Evaluation and optimization of FTIR spectroscopy to quantify PHA production by municipal wastewater sludge.

Polyhydroxyalkanoate (PHA) is a family of naturally-occurring biopolymers synthesized by more than 300 microorganisms in the environment. These biopolymers have been investigated as a source material to substitute fossil fuel-based polymers; hence the synthesis of biopolymers and their characterization is a critical step in optimizing the process. Because of this, the biological production of PHA using PHA-producing microorganisms is currently the dominating process; however, the use of microbial mixed culture (MMC), such as wastewater sludge, is gaining attention. Different than pure cultures, MMC has higher culturing condition tolerance since the complex species composition and is easily obtained from wastewater treatment plants, which shortens the culturing time, lowers the cost, and promotes the application. The main constraint in MMC-based PHA is the extraction and quantification of PHA from the more complex matrix. In this paper, Fourier-transform infrared (FTIR) spectroscopy is evaluated to be used as a quantification method of PHA in MMC systems. Firstly, commercially available analytical standards, which consist of PHA/PHB, and two different solvents (chloroform and dichloromethane), were used and tested by this method, with KBr card and liquid cell methods, and the results are validated by gas chromatography mass spectrometry (GC/MS). The method was then tested using 12 samples from wastewater treatment plants. The PHA content in biomass varied from 3.42 w/w% to 1.22 w/w% following extraction with chloroform as solvent as determined by this method. In the four different combination standards, the best one is consisted of PHB and chloroform, and FTIR-liquid cell showed higher promise for PHA quantification in complex matrices.

Producción de plásticos biodegradables a partir de bacterias de hábitats salinos aisladas de la Laguna de Ayarza / Production of biodegradable plastics from saline habitats bacteria isolated from Laguna de Ayarza

La contaminación por plásticos petroquímicos es una grave amenaza para el medio ambiente que requiere im-plementar alternativas como los bioplásticos para lograr un desarrollo sostenible. Los polihidroxialcanoatos (PHA) son polímeros utilizados para la producción de plásticos biodegradables y que han llamado la atención como sustitutos de los plásticos de base fósil. Sin embargo, el costo de producción de los PHA constituye una barrera para su producción industrial a gran escala. Las de bacterias de hábitats salinos son microorganismos prometedores para la síntesis de PHA debido a sus características tales como altos requisitos de salinidad que previenen la contaminación microbiana, la alta presión osmótica intracelular que permite una fácil lisis celular para purificar los PHA y la capacidad para usar un amplio espectro de sustratos. La presente investigación planteó determinar las cepas nativas de bacterias halófilas y halotolerantes de la Laguna de Ayarza capaces de producir PHA, establecer la capacidad que tienen de utilizar residuos agrícolas para la producción de PHA y determinar su eficiencia. Esto se logró a través de la inoculación de las cepas productoras de PHA en medios de fermentación con pulpa de café, cáscaras de plátanos y salvado de trigo lo que permitió determinar las cepas más eficientes. Se encontró que las bacterias productoras de PHA pertenecen a las especies: Alcaligenes faecalis, Bacillus idriensis, Bacillus megaterium, Exiguobacterium acetylicum, E. aurantiacum, Pseudomonas cuatrocienegasensis y Sta-phylococcus capitis y que las cepas AP21-14, AP21-10 y AP21-03 mostraron los mejores resultados que podrían ser prometedores para la producción a nivel industrial.

Pollution by petrochemical plastics is a serious threat to the environment that requires the implementation of al-ternatives such as bioplastics to achieve sustainable development. Polyhydroxyalkanoates (PHAs) are polymers used for the production of biodegradable plastics and have drawn attention as substitutes for fossil-based plastics. However, the cost of producing PHAs constitutes a barrier to their large-scale industrial production. Bacteria from saline environments bacteria are promising microorganisms for PHA synthesis due to their characteristics such as high salinity requirements that prevent microbial contamination, high intracellular osmotic pressure that allows easy cell lysis to purify PHAs, and the ability to use a broad spectrum of substrates. This research project aimed to determine the native strains of halophilic and halotolerant bacteria from Laguna de Ayarza capable of producing PHA, establish their ability to use agricultural residues for the production of PHA, and determine their efficiency. This was achieved through the inoculation of the PHA-producing strains in fermentation media with coffee pulp, banana peels and wheat bran, which allowed determining the most efficient strains. It was found that the PHA-producing bacteria belong to the species: Alcaligenes faecalis, Bacillus idriensis, Bacillus mega-terium, Exiguobacterium acetylicum, E. aurantiacum, Pseudomonas cuatrocienegasensis and Staphylococcus capitis and that the strains AP21-14, AP21-10 and AP21-03 showed the best results that could be promising for production at an industrial level.

