Article

Improvement of the conversion of polystyrene to polyhydroxyalkanoate through the manipulation of the microbial aspect of the process: A nitrogen feeding strategy for bacterial cells in a stirred tank reactor

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Abstract

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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... Researchers have attempted to address both the cost of production and waste management using different types of waste, including plastic waste, as a cheap feedstock for PHA production, for example polystyrene (Goff et al., 2007), polyethylene terephthalate (PET) (Kenny et al., 2012), waste glycerol (Cavalheiro et al., 2009), animal-based waste streams (Titz et al., 2012), syngas obtained by municipal solid waste (MSW) pyrolysis (Revelles et al., 2016) as well as using low cost biomass (Cerrone et al., 2015;Walsh et al., 2015). ...
... Of particular interest to the emerging circular economy is the upcycling of plastic waste into biodegradable plastic (Goff et al., 2007;Kenny et al., 2008Kenny et al., , 2012Wierckx et al., 2015). While conventional recycling technologies are available, there are several limitations, including cost and relatively low quality of the recycled polymers. ...
... Employing the microbial cell factory to convert plastic waste into high value product provides an alternative to conventional recycling. Due to extreme recalcitrance of plastics to microbial degradation, this biotechnological process currently employs pyrolysis to produce oils, which are subsequently fed to bacteria (Goff et al., 2007;Kenny et al., 2012). However, microbial hydrolases capable of modifying or degrading plastics have emerged recently as a potential technology for plastic biodepolymerization (Wei and Zimmermann, 2017) allowing for a completely biological recycling of plastics. ...
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Oceans are a major source of biodiversity, they provide livelihood, and regulate the global ecosystem by absorbing heat and CO2 . However, they are highly polluted with plastic waste. We are discussing here microbial biotechnology advances with the view to improve the start and the end of life of biodegradable polymers, which could contribute to the sustainable use of marine and coastal ecosystems (UN Sustainability development goal 14).
... Recent legislation on waste diversion from landfill (2008/ 98/EC), has driven the search for technologies to recycle wastes such as plastic (Aguardo et al., 2008; Panda et al., 2010 ). We have previously developed a two-step chemobiotechnological conversion of polystyrene, a major postconsumer waste, to mcl-PHA whereby polystyrene is converted to styrene by pyrolysis and the styrene monomer is fermented by bacteria that accumulate mcl-PHA intracellularly (Goff et al., 2007; O'Connor et al., 1995; Ward et al., 2006). Early experiments in stirred bioreactors with styrene as the sole carbon source yielded cell dry weight concentrations of approximately 1 g L À1 (Ward et al., 2006). ...
... Early experiments in stirred bioreactors with styrene as the sole carbon source yielded cell dry weight concentrations of approximately 1 g L À1 (Ward et al., 2006). The process was improved through the controlled feeding of nitrogen to the growth medium which resulted in a twofold increase in biomass and a 1.4-fold increase in mcl-PHA accumulation (Goff et al., 2007). To date we have reported on the growth and mcl-PHA accumulation by P. putida CA- 3 from gaseous styrene (Goff et al., 2007; Ward et al., 2006). ...
... The process was improved through the controlled feeding of nitrogen to the growth medium which resulted in a twofold increase in biomass and a 1.4-fold increase in mcl-PHA accumulation (Goff et al., 2007). To date we have reported on the growth and mcl-PHA accumulation by P. putida CA- 3 from gaseous styrene (Goff et al., 2007; Ward et al., 2006). The supply of styrene through a liquid feed should increase its rate of supply to the bioreactor, thus providing the potential for increased biomass production. ...
Article
Biofilm-related research using 96-well microtiter plates involves static incubation of plates indiscriminate of environmental conditions, making oxygen availability an important variable which has not been considered to date. By directly measuring dissolved oxygen concentration over time we report here that dissolved oxygen is rapidly consumed in Staphylococcus epidermidis biofilm cultures grown in 96-well plates irrespective of the oxygen concentration in the gaseous environment in which the plates are incubated. These data indicate that depletion of dissolved oxygen during growth of bacterial biofilm cultures in 96-well plates may significantly influence biofilm production. Furthermore higher inoculum cell concentrations are associated with more rapid consumption of dissolved oxygen and higher levels of S. epidermidis biofilm production. Our data reveal that oxygen depletion during bacterial growth in 96-well plates may significantly influence biofilm production and should be considered in the interpretation of experimental data using this biofilm model.
... These two abilities have been employed in the two-step chemo-biotechnological conversion of polystyrene, a non-biodegradable polymer, to mclPHA at laboratory scale (Ward et al., 2006). The process has been improved through the controlled feeding of N to the growth medium, which results in a twofold increase in biomass and a 1.4-fold increase in mclPHA accumulation (Goff et al., 2007). ...
... P. putida CA-3 (NCIMB 41162) had been isolated previously from a bioreactor containing styrene (O'Connor et al., 1995). Cultures were grown in a 5 l stirred tank reactor (Electrolab) with the styrene supplied continuously as a vapour, as described previously (Goff et al., 2007). The growth medium used was minimal mineral salts (MSM) containing, per litre: 9 g Na 2 HPO 4 .12H 2 O, 1.5 g KH 2 PO 4 , 0.2 g MgSO 4 .7H 2 O, 0.002 g CaCl 2 and 1 ml trace element solution (Schlegel et al., 1961). ...
... The N source was NH 4 Cl (Sigma-Aldrich). For mclPHA accumulation, N was limited with a starting concentration of 65 mg l 21 and a feeding rate of 1.5 mg l 21 h 21 (Goff et al., 2007). To prevent mclPHA accumulation, N was supplied at an initial concentration of 525 mg l 21 and maintained above 400 mg l 21 using a N feeding rate of 45 mg l 21 h 21 . ...
Article
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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).
... Recent legislation on waste diversion from landfill (2008/ 98/EC), has driven the search for technologies to recycle wastes such as plastic (Aguardo et al., 2008; Panda et al., 2010 ). We have previously developed a two-step chemobiotechnological conversion of polystyrene, a major postconsumer waste, to mcl-PHA whereby polystyrene is converted to styrene by pyrolysis and the styrene monomer is fermented by bacteria that accumulate mcl-PHA intracellularly (Goff et al., 2007; O'Connor et al., 1995; Ward et al., 2006). Early experiments in stirred bioreactors with styrene as the sole carbon source yielded cell dry weight concentrations of approximately 1 g L À1 (Ward et al., 2006). ...
... Early experiments in stirred bioreactors with styrene as the sole carbon source yielded cell dry weight concentrations of approximately 1 g L À1 (Ward et al., 2006). The process was improved through the controlled feeding of nitrogen to the growth medium which resulted in a twofold increase in biomass and a 1.4-fold increase in mcl-PHA accumulation (Goff et al., 2007). To date we have reported on the growth and mcl-PHA accumulation by P. putida CA- 3 from gaseous styrene (Goff et al., 2007; Ward et al., 2006). ...
... The process was improved through the controlled feeding of nitrogen to the growth medium which resulted in a twofold increase in biomass and a 1.4-fold increase in mcl-PHA accumulation (Goff et al., 2007). To date we have reported on the growth and mcl-PHA accumulation by P. putida CA- 3 from gaseous styrene (Goff et al., 2007; Ward et al., 2006). The supply of styrene through a liquid feed should increase its rate of supply to the bioreactor, thus providing the potential for increased biomass production. ...
Article
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.
... In this study, we attempt to feed two substrates (sodium terephthalate and glycerol) to bacteria for the production of the well-known biodegradable polymer polyhydroxyalkanoate (PHA) in an attempt to increase biomass and PHA productivity for the terephthalic acid to PHA process and integrate the conversion of two waste products to PHA. PHA has been produced from a broad range of substrates including petrochemical (Ward et al. 2006;Goff et al. 2007;Kenny et al. 2008;Nikodinovic et al. 2008), biological (Doi et al. 1992;Steinbüchel and Valentin 1995;Reddy et al. 2003) and waste based resources (Fernández et al. 2005). Terephthalic acid is a pyrolysis product of PET which is a major plastic waste NAPCOR 2011). ...
... For the subsequent 24 h, the NH 4 Cl concentration was reduced in order to limit nitrogen availability. Nitrogen limitation rather than starvation is a preferred approach as a previous study has shown it can lead to improvements in PHA accumulation (Goff et al. 2007). ...
... Several studies have previously achieved an increase in PHA and biomass levels from a variety of substrates through bioprocess manipulation (Lee et al. 1999a, b;Goff et al. 2007;Sun et al. 2007a, b;Elbahloul and Steinbuchel 2009;Ibrahim and Steinbuchel 2009). Furthermore, several studies have previously investigated the co-feeding of different substrates in order to both improve growth and PHA accumulation or to tailor PHA composition (Curley et al. 1996;Madden et al. 1998;Lee et al. 1999bLee et al. , 2008Majid et al. 1999;Sun et al. 2007aSun et al. , 2009Bengtsson et al. 2010). ...
