growth conditions tested. A higher cell density was observed in cells supplied with nitrogen and thus further increases in the overall production of PHA were observed through nitrogen feeding. The highest yield of PHA was 0.28 g PHA per g styrene supplied with a nitrogen feeding rate of 1.5 mg/l/h.
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Discusses a biochemical engineering course that is offered as part of a chemical engineering curriculum and includes topics that influence the behavior of man-made or natural microbial or enzyme reactors. (MLH)
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Production of polyhydroxyalkanoates (PHAs) has been investigated for more than eighty years but recently a number of factors including increase in the price of crude oil and public awareness of the environmental issues have become a notable driving force for extended research on biopolymers. The versatility of PHAs has made them good candidates for the study of their potential in a variety of areas from biomedical/medical fields to food, packaging, textile and household material. While production costs are still a drawback to wider usage of these biopolymers, their application as low volume high cost items is becoming a reality. The future trend is to focus on the development of more efficient and economical processes for PHA production, isolation, purification and improvement of PHA material properties.
Article
Biopolyesters polyhydroxyalkanoates (PHA) produced by many bacteria have been investigated by microbiologists, molecular biologists, biochemists, chemical engineers, chemists, polymer experts and medical researchers. PHA applications as bioplastics, fine chemicals, implant biomaterials, medicines and biofuels have been developed and are covered in this critical review. Companies have been established or involved in PHA related R&D as well as large scale production. Recently, bacterial PHA synthesis has been found to be useful for improving robustness of industrial microorganisms and regulating bacterial metabolism, leading to yield improvement on some fermentation products. In addition, amphiphilic proteins related to PHA synthesis including PhaP, PhaZ or PhaC have been found to be useful for achieving protein purification and even specific drug targeting. It has become clear that PHA and its related technologies are forming an industrial value chain ranging from fermentation, materials, energy to medical fields (142 references).
Article
Although microbial growth on substrate mixtures is commonly encountered in bioremediation, wastewater treatment, and fermentation, mathematical modeling of mixed substrate kinetics has been limited. We report the kinetics of Pseudomonas putida F1 growing on benzene, toluene, phenol, and their mixtures, and compare mathematical models to describe these results. The three aromatics are each able to act as carbon and energy sources for this strain. Biodegradation rates were measured in batch cultivations following a protocol that eliminated mass transfer limitations for the volatile substrates and considered the culture history of the inoculum and the initial substrate to inoculum mass ratio. Toluene and benzene were better growth substrates than phenol, resulting in faster growth and higher yield coefficients. In the concentration ranges tested, toluene and benzene biodegradation kinetics were well described by the Monod model. The Monod model was also used to characterize phenol biodegradation by P. putida F1, although a small degree of substrate inhibition was noted. In mixture experiments, the rate of consumption of one substrate was found to be affected by the presence of the others, although the degree of influence varied widely. The substrates are catabolized by the same enzymatic pathway, but purely competitive enzyme kinetics did not capture the substrate interactions well. Toluene significantly inhibited the biodegradation rate of both of the other substrates, and benzene slowed the consumption of phenol (but not of toluene). Phenol had little effect on the biodegradation of either toluene or benzene. Of the models tested, a sum kinetics with interaction parameters (SKIP) model provided the best description of the paired substrate results. This model, with parameters determined from one- and two-substrate experiments, provided an excellent prediction of the biodegradation kinetics for the three-component mixture.
