Halomonas alkaliantarctica as a platform for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production from biodiesel-derived glycerol.

Polyhydroxyalkanoates (PHAs) are biodegradable polyesters produced by a wide range of microorganisms, including extremophiles. These unique microorganisms have gained interest in PHA production due to their ability to utilise low-cost carbon sources under extreme conditions. In this study, Halomonas alkaliantarctica was examined with regards to its potential to produce PHAs using crude glycerol from biodiesel industry as the only carbon source. We found that cell dry mass concentration was not dependent on the applying substrate concentration. Furthermore, our data confirmed that the analysed halophile was capable of metabolising crude glycerol into poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer within 24 h of the cultivation without addition of any precursors. Moreover, crude glycerol concentration affects the repeat units content in the purified PHAs copolymers and their thermal properties. Nevertheless, a differential scanning calorimetric and thermogravimetric analysis showed that the analysed biopolyesters have properties suitable for various applications. Overall, this study described a promising approach for the valorisation of crude glycerol as a future strategy of industrial waste management to produce high value microbial biopolymers.

Production and characterization of polyhydroxyalkanoates by Halomonas alkaliantarctica utilizing dairy waste as feedstock.

Currently, the global demand for polyhydroxyalkanoates (PHAs) is significantly increasing. PHAs are produced by several bacteria that are an alternative source of synthetic polymers derived from petrochemical refineries. This study established a simple and more feasible process of PHA production by Halomonas alkaliantarctica using dairy waste as the only carbon source. The data confirmed that the analyzed halophile could metabolize cheese whey (CW) and cheese whey mother liquor (CWML) into biopolyesters. The highest yield of PHAs was 0.42 g/L in the cultivation supplemented with CWML. Furthermore, it was proved that PHA structure depended on the type of by-product from cheese manufacturing, its concentration, and the culture time. The results revealed that H. alkaliantarctica could produce P(3HB-co-3HV) copolymer in the cultivations with CW at 48 h and 72 h without adding of any precursors. Based on the data obtained from physicochemical and thermal analyses, the extracted copolymer was reported to have properties suitable for various applications. Overall, this study described a promising approach for valorizing of dairy waste as a future strategy of industrial waste management to produce high value microbial biopolymers.

Recent Challenges and Trends of Polyhydroxyalkanoate Production by Extremophilic Bacteria Using Renewable Feedstocks.

Polyhydroxyalkanoates (PHAs) are biodegradable polymers with immense potential in addressing the global plastic pollution crisis and advancing sustainable bioplastics production. Among the various microbes known for PHA production, extremophilic bacteria possess unique capabilities to thrive under extreme conditions, making them attractive candidates for PHA synthesis. Furthermore, the utilization of renewable feedstocks for PHA production aligns with the growing demand for sustainable bioplastic alternatives. A diverse range of extremophilic bacteria, especially halophiles and thermophiles, has provided cost-competitive platforms for producing customized PHA polymers. Extremophilic bacteria offer unique advantages over mesophiles due to their contamination resistance, high cell density growth, and unique culture conditions. The current status of Halomonas spp. as a chassis further allows exploration of metabolic engineering approaches to overcome the challenges associated with current industrial biotechnology. This article especially focuses on extremophilic bacteria and explores recent advances in utilizing renewable feedstocks such as lignocellulosic biomass, agro-industrial residues, and waste streams for PHA production. The integration of biorefinery concepts and circular economy principles in PHA manufacturing is also examined. This review is an attempt to provide an understanding of renewable substrates as feedstocks and emerging trends in PHA production by extremophilic bacteria. It underscores the pivotal role of extremophiles and sustainable feedstock sources in advancing the feasibility and eco-friendliness of PHAs as a promising biopolymer alternative.

Effect of short- and medium-chain fatty acid mixture on polyhydroxyalkanoate production by Pseudomonas strains grown under different culture conditions.