Article
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Sodium terephthalate (TA) produced from a PET pyrolysis product and waste glycerol (WG) from biodiesel manufacture were supplied to Pseudomonas putida GO16 in a fed-batch bioreactor. Six feeding strategies were employed by altering the sequence of TA and WG feeding. P. putida GO16 reached 8.70 g/l cell dry weight (CDW) and 2.61 g/l PHA in 48 h when grown on TA alone. When TA and WG were supplied in combination, biomass productivity (g/l/h) was increased between 1.3- and 1.7-fold and PHA productivity (g/l/h) was increased 1.8- to 2.2-fold compared to TA supplied alone. The monomer composition of the PHA accumulated from TA or WG was predominantly composed of 3-hydroxydecanoic acid. PHA monomers 3-hydroxytetradeeanoic acid and 3-hydroxytetradecenoic acid were not present in PHA accumulated from TA alone but were present when WG was supplied to the fermentation. When WG was either the sole carbon source or the predominant carbon source supplied to the fermentation the molecular weight of PHA accumulated was lower compared to PHA accumulated when TA was supplied as the sole substrate. Despite similarities in data for the properties of the polymers, PHAs produced with WG present in the PHA accumulation phase were tacky while PHA produced where TA was the sole carbon substrate in the polymer accumulation phase exhibited little or no tackiness at room temperature. The co-feeding of WG to fermentations allows for increased utilisation of TA. The order of feeding of WG and TA has an effect on TA utilisation and polymer properties.
... 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, ...
... A single pyrolysis run and four fermentation runs resulted in the conversion of 64 g of PS to 6.4 g of PHA and this corresponds to a yield of 0.1 g of PHA per gram of PS. 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). ...
... The products of PET, PS, and mixed plastic pyrolysis are PHA unrelated carbon substrates resulting in (R)-3-hydroxydecanoic acid as the predominant monomer in all of the PHA-producing strains reported (Goff et al., 2007;Kenny et al., 2012;Nikodinovic-Runic et al., 2011;Ward et al., 2006). The purified polymer arising from BTEXS was partially crystalline with an average molecular weight of 86.9 kDa and had a thermal degradation temperature of 350 C with a glass transition temperature of À48.5 C . ...
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.
... While the products of pyrolysis are fuels, they can also be used as feedstock for bacteria that can make biodegradable plastic ( (Ward et al., 2006;Goff et al., 2007;Kenny et al., 2008). ...
... A critical stimulus for PHA production by many bacteria is a limitation in a critical nutrient such as nitrogen or phosphorous. Due to this knowledge many substratefeeding strategies for fed-batch fermentations incorporate an initial phase of fast biomass accumulation followed by nutrient-limiting stage for PHA accumulation (Goff et al., 2007;Kellerhals et al., 1999a;Bourque et al., 1995). However, some mcl-PHA producers do not require such a nutrient limitation and are able to accumulate significant levels of polymer (Durner et al., ...
... Thermochemical depolymerization of plastics is a rapidly developing field, but often suffers from a lack of added value since the focus lies mostly on liquid and gaseous fuel [synthesis gas (syngas)] production or the re-polymerization to the original polymer. The thermochemical depolymerization of plastic wastes to monomers or syngas, followed by microbial fermentation to generate, for example, higher value biodegradable polymers has emerged as an exciting approach for plastic upcycling (Ward et al., 2006; Goff et al., 2007; Kenny et al., 2008; Guzik et al., 2014; Drzyzga et al., 2015). However improvements in the growth and levels of biodegradable polymer accumulation by bacteria from these substrates are required. ...
... Pseudomonas is a promising host for biotechnology with a wide variety of possible products including biodegradable polymers (Ward et al., 2006; Goff et al., 2007; Kenny et al., 2008; Guzik et al., 2014; Tiso et al., 2015). Specific opportunities may be found in the increasing bioplastic and biosurfactant markets. ...
Article
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Two hundred and seventy five million tons of plastic waste were produced in 2010 alone (Jambeck et al., 2015), with Europe accounting for about 55 million tons per year. The environmental impact of these, primarily fossil-based, plastics has been broadly discussed. While the vast majority of these polymers are not biodegradable, their strength and light weight provide comparative advantages. Poly(ethylene terephthalate) (PET), for instance, has contributed significantly to reducing energy expenditure during transport, especially in the beverage industry. Due to its thermoplastic nature PET is also easy to recycle. However, recycled PET products struggle to compete with virgin PET on price and quality, leading to an overall European recycling rate of less than 30% (PlasticsEurope, 2015). Polyurethanes (PU) are used extensively in a wide range of applications including construction, transportation, furniture and medicine. Since many PU types have a thermoset nature with covalent cross links, one of the main concerns for this plastic is the notable lack of end-of-life recycling (< 5%). Finally, polyethylene (the most used plastic, ca. 140 million tons per year), is considered to be practically inert and its recycling (other than its downcycling into lumber) is economically unfavourable (Sivan, 2011), thereby creating a phenomenal environmental impact, especially in marine environments (Cozar et al., 2014; 2015). While a few countries manage major fractions of plastic waste through incineration in controlled industrial facilities, release of recalcitrant post-consumer plastic into nature remains a major problem globally. Significant amounts of plastic waste contribute to the large-scale pollution of the oceans (Katsnelson, 2015), with terms such as ‘the Great Pacific Garbage Patch’ and ‘the Trash Vortex’ mobilizing public opinion. The widespread distribution of microplastics in the food chain, with as yet unknown effects on biodiversity and human health is also appearing more in the scientific and general literature (Allsopp et al., 2006; Setala et al., 2014; Avio et al., 2015). The momentum for change away from current practices and towards a sustainable model of exploitation of waste and renewable resources is growing. In order to counteract the pollutant/recycling problems, the revised European Union (EU) Waste Framework Directive has set a minimum plastic recycling target of 50% for household waste and 70% for building and construction waste, which must be reached by all EU member states by 2020. However, without a clear technology roadmap – not to mention an attractive market strategy, the increase in recycling rates will in our opinion not be achievable. Given this background, we propose to use plastic wastes as substrates for the synthesis of added value products, which will empower the recycling industry to a qualitatively new dimension. How can microbial biotechnology contribute now to this enormous challenge? The advances in our understanding of microbial functions from enzyme, to pathways, and entire metabolic networks now allow the engineering of complex metabolic functions in microbes (Blank and Ebert, 2013). With the ever-advancing tools from synthetic biology e.g. for genome editing (Nikel et al., 2014), we can overcome the major challenges for a biotechnological plastic waste-based value chain. Some of the challenges and recent developments are highlighted below.
... ), molasses (Albuquerque et al., 2007; Solaiman et al., 2006), starch (Chen et al., 2006; Haas et al., 2008), whey (Koller et al., 2008), and industrial wastes (Nielsen, 2007, Goff et al., 2007; Yu and Heiko, 2008; Bengtsson and Werker, 2008). The detailed results are showed on Table 2. ...
... Although there are considerable industrial interests in PHAs, their cost of production is a major issue. To make PHAs production more economical, some researchers have tried to produce PHAs from inexpensive carbon sources such as plant oils (Shang et al., 2008; Bhubalan et al., 2008; Lee et al., 2008; Kek et al., 2008), molasses (Albuquerque et al., 2007; Solaiman et al., 2006), starch (Chen et al., 2006; Haas et al., 2008), whey (Koller et al., 2008), and industrial wastes (Nielsen, 2007, Goff et al., 2007 Yu and Heiko, 2008; Bengtsson and Werker, 2008). The detailed results are showed onTable 2. The highest cell density reached 179 g/L with a PHAs content of 55% when waste tomato starch was used. ...
Article
Full-text available
Polyhydroxyalkanoates (PHAs) as an attractive biopolymer with a wide range of applications have been extensively studied. Many new strategies have been employed to effectively and economically produce PHAs. This mini-review mainly focuses on the research and development of new PHAs-producing strains, utilization of renewable materials or industrial wastes, and high cell density culture technologies for PHAs production developed in recent years. The status of PHAs mass production is also introduced here.
... Previously, we reported a chemo-biotechnology processes that enable the conversion of polystyrene, PET, and PE to medium chain ☆ We dedicate this manuscript to our friend Dr Shane T. Kenny, who sadly left us too early. length polyhydroxyalkanoates (mcl-PHA) (Goff et al., 2007;Kenny et al., 2008;Guzik et al., 2014). Mcl-PHA is a partially crystalline biodegradable and biocompatible polymer with a wide range of applications, from packaging to medical use . ...
... While we have reported on the conversion of polystyrene (Goff et al., 2007), polyethene terephthalic acid (Kenny et al., 2008) and polyethene (Guzik et al., 2014) the biomass and PHA productivity in the current study is an order of magnitude higher. This conversion technology is comparable to the best reports for mcl-PHA production from virgin biological substrates, such as fatty acids (Lee et al., 2000;Maclean et al., 2008). ...