High natural PHA production from acetate in Cobetia sp. MC34 and Cobetia marina DSM 4741T and in silico analyses of the genus specific PhaC2 polymerase variant.

BACKGROUND: Several members of the bacterial Halomonadacea family are natural producers of polyhydroxyalkanoates (PHA), which are promising materials for use as biodegradable bioplastics. Type-strain species of Cobetia are designated PHA positive, and recent studies have demonstrated relatively high PHA production for a few strains within this genus. Industrially relevant PHA producers may therefore be present among uncharacterized or less explored members. In this study, we characterized PHA production in two marine Cobetia strains. We further analyzed their genomes to elucidate pha genes and metabolic pathways which may facilitate future optimization of PHA production in these strains. RESULTS: Cobetia sp. MC34 and Cobetia marina DSM 4741T were mesophilic, halotolerant, and produced PHA from four pure substrates. Sodium acetate with- and without co-supplementation of sodium valerate resulted in high PHA production titers, with production of up to 2.5 g poly(3-hydroxybutyrate) (PHB)/L and 2.1 g poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)/L in Cobetia sp. MC34, while C. marina DSM 4741T produced 2.4 g PHB/L and 3.7 g PHBV/L. Cobetia marina DSM 4741T also showed production of 2.5 g PHB/L from glycerol. The genome of Cobetia sp. MC34 was sequenced and phylogenetic analyses revealed closest relationship to Cobetia amphilecti. PHA biosynthesis genes were located at separate loci similar to the arrangement in other Halomonadacea. Further genome analyses revealed some differences in acetate- and propanoate metabolism genes between the two strains. Interestingly, only a single PHA polymerase gene (phaC2) was found in Cobetia sp. MC34, in contrast to two copies (phaC1 and phaC2) in C. marina DSM 4741T. In silico analyses based on phaC genes show that the PhaC2 variant is conserved in Cobetia and contains an extended C-terminus with a high isoelectric point and putative DNA-binding domains. CONCLUSIONS: Cobetia sp. MC34 and C. marina DSM 4741T are natural producers of PHB and PHBV from industrially relevant pure substrates including acetate. However, further scale up, optimization of growth conditions, or use of metabolic engineering is required to obtain industrially relevant PHA production titers. The putative role of the Cobetia PhaC2 variant in DNA-binding and the potential implications remains to be addressed by in vitro- or in vivo methods.

High cell density culture of Paracoccus sp. LL1 in membrane bioreactor for enhanced co-production of polyhydroxyalkanoates and astaxanthin.

A cell retention culture of Paracoccus sp. LL1 was performed in a membrane bioreactor equipped with an internal ceramic filter module to reach high cell density and thus enhance the co-production of polyhydroxyalkanoates (PHA) and astaxanthin as growth-associated products. Cell retention culture results showed that PHA accumulation increased with increasing dry cell weight (DCW), giving rise to a maximum of 113 ± 0.92 g/L of DCW with 43.9 ± 0.91 g/L of PHA (38.8% of DCW) at 48 h. A significant increase in both intracellular and extracellular astaxanthin concentrations was also recorded during fermentation process achieving a maximum of 8.51 ± 0.20 and 10.2 ± 0.24 mg/L, respectively. Amounts of PHA and total astaxanthin produced by cell retention culture were 6.29 and 19.7-folds higher, respectively, than those recorded under batch cultivation. PHA and total astaxanthin productivities by cell retention culture also increased up to 0.914 g/L/h and 0.781 mg/L/h, respectively, which were 3.54 and 11.1-folds higher than those of batch culture. Based on gas chromatography, Fourier transform infrared spectroscopy, and 1H nuclear magnetic resonance spectroscopy, the extracted PHA was identified as a copolymer of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with a 3-hydroxyvalerate content of 3.78 mol%.

PHA granule formation and degradation by Cupriavidus necator under different nutritional conditions.

Polyhydroxyalkanoates (PHA) are polymers produced by microorganisms with increasing commercialization potential; Cupriavidus necator has been the model microorganism to research PHA production. Despite many contributions concerning the formation and degradation of PHA granules, as well as the morphological changes in cells, these phenomena have not been univocally explained yet. Thus, this study aims to integrate the microscopic and analytical analysis to characterize changes in bacterial cell/PHA granules morphology, PHA content, and yield coefficients under different cultivation strategies of C. necator ATCC 17697. The cell size and morphology, granule size and amount, residual biomass, and PHA concentration along the fermentation and degradation depend greatly on nutritional conditions and cultivation time of C. necator. It was proposed to calculate a yield coefficient for the residual biomass production in the PHA utilization stage, related to the bacteria's ability to survive without a carbon source in the culture medium by utilizing the accumulated PHA previously. Maximum granule length reached 1.07 µm after 72 h of PHA accumulation stage under optimum nutritional conditions. This value is twice the values previously reported for C. necator. It is important since the larger PHA granules facilitate the recovery of PHA and different application development.