Short- and medium-chain fatty acids (SMCFAs) derived from the acidogenic anaerobic mixed culture fermentation of acid whey obtained from a crude cheese production line and their synthetic mixture that simulates a real SMCFA-rich stream were evaluated for polyhydroxyalkanoate (PHA) production. Three individual Pseudomonas sp. strains showed different capabilities of growing and producing PHAs in the presence of a synthetic mixture of SMCFAs. Pseudomonas sp. GL06 exhibited the highest SMCFA tolerance and produced PHAs with the highest productivity (2.7 mg/L h). Based on these observations, this strain was selected for further investigations on PHA production in a fed-batch bioreactor with a SMCFA-rich stream extracted from the effluent. The results showed that PHA productivity reached up to 4.5 mg/L h at 24 h of fermentation together with the ammonium exhaustion in the growth medium. Moreover, the PHA monomeric composition varied with the bacterial strain and the type of the growth medium used. Furthermore, a differential scanning calorimetric and thermogravimetric analysis showed that a short- and medium-chain-length PHA copolymer made of 3-hydroxybutyric, -hexanoic, -octanoic, -decanoic, and -dodecanoic has promising properties. The ability of Pseudomonas sp. to produce tailored PHA copolymers together with the range of possible applications opens new perspectives in the development of PHA bioproduction as a part of an integrated valorization process of SMCFAs derived from waste streams.

Conversion of Short and Medium Chain Fatty Acids into Novel Polyhydroxyalkanoates Copolymers by Aeromonas sp. AC_01.

Polyhydroxyalkanoates (PHAs) production by Aeromonas sp. AC_01 was investigated using synthetic and waste derived short and medium chain fatty acids (SMCFAs). The obtained results revealed that the analyzed bacterial strain was able to grow and synthesize PHAs using SMCFAs. The highest PHA productivity was observed in the cultivation supplemented with a mixture of acetic acid and butyric acid (3.89 mg/L·h). Furthermore, SMCFAs-rich stream, derived from acidogenic mixed culture fermentation of acid whey, was found to be less beneficial for PHA productivity than its synthetic mixture, however the PHA production was favored by the nitrogen-limited condition. Importantly, Aeromonas sp. AC_01 was capable of synthesizing novel scl-mcl copolymers of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 3-hydroxytridecanoate (3HtriD) and/or 3-hydroxytetradecaonate (3HTD) with high 3HB and 3HV fractions. They were identified with alterable monomers composition depending on the culture conditions used. Moreover, in-depth thermal analyses proved that they are highly resistant to thermal degradation regardless of their monomeric composition. The obtained results confirm that Aeromonas sp. AC_01 is a promising candidate for the biotechnological production of PHAs from SMCFAs with thermal properties that can be tuned together with their chemical composition by the corresponding adjustment of the cultivation process.

Polyhydroxyalkanoates production from short and medium chain carboxylic acids by Paracoccus homiensis.

The aim of this study was to evaluate an effect of short and medium chain carboxylic acids (CAs) rich stream derived from acidogenic mixed culture fermentation of acid whey on polyhydroxyalkanoates (PHAs) synthesis by Paracoccus homiensis and compare it with the impact of individual synthetic CAs. The obtained results confirmed that the analyzed bacterium is able to metabolize synthetic CAs as the only carbon sources in the growth medium with maximum PHAs production yields of 26% of cell dry mass (CDM). The replacement of the individual CAs by a CAs-rich residual stream was found to be beneficial for the Paracoccus homiensis growth. The highest biomass concentration reached about 2.5 g/L with PHAs content of 17% of CDM. The purified PHAs were identified as poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by applying gas chromatography coupled with mass spectrometry, Fourier transform infrared spectroscopic spectra and UV-Vis spectra. Furthermore, a differential scanning calorimetric, thermogravimetric and water contact angle analysis proved that the extracted copolymers have useful properties. The obtained data are promising in the perspective of developing a microbial PHAs production as a part of an integrated valorization process of high CAs content waste-derived streams.

What Is New in the Field of Industrial Wastes Conversion into Polyhydroxyalkanoates by Bacteria?