Article
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In this study, the optimisation of a process for producing medium-chain-length polyhydroxyalkanoate (mcl-PHA) by Pseudomonas putida KT2440 when fed with a polyethene (PE)-derived fatty acid mixture was investigated. The PE was pyrolysed to produce a hydrocarbon wax that was subsequently oxidised to produce a mixture of fatty acids, purified, and used as a PHA substrate for the growth and selection of microorganisms. Based on the shaken flask screening, a production strain, i.e., Pseudomonas putida KT2440, was selected for conducting bioreactor studies. Feeding PE-derived fatty acids in a 20-L setup resulted in high mcl-PHA yields (83.0 g L − 1 CDW with 65% PHA in 25 h). Furthermore, life-cycle assessment (LCA) was conducted to determine the environmental advantages of the proposed process and its impacts compared to those of other technologies for treating PE-derived waste streams. We conclude that processing waste PE into PHA, rather than incineration, produces biodegradable material while also reducing the additional emissions that arise from traditional PE waste treatment processes, such as incineration to gain energy.
... 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]. ...
... Ward et al. [29], has reported that P. putida CA-3 was capable of converting styrene, its metabolic intermediate phenylacetic acid and glucose into mcl-PHA under nitrogen limited conditions, characterized by conversion yields of 0.11, 0.17, and 0.22 g/g, respectively. 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]. ...
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.
... We have reported on the combination of pyrolysis and microbiology to convert non-degradable plastics such as PET, polyethylene (PE), and polystyrene (PS) into biodegradable counterparts, namely polyhydroxyalkanoates (PHA), offering an unconventional route to connect technical and biological parts of the circular economy [12,[43][44][45]. ...
Article
The envisaged circular economy requires absolute carbon efficiency and in the long run abstinence from fossil feedstocks, and integration of industrial production with end-of-life waste management. Non-conventional feedstocks arising from industrial production and societal consumption such as CO2 and plastic waste may soon enable manufacture of multiple products from simple bulk chemicals to pharmaceuticals using biotechnology. The change to these feedstocks could be faster than expected by many, especially if the true cost, including the carbon footprint of products, is considered. The efficiency of biotechnological processes can be improved through metabolic engineering, which can help fulfill the promises of the Paris agreement.
... In addition, it was observed that PHA supports cell survival under various stress conditions such as extreme temperatures, freezing, osmotic and oxidative pressure, UV irradiation, ethanol, desiccation or heavy metals. Furthermore, some bacteria are even capable of direct conversion of toxic substances such as methanol (Khosravi-Darani et al., 2013;Mokhtari-Hosseini et al., 2009) or styrene (Goff et al., 2007) into PHA. ...
Article
Polyhydroxyalkanoates (PHA) are polyesters accumulated by numerous prokaryotes as storage materials; they attract attention as "green" alternatives to petrochemical plastics. Recent research has demonstrated that their biological role goes beyong their storage function, since they presence in cytoplasm enhances stress resistance of microorganisms. To address these complex functions, this review summarizes the protective effects of PHA for microrganisms; the involvement of PHA in stress resistance is discussed also from a praxis-oriented perspective. The review discourses the controlled application of stress to improve PHA productivity. Also the manifold advantages of using stress adapted microbes - extremophiles as PHA producers are discussed.
... Such upcycling of plastic waste creates an opportunity to improve the efficiency of resource usage and contribute to a circular economy (European Commission 2017), likely resulting in a transformative technology with an outstanding potential to deliver social and economic benefits. The feasibility of this approach has already been demonstrated in labscale processes for the chemo-biotechnological conversion of PS (Goff et al. 2007), PET (Kenny et al. 2008(Kenny et al. , 2012, and PE (Guzik et al. 2014). In these twostep technologies, the oil obtained by plastic waste pyrolysis is used as a feedstock for microbial production of PHA. ...
Chapter
Plastics are extremely useful materials that have transformed our society in a myriad of ways. However, the widespread use of these materials has led to a staggering amount of plastic pollution in man-made and natural environments. The biodegradation of plastics is a key factor to reduce the impact of this plastic pollution. On the one hand, organisms are emerging that can degrade relatively recalcitrant plastics. On the other hand, biodegradable plastics are being developed that are intrinsically more amenable to microbial attack. In this chapter we provide an overview of the natural fates of these two types of plastics, the molecular bonds that occur in them, and the enzymatic activities associated with their degradation. Finally, an outlook is provided for the biotechnological utilization of plastics waste as a substrate, either using these enzymes or through thermochemical pretreatment.
... Furthermore, the biotechnological development contributes to replace fossil fuels with biomass (organic waste and/or energy crops) as source of energy and biomaterials, thus preventing the increase of CO 2 in the atmosphere and indirectly taking part to mitigate the global warming (Bauer et al., 2010). For instance, the organic fraction of the municipal solid waste (OFMSW) is successfully and worldwide used for producing enzymes (Clanet et al., 1988), biohythane (Escamilla-Alvarado et al., 2017) and ethanol (Ballesteros et al., 2010); agricultural biomass including corn, woods, sugar, rice and wheat straw, has found a wide use in generating bioalcohols, bio-oil, biogas and biohydrogen (Poggi-Varaldo et al., 2014;Mancini et al., 2016Mancini et al., , 2018; and even not readily biodegradable C-based wastes, such as polystyrene (Goff et al., 2007) and polyethylene terephthalate (PET) (Kenny et al., 2012), have resulted to be suitable for polyhydroxyalkanoates (PHA) production. Potential substrates for bioenergy production are cheese whey and buttermilk, by-products of cheese, yogurt, milk and butter processing in dairy factories. ...
Article
Dairy wastes can be conveniently processed and valorized in a biorefinery value chain since they are abundant, zero-cost and all year round available. For a comprehensive knowledge of the microbial species involved in producing biofuels and valuable intermediates from dairy wastes, the changes in bacterial and archaeal population were evaluated when H2, CH4and chemical intermediates were produced. Batch anaerobic tests were conducted with a mixture of mozzarella cheese whey and buttermilk as organic substrate, inoculated with 1% and 3% w/v industrial animal manure pellets. The archaeal methanogens concentration increased in the test inoculated at 3% (w/v) when H2and CH4production occurred, being 1 log higher than that achieved in the test inoculated at 1% (w/v). Many archaeal species, mostly involved in the production of CH4, were identified by sequencing denaturing gradient gel electrophoresis (DGGE) bands. Methanoculleus, Methanocorpusculum and Methanobrevibacter genera were dominant archaea involved in the anaerobic process for bioenergy production from mozzarella cheese whey and buttermilk mixture.
... Such upcycling of plastic waste creates an opportunity to improve the efficiency of resource usage and contribute to a circular economy (European Commission 2017), likely resulting in a transformative technology with an outstanding potential to deliver social and economic benefits. The feasibility of this approach has already been demonstrated in lab-scale processes for the chemobiotechnological conversion of PS (Goff et al. 2007), PET (Kenny et al. 2008(Kenny et al. ,2012, and PE (Guzik et al. 2014). In these two-step technologies, the oil obtained by plastic waste pyrolysis is used as a feedstock for microbial production of PHA. ...
Chapter
Plastics are extremely useful materials that have transformed our society in a myriad of ways. However, the widespread use of these materials has led to a staggering amount of plastic pollution in man-made and natural environments. The biodegradation of plastics is a key factor to reduce the impact of this plastic pollution. On the one hand, organisms are emerging that can degrade relatively recalcitrant plastics. On the other hand, biodegradable plastics are being developed that are intrinsically more amenable to microbial attack. In this chapter we provide an overview of the natural fates of these two types of plastics, the molecular bonds that occur in them, and the enzymatic activities associated with their degradation. Finally, an outlook is provided for the biotechnological utilization of plastics waste as a substrate, either using these enzymes, or through thermochemical pretreatment.
... Ward et al. conducted a serious of studies to convert aromatics (styrene and phenylalkanoic acids) into PHA using strain Pseudomonas putida CA-3 (Ward et al., , 2006Ward and O'Connor, 2005;Goff et al., 2007). They obtained a yield of 0.1-0.42 ...
Article
Microbial intracellular biopolymer PHA was synthesized from toxic pollutant phenol by an acclimated consortium. Various operational conditions were experimented for their effects on biomass growth and PHA accumulation. Carbon to nitrogen ratios from 5 to 40 (w/w) showed little impact, as did the levels of Fe, Ca and Mg in a short term. Acidic pH inhibited both growth and PHA synthesis, and an optimal dissolved oxygen level of 1-4 mg L-1 was identified. Low temperature (7 °C) significantly slowed but did not totally repress microbial activities. A 2% NaCl shock retarded reactions and 4% NaCl caused irreversible damage. Various initial phenol (S0) and biomass concentrations (X0) were combined to study the effect of food to microbe (F/M) ratio. High S0 and F/M exerted toxicity, reducing reaction rates but generating higher ultimate PHA wt% in biomass. Increasing X0 alleviated phenol inhibition and improved productivity and carbon conversion from phenol. A pseudo-optimized F/M ratio of 0.2-0.4 and a maximum PHA% rate of 1.15% min-1 was identified under medium S0/high X0. This study is the first to systematically investigate the feasibility of toxic industrial waste as the carbon source for PHA production, and likely the only one indicating potential for scaling-up and industrialization.