Article
Functionalized medium-chain-length polyhydroxyalkanoates (mclPHAs) have gained much interest in research on biopolymers because of their ease of chemical modification. Tailored olefinic mclPHA production from mixtures of octanoic acid and 10-undecenoic acid was investigated in batch and dual (C,N) nutrient limited chemostat cultures of Pseudomonas putida GPo1 (ATCC 29347). In a batch culture, where P. putida GPo1 was grown on a mixture of octanoic acid (58 mol%) and 10-undecenoic acid (42 mol%), it was found that the fraction of aliphatic monomers was slightly lower in mclPHA produced during exponential growth than during late stationary phase. Thus, the total monomeric composition changed over time indicating different kinetics for the two carbon substrates. Chemostat experiments showed that the dual (C,N) nutrient limited growth regime (DNLGR) for 10-undecenoic acid coincided with the one for octanoic acid. Five different chemostats on equimolar mixtures of octanoic acid and 10-undecenoic acid within the DNLGR revealed that the monomeric composition of mclPHA was not a function of the carbon to nitrogen (C(0)/N(0)) ratio in the feed medium but rather of the dilution rate. The fraction of aliphatic monomers in the accumulated mclPHA was slightly lower at high dilution rates and increased towards low dilution rates, again indicating different kinetics for the two carbon substrates in P. putida GPo1.
Article
The urinary excretion of unmetabolized styrene can be a very good indicator for biomonitoring styrene in occupationally exposed people. The use of a new urine sampling system, involving a solid-phase extraction cartridge, offers several advantages for determining styrene. The advantages are especially related to the pre-analytical phase of styrene determination, which may be influenced by many variables. The effect on styrene recovery of sorbent type, eluting solvent, elution volume, elution flow-rate, and the addition of methanol to the washing solvent, was evaluated by experimental design methodology. As a result, Oasis HLB cartridges were selected for urine sampling, as well as 1.5 mL of ethyl acetate at 0.5 mL/min for eluting the retained styrene. These conditions were then applied to the validation of the solid-phase extraction combined with GC-MS method for the sampling and analysis of unmetabolized styrene in urine. The overall uncertainty was in the 12-22% range and the limit of detection was 2.2 microg/L for a 4 mL urine sample. The stability of styrene has been studied both in cartridges and in vials under different storage periods. After 1 month period the styrene stored on cartridges at room temperature remained stable, whereas this is not the case for styrene recovery from vials. The results obtained indicate that on-site solid-phase extraction of urine can provide a simple, accurate and reproducible sampling and analytical method for the biomonitoring of styrene in urine.
Article
A novel approach to the recycling of polystyrene is reported here; polystyrene is converted to a biodegradable plastic, namely polyhydroxyalkanoate (PHA). This unique combinatorial approach involves the pyrolysis of polystyrene to styrene oil, followed by the bacterial conversion of the styrene oil to PHA by Pseudomonas putida CA-3 (NCIMB 41162). The pyrolysis (520 degrees C) of polystyrene in a fluidized bed reactor (Quartz sand (0.3-0.5 mm)) resulted in the generation of an oil composed of styrene (82.8% w/w) and low levels of other aromatic compounds. This styrene oil, when supplied as the sole source of carbon and energy allowed for the growth of P. putida CA-3 and PHA accumulation in shake flask experiments. Styrene oil (1 g) was converted to 62.5 mg of PHA and 250 mg of bacterial biomass in shake flasks. A 1.6-fold improvement in the yield of PHA from styrene oil was achieved by growing P. putida CA-3 in a 7.5 liter stirred tank reactor. The medium chain length PHA accumulated was comprised of monomers 6, 8, and 10 carbons in length in a molar ratio of 0.046:0.436:1.126, respectively. A single pyrolysis run and four fermentation runs resulted in the conversion of 64 g of polystyrene to 6.4 g of PHA.
Pseudomonasoleovoransasa source of poly(b-hydroxyalkanoates) for potential applications as biodegradable polyesters
  • Brandlh
  • Grossra
  • Lenzrw
  • Fullerrc
BrandlH,GrossRA,LenzRW,FullerRC.1988.Pseudomonasoleovoransasa source of poly(b-hydroxyalkanoates) for potential applications as biodegradable polyesters. Appl Environ Microbiol 54:1977–1982
Bioprocess technology – fundamentals and applications (A textbook for introduction of the theory and practice of biotechnical processes)
  • Enfors SO
  • Häggström L.
Energetics and kinetics in biotechnology
  • Roels JA