The rising global consumption and industrialization has resulted in increased food processing demand. Food industry generates a tremendous amount of waste which causes serious environmental issues. These problems have forced us to create strategies that will help to reduce the volume of waste and the contamination to the environment. Waste from food industries has great potential as substrates for value-added bioproducts. Among them, polyhydroxyalkanaotes (PHAs) have received considerable attention in recent years due to their comparable characteristics to common plastics. These biodegradable polyesters are produced by microorganisms during fermentation processes utilizing various carbon sources. Scale-up of PHA production is limited due to the cost of the carbon source metabolized by the microorganisms. Therefore, there is a growing need for the development of novel microbial processes using inexpensive carbon sources. Such substrates could be waste generated by the food industry and food service. The use of industrial waste streams for PHAs biosynthesis could transform PHA production into cheaper and more environmentally friendly bioprocess. This review collates in detail recent developments in the biosynthesis of various types of PHAs produced using waste derived from agrofood industries. Challenges associated with this production bioprocess were described, and new ways to overcome them were proposed.

Volatile Fatty Acids as Carbon Sources for Polyhydroxyalkanoates Production.

Waste of industrial origin produced from synthetic materials are a serious threat to the natural environment. The ending resources of fossil raw materials and increasingly restrictive legal standards for the management of plastic waste have led to research on the use of biopolymers, which, due to their properties, may be an ecological alternative to currently used petrochemical polymers. Polyhydroxyalkanoates (PHAs) have gained much attention in recent years as the next generation of environmentally friendly materials. Currently, a lot of research is being done to reduce the costs of the biological process of PHA synthesis, which is the main factor limiting the production of PHAs on the industrial scale. The volatile fatty acids (VFAs) produced by anaerobic digestion from organic industrial and food waste, and various types of wastewater could be suitable carbon sources for PHA production. Thus, reusing the organic waste, while reducing the future fossil fuel, originated from plastic waste. PHA production from VFAs seem to be a good approach since VFAs composition determines the constituents of PHAs polymer and is of great influence on its properties. In order to reduce the overall costs of PHA production to a more reasonable level, it will be necessary to design a bioprocess that maximizes VFAs production, which will be beneficial for the PHA synthesis. Additionally, a very important factor that affects the profitable production of PHAs from VFAs is the selection of a microbial producer that will effectively synthesize the desired bioproduct. PHA production from VFAs has gained significant interest since VFAs composition determines the constituents of PHA polymer. Thus far, the conversion of VFAs into PHAs using pure bacterial cultures has received little attention, and the majority of studies have used mixed microbial communities for this purpose. This review discusses the current state of knowledge on PHAs synthesized by microorganisms cultured on VFAs.

Transcriptome Changes in Pseudomonas putida KT2440 during Medium-Chain-Length Polyhydroxyalkanoate Synthesis Induced by Nitrogen Limitation.

Pseudomonas putida's versatility and metabolic flexibility make it an ideal biotechnological platform for producing valuable chemicals, such as medium-chain-length polyhydroxyalkanoates (mcl-PHAs), which are considered the next generation bioplastics. This bacterium responds to environmental stimuli by rearranging its metabolism to improve its fitness and increase its chances of survival in harsh environments. Mcl-PHAs play an important role in central metabolism, serving as a reservoir of carbon and energy. Due to the complexity of mcl-PHAs' metabolism, the manner in which P. putida changes its transcriptome to favor mcl-PHA synthesis in response to environmental stimuli remains unclear. Therefore, our objective was to investigate how the P. putida KT2440 wild type and mutants adjust their transcriptomes to synthesize mcl-PHAs in response to nitrogen limitation when supplied with sodium gluconate as an external carbon source. We found that, under nitrogen limitation, mcl-PHA accumulation is significantly lower in the mutant deficient in the stringent response than in the wild type or the rpoN mutant. Transcriptome analysis revealed that, under N-limiting conditions, 24 genes were downregulated and 21 were upregulated that were common to all three strains. Additionally, potential regulators of these genes were identified: the global anaerobic regulator (Anr, consisting of FnrA, Fnrb, and FnrC), NorR, NasT, the sigma54-dependent transcriptional regulator, and the dual component NtrB/NtrC regulator all appear to play important roles in transcriptome rearrangement under N-limiting conditions. The role of these regulators in mcl-PHA synthesis is discussed.