Rapid analysis of polyhydroxyalkanoate contents and its monomer compositions by pyrolysis-gas chromatography combined with mass spectrometry (Py-GC/MS).

Here, we report an analysis method for determining PHA (polyhydroxyalkanoates) contents and their monomer composition in microbial cells based on pyrolysis gas chromatography combined with mass spectrometry (Py-GC/MS). Various kinds of microbial cells accumulating different PHA contents and monomer compositions were prepared through the cultivation of Ralstonia eutropha and recombinant Escherichia coli. Py-GC/MS could analyse these samples in a short time without complicated pretreatment steps. Characteristic peaks such as 2-butenoic acid, 2-pentenoic acid, and hexadecanoic acid regarding PHA compositions and cell components were identified. Considering constituents of cells and ratios of peak areas of dehydrated monomers to hexadecanoic acid, a simple equation for estimation of PHA contents in microbial cells was derived. Also, monomer compositions of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) in R. eutropha could be successfully determined based on peak area of 2-butenoic acid and 2-pentenoic acid of Py-GC/MS, which are the corresponding species of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) in PHBV. Correlation of results between GC-FID and Py-GC/MS could be fitted very well. This method shows similar results for the samples obtained from same experimental conditions, allowing rapid and reliable analysis. Py-GC/MS can be a promising tool to rapidly screen PHA-positive strains based on polymer contents along with monomer compositions.

Low-quality animal by-product streams for the production of PHA-biopolymers: fats, fat/protein-emulsions and materials with high ash content as low-cost feedstocks.

OBJECTIVE: The rapid accumulation of crude-oil based plastics in the environment is posing a fundamental threat to the future of mankind. The biodegradable and bio-based polyhydroxyalkanoates (PHAs) can replace conventional plastics, however, their current production costs are not competitive and therefore prohibiting PHAs from fulfilling their potential. RESULTS: Different low-quality animal by-products, which were separated by thermal hydrolysis into a fat-, fat/protein-emulsion- and mineral-fat-mixture- (material with high ash content) phase, were successfully screened as carbon sources for the production of PHA. Thereby, Ralstonia eutropha Re2058/pCB113 accumulated the short- and medium-chain-length copolymer poly(hydroxybutyrate-co-hydroxyhexanoate) [P(HB-co-HHx)]. Up to 90 wt% PHA per cell dry weight with HHx-contents of 12-26 mol% were produced in shake flask cultivations. CONCLUSION: In future, the PHA production cost could be lowered by using the described animal by-product streams as feedstock.

Cupriavidus necator B-10646 growth and polyhydroxyalkanoates production on different plant oils.

The study addresses the growth of the wild-type strain Cupriavidus necator B-10646 and synthesis of polyhydroxyalkanoates by this strain on media containing plant oils with different compositions of fatty acids: palm, Siberian oilseed, and refined and unrefined sunflower seed oils. The study showed that the best carbon substrate was palm oil. Comparison of fatty acid compositions of the starting oils and unutilized residual substrates showed that C. necator B-10646 cells consumed the fatty acids from palm oil evenly while in experiments with other oils, they utilized polyenoic fatty acids first. Higher production parameters of the culture were obtained by preparation of emulsified oil medium using Tween 80 and sodium cocoyl glutamate as emulsifiers. All polyhydroxyalkanoate specimens were terpolymers that contained 3-hydroxybutyrate as the major component and minor amounts of 3-hydroxyvalerate (0.9-1.9 mol%) and 3-hydroxyhexanoate (0.5-1.1 mol%). Molecular weight of polyhydroxyalkanoate specimens depended on the type of plant oil and emulsifier.

Production of Polyhydroxyalkanoates by Two Halophilic Archaeal Isolates from Chott El Jerid Using Inexpensive Carbon Sources.