... Firstly, biodegradable plastics such as PLA can be mechanically recycled but also converted by biological processes where carbon can be returned to nature in a safe and sustainable way, e.g. by composting, which is central to a circular economy. We have also reported on the combination of pyrolysis and microbiology to convert nondegradable plastics into biodegradable plastics, offering an unconventional route for non-degradable plastics to cross over from the technical half of the circular economy to the biological half [31,[79][80][81]. If improvements to enzyme activity are made then one can envisage enzyme technologies entering the technical half (i.e. ...
Article
The strength, flexibility and light weight of traditional oil-derived plastics make them ideal materials for a large number of applications, including packaging, medical devices, building, transportation, etc. However, the majority of produced plastics are single-use plastics, which, coupled with a throw-away culture, leads to the accumulation of plastic waste and pollution, as well as the loss of a valuable resource. In this review we discuss the advances and possibilities in the biotransformation and biodegradation of oil-based plastics. We review bio-based and biodegradable polymers and highlight the importance of end-of-life management of biodegradables. Finally, we discuss the role of a circular economy in reducing plastic waste pollution.
... The productivity and yields of biomass and PHAs were relatively low, which suggested that optimization of the PHA production processes is needed. The examples of possible strategies for improving the productivity and the yields of biomass and PHAs are feeding adjustment and optimization of oxygen and initial inoculum concentrations in the system [26]. The performance of PHA production from hydrogenogenic effluent by Cupriavidus sp. ...
Article
This study investigated the use of the residual sugar and volatile fatty acids (VFAs) in the effluent of the hydrogen production process to produce polyhydroxyalkanoates (PHAs) by Cupriavidus sp. KKU38 in batch fermentation. VFAs in the effluent were lactic, butyric, acetic and propionic acids with a total VFA concentration of 1725 mg/L. The C/N ratio of effluent was 100:2.5, which is defined as the excess carbon and limited nitrogen condition suitable for PHA production. The experiments were conducted in 250-mL Erlenmeyer flasks with a 100 mL working volume. The inoculum size was 30% (v/v) with the initial number of cells 10(6) cells/mL. Residual sugars and acetic acid in the effluent were the major substrates used to produce PHAs while lactic and butyric acids in the effluent were used for biomass synthesis. The maximum PHA concentration and PHA content obtained were 0.85 g/L and 71.42% (w/w) of dry biomass weight, respectively. After fermentation, carbon oxygen demand (COD) in the effluent was reduced by up to 82.73%.
... In a run, the transformation rate from PS waste to PHA was 10%. In order to improve the conversion rate, Goff et al. performed a batch fermentation of P. putida CA-3 grown on styrene oil in a stirred tank reactor with an optimized nitrogen feeding strategy (Goff et al., 2007). ...
Article
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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.
... [134] This result brings the simultaneous 450 PS biodegradation and PHA production via P. putida CA-3 and paved the way for others to valorize 451 PS waste into valuable chemicals. [133,135] 452 ...
Article
Although fossil‐based plastic products have many attractive characteristics, their production has led to severe environmental burdens that require immediate solutions. Despite these plastics being non‐natural chemical compounds, they can be degraded and metabolized by some microorganisms, which suggests the potential application of biotechnologies based on the mechanism of plastic biodegradation. In this context, microbe‐based strategies for the degradation, recycling, and valorization of plastic waste offer a feasible approach for alleviating environmental challenges created by the accumulation of plastic waste. This review highlights recent advances in the biotechnology‐based biodegradation of both traditional polymers and bio‐based plastics, focusing on the mechanisms of biodegradation. From an application perspective, this review also summarizes recent progress in the recycling and valorization of plastic waste, which are feasible solutions for tackling the plastic waste dilemma.
... 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
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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.
... USMAA2-4 Kek et al. (2010) Fatty acids and waste glycerol Ralstonia eutropha Pseudomonas aeruginosa NCIB 40045 Cupriavidus necator Kahar et al. (2004) Fernández et al. (2005) Cavalheiro et al. (2009) Paper mill wastewater Activated sludge Bengtsson and Werker (2008) Swine waste liquor Azotobacter vinelandii UWD. Cho et al. (1997) Petrochemical plastic waste Pseudomonas putida CA-3 Goff et al. (2007) Sunflower cake, soy bran and olive mill Pseudomonas hydrogenovora Koller et al. (2005) Soy and malt wastes Mixed bacteria Wang et al. (2007) Waste activated sludge Bacillus sp. Thirumala et al. (2010) Dairy waste and sea water Bacillus megaterium Ram Kumar Pandian et al. (2010) Waste water from olive oil mills (called alpechín) Pseudomonas putida KT2442 Azotobacter chroococcum H23 Ribera et al. (2001) Pozo et al. (2002) Acid-hydrolysed malt waste Recombinant Bacillus subtilis strains Law et al. (2003) dNTPack kit (Roche). ...
Article
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A strain of poly--hydroxybutyrate (PHB)-accumulating bacterium was isolated and identified as Enterobacter aerogenes (designated E. aerogenes 12Bi) by using biochemical and phylogenetic characterization. The accumulation of a large amount of granules in its cells cultured in the domestic wastewater medium (DWWM) were showed by transmission electron microscopy (TEM). When PHB production by our strain was determined by Hypochlorite method, it was found that PHB production ranged from 16.66 to 96.25% (w/w). The highest PHB yield by our microorganism was up to 96.25% within 18 h in DWWM 5 (supplemented with 100% DWW). This is the first report of the use of DWW for production of PHB by E. aerogenes. The results obtained in the study demonstrated that PHB could be efficiently produced to a high concentration with high productivity by using DWWM as an inexpensive substrate. Thus, it can contribute to the reduction of high production cost of PHB.
... The properties discovered in novel PHAs, such as polythioesters (high thermal stability and putative antibacterial activity) could broaden the medical applications of these compounds (Lütke-Eversloh and . Furthermore, taking into account that PHAs can be synthesized from many different carbon sources (crop plants, bagasse hydrolysates, paper mill waste water, whey, municipal waste water, sugar cane molasses, animal fats and vegetable oils, etc.; Coats et al. 2007 ;Bengtsson et al. 2008 ;Koller et al. 2008 ;Mengmeng et al. 2008 ;van Beilen and Poirier 2008 ;Yu and Stahl 2008) , PHA-producer organisms could play important ecological roles since they can be used to eliminate contaminants (Sudesh et al. 2007) or to recycle different industrial residues, even those that are toxic or dangerous for many species (Goff et al. 2007 ;Nikodinovic et al. 2008) . ...
Chapter
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Unusual polyhydroxyalkanoates (UnPHAs) constitute a particular group of polyoxo(thio)esters belonging to the PHA family, which are tailored with uncommon monomers. Thus, unusual PHAs include (1) polyhydroxyalkanoates (PHAs) of microbial origin that have been synthesized either from natural monomers bearing different chemical functions, or from chemical derivatives of the natural ones and (2) PHAs obtained either by chemical synthesis or by physical modifications of naturally occurring polymers. Regarding their chemical structure, UnPHAs can be grouped in four different classes. Class 1 includes PHAs whose lateral chains contain double or triple bounds or/and different functional groups (methyl, methoxy, ethoxy, acetoxy, hydroxyl, epoxy, carbonyl, cyano, phenyl, nitrophenyl, phenoxy, cyanophenoxy, benzoyl, halogen atoms, etc.). Classes 2 and 3 have been established regarding the nature of the PHA backbone; whereas class 2 includes PHAs in which the length of the monomer participating in the oxoester linkage has been modified (the hydroxyl group to be esterified is not located at C-3), class 3 groups those polymers in which some oxoester linkages have been replaced by thioester functions (thioester-containing PHAs). Finally, class 4 includes those PHAs that have been manipulated chemically or physically. In this chapter we shall describe the chemical structure of unusual PHAs belonging to these four classes; we shall analyse their biosynthetic particularities (if any), and we shall discuss some of their characteristics and biotechnological applications.
... Exceptionally high-performance fiber-reinforced plastic derived from the combination of deconstructed PET (reclaimed PET) with renewable and available monomers can provide the option of upcycling PETs, which are the largest produced polyesters (Rorrer et al., 2019). Similarly, the pyrolysis products of PS, PET, and PE can act as selective feedstocks for the bacterial production of biodegradable polyhydroxyalkanoate (PHA) (Goff et al., 2007;Guzik et al., 2014;Kenny et al., 2012). For instance, the conversion of PS to PHA using styrene oil, an intermediate pyrolysis product, was achieved by Pseudomonas putida CA-3 (Ward et al., 2006). ...
Article
The accumulation of microplastics (MPs) and nanoplastics (NPs) in terrestrial and aquatic ecosystems has raised concerns because of their adverse effects on ecosystem functions and human health. Plastic waste management has become a universal problem in recent years. Hence, sustainable plastic waste management techniques are vital for achieving the United Nations Sustainable Development Goals. Although many reviews have focused on the occurrence and impact of micro- and nanoplastics (MNPs), there has been limited focus on the management of MNPs. This review summarizes the ecotoxicological impacts of plastic waste sources and issues related to the sustainable management of MNPs in the environment. Moreover, this review critically evaluates possible approaches for incorporating plastics into the circular economy in order to cope with the problem of plastics. Pollution associated with MNPs can be tackled through source reduction, incorporation of plastics into the circular economy, and suitable waste management. Appropriate infrastructure development, waste valorization, and economically sound plastic waste management techniques and viable alternatives are essential for reducing MNPs in the environment. Policymakers must pay more attention to this critical issue and implement appropriate environmental regulations to achieve environmental sustainability.