Proteomic Response of Pseudomonas putida KT2440 to Dual Carbon-Phosphorus Limitation during mcl-PHAs Synthesis.

Pseudomonas putida KT2440, one of the best characterized pseudomonads, is a metabolically versatile producer of medium-chain-length polyhydroxyalkanoates (mcl-PHAs) that serves as a model bacterium for molecular studies. The synthesis of mcl-PHAs is of great interest due to their commercial potential. Carbon and phosphorus are the essential nutrients for growth and their limitation can trigger mcl-PHAs' production in microorganisms. However, the specific molecular mechanisms that drive this synthesis in Pseudomonas species under unfavorable growth conditions remain poorly understood. Therefore, the proteomic responses of Pseudomonas putida KT2440 to the limited carbon and phosphorus levels in the different growth phases during mcl-PHAs synthesis were investigated. The data indicated that biopolymers' production was associated with the cell growth of P. putida KT2440 under carbon- and phosphorus-limiting conditions. The protein expression pattern changed during mcl-PHAs synthesis and accumulation, and during the different physiological states of the microorganism. The data suggested that the majority of metabolic activities ceased under carbon and phosphorus limitation. The abundance of polyhydroxyalkanoate granule-associated protein (PhaF) involved in PHA synthesis increased significantly at 24 and 48 h of the cultivations. The activation of proteins belonging to the phosphate regulon was also detected. Moreover, these results indicated changes in the protein profiles related to amino acids metabolism, replication, transcription, translation, stress response mechanisms, transport or signal transduction. The presented data allowed the investigation of time-course proteome alterations in response to carbon and phosphorus limitation, and PHAs synthesis. This study provided information about proteins that can be potential targets in improving the efficiency of mcl-PHAs synthesis.

Polyhydroxyalkanoates Synthesized by Aeromonas Species: Trends and Challenges.

The negative effects of petrochemical-derived plastics on the global environment and depletion of global fossil fuel supplies have paved the way for exploring new technologies for the production of bioplastics. Polyhydroxyalkanoates (PHAs) are considered an alternative for synthetic polymers because of their biodegradability, biocompatibility, and non-toxicity. Many bacteria have been reported to have the ability to synthesize PHAs. Among them, the Aeromonas species seem to be ideal hosts for the industrial production of these biopolyesters due to their robust growth, simple growth requirements, their ability for the synthesis of homopolymers, co-polymers, and terpolymers with unique material properties. Some Aeromonas strains were able to produce PHAs in satisfactory amounts from simple carbon sources. Efforts have been made to use genetically modified Aeromonas strains for enhanced PHAs and to obtain bacteria with modified compositions and improved properties. This review discusses the current state of knowledge of polyhydroxyalkanoates synthesized by Aeromonas species, with a special focus on their potential, challenges, and progress in PHA synthesis.

A 2D-DIGE-based proteomic analysis brings new insights into cellular responses of Pseudomonas putida KT2440 during polyhydroxyalkanoates synthesis.