The large use of conventional plastics has resulted in serious environmental problems. Polyhydroxyalkanoates represent a potent replacement to synthetic plastics because of their biodegradable nature. This study aimed to screen bacteria and archaea isolated from an extreme environment, the salt lake Chott El Jerid for the accumulation of these inclusions. Among them, two archaeal strains showed positive results with phenotypic and genotypic methods. Phylogenetic analysis, based on the 16S rRNA gene, indicated that polyhydroxyalkanoate (PHA)-producing archaeal isolates CEJGTEA101 and CEJEA36 were related to Natrinema altunense and Haloterrigena jeotgali, respectively. Gas chromatography and UV-visible spectrophotometric analyses revealed that the PHA were identified as polyhydroxybutyrate and polyhydroxyvalerate, respectively. According to gas chromatography analysis, the strain CEJGTEA101 produced maximum yield of 7 wt % at 37 °C; pH 6.5; 20% NaCl and the strain CEJEA36 produced 3.6 wt % at 37 °C; pH 7; 25% NaCl in a medium supplemented with 2% glucose. Under nutritionally optimal cultivation conditions, polymers were extracted from these strains and were determined by gravimetric analysis yielding PHA production of 35% and 25% of cell dry weight. In conclusion, optimization of PHA production from inexpensive industrial wastes and carbon sources has considerable interest for reducing costs and obtaining high yield.

Diclofenac inhibited the biological phosphorus removal: Performance and mechanism.

This work aims to evaluate the effect of new contaminant diclofenac (DCF) in sewage on the performance of Enhanced Biological Phosphorus Removal (EBPR) and its mechanism. The results showed that low-level DCF had no significant effect on EBPR. However, when the concentration of DCF was 2.0 mg/L, the removal efficiencies of chemical oxygen demand (COD), NH4+-N and soluble orthophosphate (SOP) decreased significantly to 71.2 ± 4.2%, 78.6 ± 2.9%, and 64.3 ± 4.2%, respectively. Mechanisms revealed that DCF promoted the ratio of protein to polysaccharide in activated sludge extracellular polymers and inhibited anaerobic phosphorus release and oxic phosphorus uptake. Intracellular polymer analysis showed that when the DCF content was 2.0 mg/L, the maximum content of polyhydroxyalkanoates (PHA) was only 2.5 ± 0.4 mmol-C/g VSS, which was significantly lower than that in the blank. Analysis of key enzyme activities indicated that the presence of DCF reduced the activities of exopolyphosphatase and polyphosphate kinase.

Utilization of waste fish oil and glycerol as carbon sources for polyhydroxyalkanoate production by Salinivibrio sp. M318.

A moderately halophilic bacterium isolated from fermenting shrimp paste, Salinivibrio sp. M318 was found capable of using fish sauce and mixtures of waste fish oil and glycerol as nitrogen and carbon sources, respectively, for poly(3-hydroxybutyrate) (PHB) production. A cell dry weight (CDW) of up to 10 g/L and PHB content of 51.7 wt% were obtained after 48 h of cultivation in flask experiment. Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] was synthesized when 1,4-butanediol, γ-butyrolactone, or sodium 4-hydroxybutyrate was added as precursors to the culture medium. The biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] was achieved by supplying precursors such as sodium valerate, sodium propionate, and sodium heptanoate. Salinivibrio sp. M318 was able to accumulate the above mentioned PHAs during the growth phase. High CDW of 69.1 g/L and PHB content of 51.5 wt% were obtained by strain Salinivibrio sp. M318 after 78 h of cultivation in fed-batch culture. The results demonstrate Salinivibrio sp. M318 to be a promising wild-type bacterium for the production of PHA from aquaculture residues.

[Effect of Carbon Sources on the Accumulation of Endogenous Polymers and Denitritation in the O/A-F/F Mode].

To accumulate endogenous polymers during the aerobic phase, the aerobic/anoxic-feast/famine (O/A-F/F) selection mode can be used. It can also be used in situ for endogenous denitrification by activated sludge during the anoxic phase. To further explore the effect of carbon sources on the activated sludge accumulation of endogenous polymers and endogenous denitrification, this study used acetic and glucose as the main carbon sources to investigate the accumulation of endogenous polymers, endogenous denitrification, and the structure and function of enriched activated sludge. The results show that acetic (Ac-SBR) and glucose (Gc-SBR) as the main carbon source systems achieved a 40 mg·L-1 nitrate removal by endogenous denitrification when the influent chemical oxygen demand (COD) was~500 mg·L-1 in the O/A-F/F selection mode. Both the Ac-SBR and Gc-SBR achieved partial denitrification, but the nitrite accumulation of the Ac-SBR was higher than that of the Gc-SBR. Acetic is favorable for the accumulation of endogenous polyhydroxyalkanoate (PHA); PHA drives the endogenous denitrification. The yield of PHA was 0.52 and the denitrification rate (DNR) was 9.65 mg·(L·h)-1. The Gc-SBR system achieved the simultaneous accumulation of PHA and glycogen (Gly). The yield of Gly was higher than that of PHA and the DNR driven by Gly was 4.35 mg·(L·h)-1. The Gly was the main driving force to achieve endogenous denitrification and contributed to 77% of the total nitrogen removal. The 16S rRNA high-throughput sequencing analysis of activated sludge flora shows that the class of ß-Proteobacteria in the Proteobacteria was dominant, with an abundance of 40.56% in the Ac-SBR. However, the abundance of ß-Proteobacteria was only 18.05% in the Gc-SBR. The class of α-Proteobacteria contributes to glycogen accumulation in the Gc-SBR. The PHA can be accumulated by ß-Proteobacteria, Unclassified Bacteroidetes, and Lgnavibacteria in the Ac-SBR.