... 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
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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.
... In the second proposed pathway, styrene is hydroxylated by styrene dioxygenase (SDO) on the aromatic ring to generate styrene cis-glycol, which can be further converted to acetyl-CoA by cis-glycol dehydrogenase (CGDH), catechol 2,3-dioxygenase (CDO), 2-hydroxymuconic acid semialdehyde (Sielicki et al., 1978) 17 species of fungi in axenic cultures Sludges, soils, manures, garbages, and decaying plastics 14 C labelled PS, 11 weeks 0.04 to 0.57% total decomposition based on 14 CO 2 production (Kaplan et al., 1979) Micromonospora species Yb-1 Soil 3 lignin-related polystyrene, NA a Changes in culture pH, UV and IR spectra, and gel permeation chromatograms confirmed the metab. of the polymers (Haraguchi et al., 1980) Aspergillus niger NCIM 1025 (ATCC 9642), Trichoderma sp. NCIM 1297 (ATCC 9645) and Pullularia pullulans NCIM 1049 (ATCC 9348) NCIM b carbohydrate-linked polystyrenes, 10 weeks Weight loss and FTIR spectra changes (Galgali et al., 2004) Pseudomonas putida CA-3 NCIMB Virgin polystyrene By combination with pyrolysis, PS was converted to PHA (Goff et al., 2007;Ward et al., 2006) (Peng et al., 2020) Land snails Achatina fulica EPS foam, 4 weeks (continued on next page) hydrolase (HMASALDH), 2-hydroxypenta-2,4-dienoate hydratase (HPDEH), 4-hydroxy-2-oxovalerate aldolase (HOA) and pyruvate dehydrogenase complex (PDHC). Similarly, acetyl-CoA will then enter the TCA cycle and participate in biomass synthesis or accumulation of other metabolites (like PHA) (Ali et al., 2021;O'Leary et al., 2005;Oelschlagel et al., 2018;Ru et al., 2020). ...
Article
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The global production of plastics has continuously been soaring over the last decades due to their extensive use in our daily life and in industries. Although synthetic plastics offer great advantages from packaging to construction and electronics, their low biodegradability induce serious plastic pollution that damage the environment, human health and make irreversible changes in the ecological cycle. In particular, plastics containing only carbon-carbon (C-C) backbone are less susceptible to degradation due to the lack of hydrolysable groups. The representative polyethylene (PE) and polystyrene (PS) account for about 40% of the total plastic production. Various chemical and biological processes with great potential have been developed for plastic recycle and reuse, but biodegradation seems to be the most attractive and eco-friendly method to combat this growing environmental problem. In this review, we first summarize the current advances in PE and PS biodegradation, including isolation of microbes and potential degrading enzymes from different sources. Next, the state-of-the-art techniques used for evaluating and monitoring PE and PS degradation, the scientific toolboxes for enzyme discovery as well as the challenges and strategies for plastic biodegradation are intensively discussed. In return, it inspires a further technological exploration in expanding the diversity of species and enzymes, disclosing the essential pathways and developing new approaches to utilize plastic waste as feedstock for recycling and upcycling.
Chapter
Major air pollutants that can be degraded biologically include both volatile organic as wellas inorganic compounds. Bioprocesses are not suitable for the removal of particle matter. The biodegradation routes depend on the nature of the pollutants. End-products are generally water and carbon dioxide under aerobic conditions, but other products may appear as well. Acids such as hydrogen chloride and sulphuric acid will be formed from the aerobic biodegradation of, respectively, halogenated and most sulphur-compounds. If oxygen is limiting, elemental sulphur may accumulate during the biodegradation of sulphur-pollutants. Anaerobic biodegradation of waste gases will yield different metabolites. Recent research trends have also been dealing with the biotransformation, rather than biodegradation, of volatile pollutants. This offers the possibility to convert volatile compounds to useful, value-added metabolites such as (bio)fuels to cite only one example.
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The production of polyhydroxyalkanoates (environmentally-friendly biopolymers) from a bacterial species isolated from poultry wastes was investigated. Bacterial isolation was carried out using synthetic medium, and the isolate was screened for its PHA-producing ability using Sudan black B dye. Both conventional and API kit were used to identify the bacterium. The ability of the bacterium to utilize agro-allied wastes materials (such as sugarcane bagasse, corn cob and wheat bran) as carbon substrates for PHA production were compared with standard substrates (such as fructose and sodium acetate).The PHA was extracted from the cell with chloroform using standard method and analyzed with Fourier transform infra red (FT-IR) spectroscopy. The results revealed that the bacterium was Gram negative, oxidase-positive, tiny rods, identified as Pseudomonas putrefaciens. The highest polymer yield of 66.67% w/w of cell dry weight was obtained when corn cob was used as carbon substrate, while 3.49%w/w of cell dry weight was obtained when sodium acetate was used. Maximum yield was obtained at 72 hours of growth. The functional groups of hydroxyl and the carbonyl were identified in the dried crystal extracts of the polymer from the FTIR spectra. PHA production from cheap and renewable substrates reduces cost of production, since fermenting substrate contributes significantly to the overall production cost.
Chapter
The increased use of the “omic” techniques, e.g., genomics, proteomics, metabolomics, and fluxomics, as well as the systems biology approaches for addressing biological complexity from a holistic perspective, has contributed significantly to accelerate and complete our understanding on different aspects of the physiology, ecology, biochemistry, and regulatory mechanisms underlying the catabolism of aromatic compounds in bacteria of the Pseudomonas genus. Toxic aromatic compounds simultaneously serve as potential nutrients to be metabolized by bacteria but also as cellular stressors. When bacteria are exposed to these compounds they exhibit a multifactorial response that comprises three major intimately connected programs: (i) metabolic programs that involve not only the compound-specific pathways but also their integration within the global metabolism of the host cell; (ii) stress-response programs, e.g., changes in lipid metabolism, efflux pumps, or molecular chaperones, for adaptation to sub-optimal growth conditions; and (iii) a social program, including cell motility and chemotaxis, reorganization of the cell envelope, biofilm formation, and cell-to-cell interactions. As individual cells rarely metabolize a wide range of substrates, metabolic specialization within the bacterial population becomes a relevant trait in the assembly of efficient microbial biodegrader communities. Genome-scale metabolic models of several Pseudomonas strains have been performed. These models, when combined with the emergent synthetic biology approaches, can be used to explore the potential of Pseudomonas as cell factories in different biotechnological applications. Therefore, Pseudomonas becomes a paradigmatic bacterial genus both for increasing basic knowledge on the catabolism of aromatic compounds and for the bioremediation and/or biosensing of toxic pollutants and the valorization of aromatic compounds present in biowaste toward a sustainable knowledge-based bioeconomy with social and environmental rewards.
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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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The production of polyethylene terephthalate (PET) has drastically increased in the past half-century, reaching 30 million tons every year. The accumulation of this recalcitrant waste now threatens diverse ecosystems. Despite efforts to recycle PET wastes, its rate of recycling remains limited, as the current PET downcycling is mostly unremunerative. To address this problem, PET bio-upcycling, which integrates microbial depolymerization of PET followed by repolymerization of PET-derived monomers into value-added products, has been suggested. This article critically reviews current understanding of microbial PET hydrolysis, the metabolic mechanisms involved in PET degradation, PET hydrolases, and their genetic improvement. Furthermore, this review includes the use of meta-omics approaches to search PET-degrading microbiomes, microbes, and putative hydrolases. The current development of biosynthetic technologies to convert PET-derived materials into value-added products is also comprehensively discussed. The integration of various depolymerization and repolymerization biotechnologies enhances the prospects of a circular economy using waste PET.
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It is well known that high-nitrogen content inhibits cell growth and docosahexaenoic acid (DHA) biosynthesis in heterotrophic microalgae Crypthecodinium cohnii. In this study, two nitrogen feeding strategies, pulse-feeding and continuous-feeding, were evaluated to alleviate high-nitrogen inhibition effects on C. cohnii. The results showed that continuous-feeding with a medium solution containing 50% (w/v) yeast extract at 2.1 mL/h during 12-96 h was the optimal nitrogen feeding strategy for the fermentation process, when glucose concentration was maintained at 15-27 g/L during the same period. With the optimized strategy, 71.2 g/L of dry cell weight and DHA productivity of 57.1 mg/L/h were achieved. In addition, metabolomic analysis was applied to determine the metabolic changes during different nitrogen feeding conditions, and the changes in amino acids, polysaccharides, purines and pentose phosphate pathway were observed, providing valuable metabolite-level information for exploring mechanism of high-nitrogen inhibition effect, and further improving DHA productivity in C. cohnii.