BACKGROUND: Polyhydroxyalkanoates (PHAs) have attracted much attention in recent years as natural alternatives to petroleum-based synthetic polymers that can be broadly used in many applications. Pseudomonas putida KT2440 is a metabolically versatile microorganism that is able to synthesize medium-chain-length PHAs (mcl-PHAs). The phenomena that drive mcl-PHAs synthesis and accumulation seems to be complex and are still poorly understood. Therefore, here we determine new insights into cellular responses of Pseudomonas putida KT2440 during biopolymers production using two-dimensional difference gel-electrophoresis (2D-DIGE) followed by MALDI TOF/TOF mass spectrometry. RESULTS: The maximum mcl-PHAs content in Pseudomonas putida KT2440 cells was 24% of cell dry weight (CDW) and was triggered by nitrogen depletion. Proteomic analysis allowed the detection of 150 and 131 protein spots differentially regulated at 24 h and 48 h relative to the cell growth stage (8 h), respectively. From those, we successfully identified 84 proteins that had altered expression at 24 h and 74 proteins at 48 h of the mcl-PHAs synthesis process. The protein-protein interactions network indicated that the majority of identified proteins were functionally linkage. The abundance of proteins involved in carbon metabolism were significantly decreased at 24 h and 48 h of the cultivations. Moreover, proteins associated with ATP synthesis were up-regulated suggesting that the enhanced energy metabolism was necessary for the mcl-PHAs accumulation. Furthermore, the induction of proteins involved in nitrogen metabolism, ribosome synthesis and transport was observed. Our results indicate that mcl-PHAs accumulated in the bacterial cells changed the protein abundance involved in stress response and cellular homeostasis. CONCLUSIONS: The presented data allow us to investigate time-course proteome rearrangement in response to nitrogen limitation and biopolyesters accumulation. Our results have pointed out novel proteins that might take part in cellular responses of mcl-PHA-accumulated bacteria. The study provides an additional knowledge that could be helpful to improve the efficiency of the bioprocess and make it more economically feasible.

Time-Course Proteomic Analysis of Pseudomonas putida KT2440 during Mcl-Polyhydroxyalkanoate Synthesis under Nitrogen Deficiency.

Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) have gained great attention as a new green alternative to petrochemical-derived polymers. Due to their outstanding material properties they can be used in a wide range of applications. Pseudomonas putida KT2440 is a metabolically versatile producer of mcl-polyhydroxyalkanoates. Although the metabolism of polyhydroxyalkanoate synthesis by this bacterium has been extensively studied, the comparative proteome analysis from three growth stages of Pseudomonas putida KT2440 cultured with oleic acid during mcl-PHA synthesis has not yet been reported. Therefore; the aim of the study was to compare the proteome of Pseudomonas putida KT2440 at different time points of its cultivation using the 2D difference gel electrophoresis (2D-DIGE) technique. The analyses showed that low levels of a nitrogen source were beneficial for mcl-PHA synthesis. Proteomic analysis revealed that the proteins associated with carbon metabolism were affected by nitrogen starvation and mcl-PHA synthesis. Furthermore, the induction of proteins involved in nitrogen metabolism, ribosome synthesis, and transport was observed, which may be the cellular response to stress related to nitrogen deficiency and mcl-PHA content in bacterial cells. To sum up; this study enabled the investigators to acquire a better knowledge of the molecular mechanisms underlying the induction of polyhydroxyalkanoate synthesis and accumulation in Pseudomonas putida KT2440 that could lead to improved strategies for PHAs in industrial production.

Exploring nutrient limitation for polyhydroxyalkanoates synthesis by newly isolated strains of Aeromonas sp. using biodiesel-derived glycerol as a substrate.

Aeromonas spp. strains isolated from activated sludge in a municipal wastewater treatment plant were found to be able to synthesize polyhydroxyalkanoates (PHA) utilizing pure and crude glycerol. The 16S rRNA gene sequence of the isolates exhibited similarity to Aeromonas hydrophila, A. aquatica, and A. salmonicida. Our results confirmed that the adequate supply of nitrogen and phosphorus during culture in 250-ml shake flasks did not stimulate the synthesis of PHAs. The results indicate that the PHA content of cells was higher under a phosphorus-limiting environment compared to nitrogen starvation. In the two-stage cultivation using glucose (in the first step) and crude glycerol from biodiesel industry (in the second step) as a component of the growth medium, the analyzed strains grew to 3.06 g/l of cell dry weight containing up to 22% of PHAs. Furthermore, during the same culture strategy up to 42% of PHAs were extracted, when in the second step of the process, Aeromonas sp. AC_03 was grown on pure glycerol under phosphorus limitation. The purified biopolymer was confirmed to be polyhydroxybutyrate. Aeromonas sp. AC_02 was also capable to accumulate the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer when pure glycerol was added as a substrate under nitrogen-deficiency one-step bioprocess. Our results confirm that due to the biopolymer productivity, newly isolated strains could be exploited for obtaining valuable biopolymers using wastes generated from biodiesel industry.

Transcriptome remodeling of Pseudomonas putida KT2440 during mcl-PHAs synthesis: effect of different carbon sources and response to nitrogen stress.

Bacterial response to environmental stimuli is essential for survival. In response to fluctuating environmental conditions, the physiological status of bacteria can change due to the actions of transcriptional regulatory machinery. The synthesis and accumulation of polyhydroxyalkanoates (PHAs) are one of the survival strategies in harsh environments. In this study, we used transcriptome analysis of Pseudomonas putida KT2440 to gain a genome-wide view of the mechanisms of environmental-friendly biopolymers accumulation under nitrogen-limiting conditions during conversion of metabolically different carbon sources (sodium gluconate and oleic acid). Transcriptomic data revealed that phaG expression is associated with medium-chain-length-PHAs' synthesis not only on sodium gluconate but also on oleic acid, suggesting that PhaG may play a role in this process, as well. Moreover, genes involved in the ß-oxidation pathway were induced in the PHAs production phase when sodium gluconate was supplied as the only carbon and energy source. The transition from exponential growth to stationary phase caused a significant expression of genes involved in nitrogen metabolism, energy supply, and transport system. In this study, several molecular mechanisms, which drive mcl-PHAs synthesis, have been investigated. The identified genes may provide valuable information to improve the efficiency of this bioprocess and make it more economically feasible.

Medium-chain-length polyhydroxyalkanoates synthesis by Pseudomonas putida KT2440 relA/spoT mutant: bioprocess characterization and transcriptome analysis.

Pseudomonas putida KT2440 is a model bacteria used commonly for medium-chain-length polyhydroxyalkanoates (mcl-PHAs) production using various substrates. However, despite many studies conducted on P. putida KT2440 strain, the molecular mechanisms of leading to mcl-PHAs synthesis in reaction to environmental stimuli are still not clear. The rearrangement of the metabolism in response to environmental stress could be controlled by stringent response that modulates the transcription of many genes in order to promote survival under nutritional deprivation conditions. Therefore, in this work we investigated the relation between mcl-PHAs synthesis and stringent response. For this study, a relA/spoT mutant of P. putida KT2440, unable to induce the stringent response, was used. Additionally, the transcriptome of this mutant was analyzed using RNA-seq in order to examine rearrangements of the metabolism during cultivation. The results show that the relA/spoT mutant of P. putida KT2440 is able to accumulate mcl-PHAs in both optimal and nitrogen limiting conditions. Nitrogen starvation did not change the efficiency of mcl-PHAs synthesis in this mutant. The transition from exponential growth to stationary phase caused significant upregulation of genes involved in transport system and nitrogen metabolism. Transcriptional regulators, including rpoS, rpoN and rpoD, did not show changes in transcript abundance when entering the stationary phase, suggesting their limited role in mcl-PHAs accumulation during stationary phase.

Bacterial polyhydroxyalkanoates: Still fabulous?

Bacterial polyhydroxyalkanoates (PHA) are polyesters accumulated as carbon and energy storage materials under limited growth conditions in the presence of excess carbon sources. They have been developed as biomaterials with unique properties for the past many years being considered as a potential substitute for conventional non-degradable plastics. Due to the increasing concern towards global climate change, depleting petroleum resource and problems with an utilization of a growing number of synthetic plastics, PHAs have gained much more attention from industry and research. These environmentally friendly microbial polymers have great potential in biomedical, agricultural, and industrial applications. However, their production on a large scale is still limited. This paper describes the backgrounds of PHAs and discussed the current state of knowledge on the polyhydroxyalkanoates. Ability of bacteria to convert different carbon sources to PHAs, the opportunities and challenges of their introduction to global market as valuable renewable products have been also discussed.