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Cupriavidus necator, a versatile microorganism found in both soil and water, can have both heterotrophic and lithoautotrophic metabolisms depending on environmental conditions. C. necator has been extensively examined for producing Polyhydroxyalkanoates (PHAs), the promising polyester alternatives to petroleum-based synthetic polymers because it has a superior ability for accumulating a considerable amount of PHAs from renewable resources. The development of metabolically engineered C. necator strains has led to their application for synthesizing biopolymers, biofuels and biochemicals such as ethanol, isobutanol and higher alcohols. Bio-based processes of recombinant C. necator have made much progress in production of these high-value products from biomass wastes, plastic wastes and even waste gases. In this review, we discuss the potential of C. necator as promising platform host strains that provide a great opportunity for developing a waste-based circular bioeconomy.
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There is an urgent need for new technologies to enable circularity for synthetic polymers, spurred by the accumulation of waste plastics in landfills and the environment and the contributions of plastics manufacturing to climate change. Chemical recycling is a promising means to convert waste plastics into molecular intermediates that can be remanufactured into new products. Given the growing interest in the development of new chemical recycling approaches, it is critical to evaluate the economics, energy use, greenhouse gas emissions, and other life cycle inventory metrics for emerging processes, relative to the incumbent, linear manufacturing practices employed today. Here we offer specific definitions for classes of chemical recycling and upcycling and describe general process concepts for the chemical recycling of mixed plastics waste. We present a framework for techno-economic analysis and life cycle assessment for both closed- and open-loop chemical recycling. Rigorous application of these process analysis tools will be required to enable impactful solutions for the plastics waste problem. Expected final online publication date for the Annual Review of Chemical and Biomolecular Engineering, Volume 13 is October 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
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PHAs are biodegradable and biocompatible polymers produced by a wide range of bacteria. In the present study, the wild strain of Bacillus cereus producing polyhydroxybutyrate-co-valrate (PHBV) copolymer with high productivity, isolated from starch effluent was used. To produce, starch effluent was studied as a cheap medium. Then, the optimal composition of the culture medium was evaluated and in order to achieve higher productivity, the high cell density method was evaluated. The results showed the maximum production of PHBV using Plackett-Burman test design was about 59.5% of CDW. Subsequently, by optimizing culture with high cell density, the production rate increased to more than 72% of CDW. Therefore, the high cell density has a significant effect on increasing the productivity of PHBV production by B. cereus. Due to the use of cheap culture medium, the importance of these results is twofold and shows the potential for production on an industrial scale.
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Background and Objective: Polyhydroxyalkanoates (PHAs) are polymers with biodegradable and biocompatible properties that are produced by some bacteria. In the present study, petroleum sediments were applied to screen PHA-producing bacteria. Method: The industrial culture medium of petroleum effluent was used as a low-cost and economical medium for isolating and identifying the superior PHA-producing strain. Finally, the chemical and physical properties of the extracted biopolymer were investigated by Fouriertransform infrared spectroscopy and proton nuclear magnetic resonance. Results: In general, 11 out of 76 isolated bacterial strains could produce biopolymers among which, the Sb8 strain was selected as the best PHAproducing strain in the industrial medium with the cell dry weight of 44.13% and 1.2 g/l in 27 h. This strain was identified as Citreicella thiooxidans by sequencing determination. Eventually, the results of physicochemical analyses revealed that polyhydroxybutyrate (PHB) was the extracted biopolymer. Conclusion: The present study is the first report on PHB production by Iranian native Citreicella thiooxidans strain by focusing on identifying and separating producing bacteria, as well as determining the type of the produced biopolymer and the production capability in a low-cost culture medium of the petroleum effluent. Considering the production of the biopolymer with a relatively high yield percentage without adding any supplement to the petroleum effluent medium, the isolated wild strain has the potential to produce PHB.
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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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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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The current period is facing the hazardous effects of synthetic plastics or polymers, reported as non-degradable with the big quantity of its accumulation in our healthy environments. Synthetic plastics are used for packaging of food items, medicine, water, cloths pharmaceutical, and also carrying vegetables or fruits or others application. To minimize or control this issue or problem, we need to utilize the biodegradable plastics or bio-plastic for various applications in our modern life. For the last two decades, microbial-derived polyhydroxyalkanoates (PHAs) gained more attention for various industrial applications. An effective microbial system (i.e., bacterial strain) is required for the maximum quantity of PHAs production at optimized cultural conditions via D-optimal statistical design. PHA-producing bacterial strain are isolated from various soil and spring samples and characterized using morphological, biochemical, and 16S rDNA sequencing method. Crude glycerin concentration and carbon to nitrogen ratio (C:N) in the culture medium for PHB growth were optimized by Response Surface Methodology (RSM). Accumulation of PHAs is found in mixed microb ial culture (MMC), and active-biomass yield coefficient (Y), observed PHA yield coefficient (Y), biomass PHAs content (X), and volumetric productivity (Pr) are four indicators influenced by culture media. Polyhydroxybutyrate (PHB) is reported in a combined form with good plastic nature with the natural nature of terpene and D-limonene organic compound. It has exhibited the dual objective of increasing PLA crystalline nature and also obtained flexible films for food packaging application tasks. The functional properties of PHB are examined through colorimetric variables, oxygen permeability, water-resistance nature evaluation, thermal stability, crystalline response, mechanical, and nano-material characteristics. FTIR spectra have revealed for their characteristics bands equivalent in PLA and PHB, provided the information for their relative molecular interaction. PyGC-MS demonstrated the D-limonene characteristic peaks along with PLA and PHB thermal degradation product profiles. In this chapter, we emphasize recent development in the various types of PHB synthesis processes along with their characterization and applications.
Chapter
Most bacteria belonging to the genus Pseudomonas can accumulate medium-chain poly-3-hydroxy-alkanoates (mcl-PHA) as storage materials. For this reason, these bacteria have a well-preserved enzymatic mechanism which involves the participation of a polymerization system (consisting of two different polymerases, PhaC1 and PhaC2), two proteins needed for the structural organization of the mcl-PHA granules (the phasins PhaI and PhaF); a regulatory protein (PhaD), and finally, a polymer-hydrolyzing enzyme (depolymerase, PhaZ) which releases the monomeric units from the granule. In the first stage, PhaC1 and PhaC2 synthesize the polymer using (R)-3-hydroxyacyl-CoA precursors obtained throughout the β-oxidation of longer fatty acids or from acetyl-CoA following the de novo fatty acid biosynthetic pathway. All the polymerases described in Pseudomonas have a wide substrate specificity, which allows for the collection of a huge number of PHAs (homopolymers and copolymers), having varied physicochemical characteristics and interesting biotechnological applications. Therefore, when considering the production of PHAs, there are some highly relevant aspects that must be taken into account. Thus, the monomer precursor selection (raw material sources), the precise characterization of their biosynthetic pathways, its metabolic networking, and the manipulations needed to ensure an efficient flow of intermediaries are essential aspects to achieve both a good bacterial growth and a high production of the desired polymer(s). This chapter focuses on describing the different mechanisms by which pseudomonads can generate useful PHA monomers as well as the strategies followed to improve the efficiency of these processes. Additionally, different approaches to allow for industrial PHA production will be discussed.
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The use of fossil-based plastics has become unsustainable because of the polluting production processes, difficulties for waste management sectors, and high environmental impact. Polyhy-droxyalkanoates (PHA) are bio-based biodegradable polymers derived from renewable resources and synthesized by bacteria as intracellular energy and carbon storage materials under nutrients or oxygen limitation and through the optimization of cultivation conditions with both pure and mixed culture systems. The PHA properties are affected by the same principles of oil-derived polyolefins, with a broad range of compositions, due to the incorporation of different monomers into the polymer matrix. As a consequence, the properties of such materials are represented by a broad range depend-ing on tunable PHA composition. Producing waste-derived PHA is technically feasible with mixed microbial cultures (MMC), since no sterilization is required; this technology may represent a solution for waste treatment and valorization, and it has recently been developed at the pilot scale level with different process configurations where aerobic microorganisms are usually subjected to a dynamic feeding regime for their selection and to a high organic load for the intracellular accumulation of PHA. In this review, we report on studies on terrestrial and marine bacteria PHA-producers. The available knowledge on PHA production from the use of different kinds of organic wastes, and otherwise, petroleum-polluted natural matrices coupling bioremediation treatment has been explored. The advancements in these areas have been significant; they generally concern the terrestrial environment, where pilot and industrial processes are already established. Recently, marine bacteria have also offered interesting perspectives due to their advantageous effects on production practices, which they can relieve several constraints. Studies on the use of hydrocarbons as carbon sources offer evidence for the feasibility of the bioconversion of fossil-derived plastics into bioplastics.
Chapter
For the last few decades, PHBs have been used to address issues around environmental pollution. This is due to their biodegradable nature when compared with conventional plastics. Moreover, their 100% biodegradability combined with biocompatibility has secured a place for PHBs in many industrial applications such as biomedical, packaging, tissue engineering and drug delivery. However, properties such as brittleness, thermal instability and narrow processing window limit the application of PHBs in many industrial applications. Therefore, in order to overcome this setback, PHBs are usually blended with other polymers, fibres or additives to obtain PHB-based composites with optimum properties. PHBs are a group of PHAs that are synthesized and stored by many microorganisms as a source of carbon and energy. With the growing environmental concerns, there is an increasing demand for the production of PHB-based materials on a larger scale. However, the major problem facing by large-scale production of PHB right now is the high cost of production. In spite of the many attempts to reduce the cost of production, there is still a need for more ways of significantly reducing the production cost. Some of the ways are briefly mentioned in this chapter. This chapter is aimed at providing a detailed discussion on the production and applications of microbial PHBs.
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Chemical recycling of solid plastic waste (SPW) is a paramount opportunity to reduce marine and land pollution and to enable the incorporation of the circular economy principle in today's society. In addition to more conscious behaviors and wiser product design (“design for recycling”), a key challenge is the identification of the leading recycling technologies, minimizing the global warming potential in an industrially relevant context. Chemical recycling technologies based on pyrolysis and gasification are leading the way because of their robustness and good economics, but an improved understanding of the chemistry and more innovative reactor designs are required to realize a potential reduction of greenhouse gas emissions of more than 100 million tonnes of CO2-eq., primarily by more efficient use of valuable natural resources. The feed flexibility of thermal processes supports the potential of pyrolysis and gasification, however, the strong variability in time and space of blending partners such as multiple and co-polymers, additives, and contaminants (such as inorganic materials) calls for accurate assessment through fundamental experiments and models. Such complex and variable mixtures are strongly sensitive to the process design and conditions: temperature, residence time, heating rates – severity, mixing level, heat and mass transfer strongly affect the thermal degradation of SPW and its selectivity to valuable products. A prerequisite in improving design and performance is the ability to model conversion profiles and product distributions based on accurate rate coefficients for the dominating reaction families established using first-principle derived transport and thermodynamic properties. These models should also help with the “design for recycling” strategy to increase recyclability, for example by identifying additives that make chemical recycling difficult. Fundamental experiments of increased quality (accuracy, integrity, validity, replicability, completeness) together with improved deterministic kinetic models, systematically developed according to the reaction classes and rate rules approach, provide insights to identify optimal process conditions. This will allow shedding some light upon the important pathways involved in the thermal degradation of the feedstock and the formation/disappearance of desired or unwanted products. In parallel, the intrinsic kinetics of the dominating elementary reaction steps should be determined with higher accuracy, moving beyond single step kinetics retrieved from thermogravimetric analysis experiments. Together with more accurate kinetic parameters, better models to account for heat and mass transfer limitations also need to be further developed, since plastic degradation involves at least three phases (solid, liquid, gas), whose interactions should be accounted for in a more rigorous way. Novel experimental approaches (e.g. detailed feedstock and product characterization using comprehensive chromatographic techniques and photoionization mass spectrometry) and available computational tools (e.g. kinetic Monte Carlo, liquid phase, and heterogeneous theoretical kinetics) are needed to tackle these problems and improve our fundamental understanding of chemical recycling of SPW.
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Background: Garbage or waste material are the substances discarded by human beings due to a perceived lack of utility or usefulness. An increase in the level of these waste materials may lead to increased levels of pollution, global warming or even cause major health issues as they may contain toxic, non-biodegradable substances as well. Thus, there arises a need for proper methods of disposal of waste. Objective: The objective of this review is to study the possibilities for degradation of non-biodegradable garbage, basically plastics and similar materials with the help of microorganisms and to find out ways to suitably manage these materials in view of their relation to global warming and other such issues. Methods: Extensive literature survey was carried out through various databases like Google Scholar, PubMed etc. and the information were collected and analyzed, and accordingly possibilities for waste management were studied with special reference to non-biodegradable waste like plastics. Results and Discussion: From the literature survey that was carried out, it has been found that researchers are trying to find out alternative packaging material and to search for microorganisms which can degrade the plastic and other such type of materials. Thus we can say that alternative packaging material could be helpful in the management of garbage. At the same time, search for microorganisms which can degrade plastic materials can also be carried out simultaneously. Conclusion: It is therefore better to degrade the existing polymeric materials with the help of suitable microorganisms. Also, the future packaging materials should be made up of biodegradable material.
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The increasing impact of plastic materials on the environment is a growing global concern. In regards to this circumstance, it is a major challenge to find new sources for the production of bioplastics. Poly-β-hydroxybutyrate (PHB) is characterized by interesting features that draw attention for research and commercial ventures. Indeed, PHB is eco-friendly, biodegradable, and biocompatible. Bacterial fermentation processes are a known route to produce PHB. However, the production of PHB through the chemoheterotrophic bacterial system is very expensive due to the high costs of the carbon source for the growth of the organism. On the contrary, the production of PHB through the photoautotrophic cyanobacterium system is considered an attractive alternative for a low-cost PHB production because of the inexpensive feedstock (CO2 and light). This paper regards the evaluation of four independent strategies to improve the PHB production by cyanobacteria: (i) the design of the medium; (ii) the genetic engineering to improve the PHB accumulation; (iii) the development of robust models as a tool to identify the bottleneck(s) of the PHB production to maximize the production; and (iv) the continuous operation mode in a photobioreactor for PHB production. The synergic effect of these strategies could address the design of the optimal PHB production process by cyanobacteria. A further limitation for the commercial production of PHB via the biotechnological route are the high costs related to the recovery of PHB granules. Therefore, a further challenge is to select a low-cost and environmentally friendly process to recover PHB from cyanobacteria.
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Advances in scientific technology in the early 20th century have facilitated the development of synthetic plastics that are lightweight, rigid, and can be easily molded to a desirable shape without changing their material properties. Thus, plastics become ubiquitous and indispensable materials that are used in various manufacturing sectors, including clothing, automotive, medical, and electronic industries. However, strong physical durability and chemical stability of synthetic plastics, most of which are produced from fossil fuels, hinders their complete degradation when they are improperly discarded after use. In addition, accumulated plastic wastes without degradation have caused severe environmental problems, such as microplastics pollution and plastic islands. Thus, the usage and production of plastics is not free from environmental pollution or resource depletion. In order to lessen the impact of climate change and reduce plastic pollution, it is necessary to understand and address the current plastic life cycles. In this review, we suggest “sustainable biopolymer” as a promising solution to current plastic crisis. Desired properties of sustainable biopolymers and bio‐based and bio/chemical hybrid technologies for the development of sustainable biopolymers are mainly discussed. This article is protected by copyright. All rights reserved
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A library of 20 000 transposon (Tn5) mutants of the gram-negative bacterium Pseudomonas putida CA-3 was generated and screened for adverse affects in polyhydroxyalkanoates (PHA) accumulation. Two mutants of interest were characterized phenotypically. CA-3-126, a mutant disrupted in a stress-related protein Clp protease subunit ClpA, demonstrated greater decreases in PHA accumulation compared with the wild type at reduced and elevated temperatures under PHA-accumulating growth conditions. CA-3-M, which is affected in the aminotransferase class I enzyme, accumulated reduced levels of PHA relative to the wild type and had lower growth yields on all carbon sources tested. Mutant CA-3-M produced up to 10-fold higher levels of lipopolysaccharide relative to the wild type and exhibited 1.2-fold lower aminotransferase activity with phenylalanine as a substrate compared with the wild-type strain. The composition of the lipopolysaccharide produced by the mutant differed from that produced by the wild-type strain. Growth and PHA accumulation by CA-3-M was the same as the wild type when the nitrogen concentration in the medium was increased to 265 mg N L(-1).
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Poly(3-hydroxyalkanoates) (PHAs) are a class of microbially produced polyesters that have potential applications as conventional plastics, specifically thermoplastic elastomers. A wealth of biological diversity in PHA formation exists, with at least 100 different PHA constituents and at least five different dedicated PHA biosynthetic pathways. This diversity, in combination with classical microbial physiology and modern molecular biology, has now opened up this area for genetic and metabolic engineering to develop optimal PHA-producing organisms. Commercial processes for PHA production were initially developed by W. R. Grace in the 1960s and later developed by Imperial Chemical Industries, Ltd., in the United Kingdom in the 1970s and 1980s. Since the early 1990s, Metabolix Inc. and Monsanto have been the driving forces behind the commercial exploitation of PHA polymers in the United States. The gram-negative bacterium Ralstonia eutropha, formerly known as Alcaligenes eutrophus, has generally been used as the production organism of choice, and intracellular accumulation of PHA of over 90% of the cell dry weight have been reported. The advent of molecular biological techniques and a developing environmental awareness initiated a renewed scientific interest in PHAs, and the biosynthetic machinery for PHA metabolism has been studied in great detail over the last two decades. Because the structure and monomeric composition of PHAs determine the applications for each type of polymer, a variety of polymers have been synthesized by cofeeding of various substrates or by metabolic engineering of the production organism. Classical microbiology and modern molecular bacterial physiology have been brought together to decipher the intricacies of PHA metabolism both for production purposes and for the unraveling of the natural role of PHAs. This review provides an overview of the different PHA biosynthetic systems and their genetic background, followed by a detailed summation of how this natural diversity is being used to develop commercially attractive, recombinant processes for the large-scale production of PHAs.
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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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Pseudomonas putida CA-3 is capable of accumulating medium-chain-length polyhydroxyalkanoates (MCL-PHAs) when growing on the toxic pollutant styrene as the sole source of carbon and energy. In this study, we report on the molecular characterization of the metabolic pathways involved in this novel bioconversion. With a mini-Tn5 random mutagenesis approach, acetyl-coenzyme A (CoA) was identified as the end product of styrene metabolism in P. putida CA-3. Amplified flanking-region PCR was used to clone functionally expressed phenylacetyl-CoA catabolon genes upstream from the sty operon in P. putida CA-3, previously reported to generate acetyl-CoA moieties from the styrene catabolic intermediate, phenylacetyl-CoA. However, the essential involvement of a (non-phenylacetyl-CoA) catabolon-encoded 3-hydroxyacyl-CoA dehydrogenase is also reported. The link between de novo fatty acid synthesis and PHA monomer accumulation was investigated, and a functionally expressed 3-hydroxyacyl-acyl carrier protein-CoA transacylase (phaG) gene in P. putida CA-3 was identified. The deduced PhaG amino acid sequence shared >99% identity with a transacylase from P. putida KT2440, involved in 3-hydroxyacyl-CoA MCL-PHA monomer sequestration from de novo fatty acid synthesis under inorganic nutrient-limited conditions. Similarly, with P. putida CA-3, maximal phaG expression was observed only under nitrogen limitation, with concomitant PHA accumulation. Thus, β-oxidation and fatty acid de novo synthesis appear to converge in the generation of MCL-PHA monomers from styrene in P. putida CA-3. Cloning and functional characterization of the pha locus, responsible for PHA polymerization/depolymerization is also reported and the significance and future prospects of this novel bioconversion are discussed.
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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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A kinetic study of the production of poly--hydroxybutyric acid (PHB) by a fed-batch culture of Protomonas extorquens showed that a nitrogen source was necessary even in the PHB production phase. The effect of ammonia feeding on PHB production was consequently investigated. The nitrogen source (ammonia water) was supplied at a low constant feeding rate after the growth phase in which cell mass concentration reached 60 g/l. Feeding with a small quantity of ammonia resulted in a more rapid increase in intracellular PHB content than was the case without ammonia feeding. Excessive feeding of ammonia, however, caused not only degradation of accumulated PHB but also reduction of microbial PHB synthetic activity.
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 Pseudomonas sp. 61-3 (isolated from soil) produced a polyester consisting of 3-hydroxybutyric acid (3HB) and of medium-chain-length 3-hydroxyalkanoic acids (3HA) of C6, C8, C10 and C12, when sugars of glucose, fructose and mannose were fed as the sole carbon source. The polyester produced was a blend of homopolymer and copolymer, which could be fractionated with boiling acetone. The acetone-insoluble fraction of the polyester was a homopolymer of 3-hydroxybutyrate units [poly (3HB)], while the acetone-soluble fraction was a copolymer [poly(3HB-co-3HA)] containing both short- and medium-chain-length 3-hydroxyalkanoate units ranging from C4 to C12:44 mol% 3-hydroxybutyrate, 5 mol% 3-hydroxyhexanoate, 21 mol% 3-hydroxyoctanoate, 25 mol% 3-hydroxydecanoate, 2 mol% 3-hydroxydodecanoate and 3 mol% 3-hydroxy-5-cis-dodecenoate. The copolyester was shown to be a random copolymer of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoate units by analysis of the 13C-NMR spectrum. The poly(3HB) homopolymer and poly (3HB-co-3HA) copolymer were produced simultaneously within cells from glucose in the absence of any nitrogen source, which suggests that Pseudomonas sp. 61-3 has two types of polyhydroxy-alkanoate syntheses with different substrate specificities.
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Optimal growth and PHB accumulation in Bacillus megaterium occurred with 5% (w/v) date syrup or beet molasses supplemented with NH4Cl. When date syrup and beet molasses were used alone without an additional nitrogen source, a cell density of about 3gl–1 with a PHB content of the cells of 50% (w/w) was achieved. NH4NO3 followed by ammonium acetate and then NH4Cl supported cell growth up to 4.8gl–1, whereas PHB accumulation was increased with NH4Cl followed by ammonium acetate, NH4NO3 and then (NH4)2SO4 to a PHB content of nearly 42% (w/w). Cultivation of B.megaterium at 30l scale gave a PHB content of 25% (w/w) of the cells and a cell density of 3.4gl–1 after 14h growth.
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Pseudomonas oleovorans was cultivated to produce medium chain length polyhydroxyalkanoates (MCL-PHAs) from octanoic acid and ammonium nitrate as carbon and nitrogen source, respectively, by a pH-stat fed-batch culture technique. The octanoate in the culture broth was maintained below 4gl–1 by feeding the mixture of octanoic acid and ammonium nitrate when the culture pH rose above 7.1. The final cell concentrations of 63, 55 and 9.5gl–1, PHA contents of 62, 75 and 67% of dry cell wt, and productivities of 1, 0.63 and 0.16gl–1h–1 were obtained when the C/N ratios in the feed were 10, 20 and 100g octanoic acidg–1 ammonium nitrate, respectively.
Article
High yield production of polyhydroxyalkanoates (PHAs) by Ralstonia eutropha H16 and its recombinant strain PHB−4/pJRDEE32d13 (a PHA-negative mutant harboring Aeromonas caviae PHA synthase gene, phaCAc) from renewable inexpensive soybean oil was investigated. The PHA production by the wild-type strain H16 was achieved with a high dry cells weight (118–126 g/l) and a high poly[(R)-3-hydroxybutyrate] [P(3HB)] content per dry cells of 72–76% (w/w). A copolymer of 3HB with 5 mol% (R)-3-hydroxyhexanoate, P(3HB-co-5 mol% 3HHx), could be produced from soybean oil as a sole carbon source by the recombinant strain PHB−4/pJRDEE32d13 with a high dry cells weight (128–138 g/l) and a high PHA content of 71–74% (w/w). The reproducible results of PHA production in the presence of soybean oil as a sole carbon source was obtained with a high yield at a range of 0.72 to 0.76 g-PHA per g-soybean oil used.
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Poly(hydroxyalkanoates) (PHAs) were isolated from Pseudomonas aeruginosa 44T1 cultivated on euphorbia oil and castor oil. With the aid of 2-D proton NMR spectra and proton-detected multiple bond coherence NMR spectra the structures of the PHAs were determined. In addition to the usual PHA constituents (C6-C14 3-hydroxy fatty acids), PHAs formed from euphorbia oil contained delta 8,9-epoxy-3-hydroxy-5c-tetradecenoate, and probably delta 6,7-epoxy-3-hydroxydodecanoate and delta 4,5-epoxy-3-hydroxydecanoate. These novel constituents account for approximately 15% of the total amount of monomers and are clearly generated via beta-oxidation of vernolic acid (delta 12,13-epoxy-9c-octadecenoic acid), the main component of euphorbia oil. In PHAs formed from castor oil, 7% of the monomers found were derived from ricinoleic acid (12-hydroxy-9c-octadecenoic acid). The presence of 3,8-dihydroxy-5c-tetradecenoate was clearly demonstrated. Furthermore, NMR analysis strongly suggested the presence of 3,6-dihydroxydodecanoate, 6-hydroxy-3c-dodecenoate, and 4-hydroxydecanoate.
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The aim of the present paper was to study the feasibility of using olive oil mill effluents (OMEs) as a substrate in biodegradable polymer production. OMEs were anaerobically fermented to obtain volatile fatty acids (VFAs), which are the most highly used substrate for polyhydroxyalkanotes (PHAs) production. The anaerobic fermentation step was studied both without pretreatment and with different pretreatments (i.e., centrifugation, bentonite addition, and bentonite addition followed by centrifugation) and at various concentrations (28.5, 36.7 and 70.4 g CODL(-1)). During fermentation, VFA concentration was determined (7-16 g CODL(-1)) as well as the corresponding yield with respect to initial COD (22-44%). At all initial concentrations, centrifugation pretreatment (with or without previous addition of bentonite) significantly increased the final VFA concentration and yield, whereas the addition of bentonite alone had no influence. Moreover, centrifugation pretreatment led to a different acid distribution, which affected the hydroxyvalerate (HV) content within the obtained copolymer poly beta-(hydroxybutyrate-hydroxyvalerate) [P(HB-HV)]. OMEs were tested for PHA production by using a mixed culture from an aerobic SBR. Centrifuged OMEs, both with or without fermentation, were tested. PHAs were produced from both matrices, but with fermented OMEs PHA production was much higher, because of the higher VFA concentration. The initial specific rate of PHA production obtained with fermented OMEs was approximately 420 mg COD g COD(-1)h(-1) and the maximum HV content within the copolymer was about 11% (on a molar basis). The HV monomer was produced only until propionic acid remained present in the medium.
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.
Tailor-made olefinic medium-chain-length poly
  • R Hartmann
  • R Hany
  • E Pletscher
  • A Ritter
  • B Witholt
  • M Zinn
Hartmann, R., Hany, R., Pletscher, E., Ritter, A., Witholt, B., Zinn, M., 2005. Tailor-made olefinic medium-chain-length poly[(R)-3-hydroxyalkanoates]