Overproduction of rhamnolipids in Pseudomonas aeruginosa PA14 by redirection of the carbon flux from polyhydroxyalkanoate synthesis and overexpression of the rhlAB-R operon.

OBJECTIVE: To construct Pseudomonas aeruginosa PA14 derivatives that overproduce rhamnolipids (RL) by blocking the synthesis of the carbon-storage polymer polyhydroxyalkanoates (PHA) and by overexpressing the rhlAB-R operon that encodes for enzymes of RL synthesis and the RhlR transcriptional regulator. RESULTS: In contrast to previous results showing that overexpression of rhlAB-R genes in two P. aeruginosa strains (PAO1 and ATCC 9027) is sufficient to overproduce RL, we show that a PA14 derivative overexpressing the rhlAB-R operon did not increase the synthesis of these biosurfactants. In addition, PA14 mutants deficient in PHA production did not overproduce RL either. However, if the rhlAB-R genes were expressed in a mutant that is completely impaired in PHA synthesis, a significant increase in RL production was observed (59%). These results show that RL production in PA14 is limited both by the availability of fatty acid precursors and by the levels of the RhlA and RhlB enzymes that are involved in the synthesis of mono-RL. CONCLUSIONS: The limitation of RL production by P. aeruginosa PA14 is multifactorial and diverse from the results obtained with other strains. Thus, the factors that limit RL production are particular to each P. aeruginosa strain, so strain-specific strategies should be developed to increase their production.

Synthesis of Short-Chain-Length and Medium-Chain-Length Polyhydroxyalkanoate Blends from Activated Sludge by Manipulating Octanoic Acid and Nonanoic Acid as Carbon Sources.

The effects of octanoic acid/nonanoic acid and acclimation time on the synthesis of short-chain-length and medium-chain-length PHA blends from activated sludge were investigated. An increased concentration (847-1366 mg/L) of PHAs resulted from 4-month acclimation compared with the concentration derived from 2-month acclimation (450-1126 mg/L). The content of octanoic acid had a positive linear relationship with the content of even-numbered carbon monomers among the PHAs. The blending products were identified mainly with scl-PHAs during the 2-month acclimation period and were thereafter dominated by mcl-PHAs until 4 months of acclimation. Thermal properties analysis demonstrated that the products derived from 4-month acclimation were a mixture of scl-PHAs and mcl-PHAs rather than a copolymer of scl-PHAs and mcl-PHAs. High-throughput sequencing results indicated that Pseudofulvimonas, Paracoccus, and Blastocatella were the dominant genera that might be responsible for scl-PHAs production during the 2-month acclimation period, whereas Comamonas and Pseudomonas that were responsible for mcl-PHAs production then became the dominant genera after 4-months acclimation.

Bioplastic reservoir of diverse bacterial communities revealed along altitude gradient of Pangi-Chamba trans-Himalayan region.

Himalaya hosts a unique environment for microbial ecology. The present study aimed to explore the bioplastic producing bacterial communities along altitude gradient of Pangi-Chamba trans-Himalayan region. A total of 411 bacteria were isolated and 70 characterized at the molecular level for potential polyhydroxyalkanoates (PHA) producers. The most abundant phylum for PHA synthesis was Proteobacteria (73%), followed by Actinobacteria (11%), Firmicutes (10%) and Bacteroidetes (6%). However, at the genus level, Pseudomonas and Janthinobacterium were dominantly reported. Also, the ability to synthesize PHA was reported for the first time for few genera such as Collimonas, Pseudarthrobacter and Paenarthrobacter. Phylogenetic analysis of partial 16S rDNA and phaC genes revealed conservation in phaC and possibility of horizontal gene transfer among distant taxa. Furthermore, GC-MS also confirmed the ability of potential bacterial isolates to synthesize PHA. In fact, we found that PHA-positive bacteria are dominant in the high altitude of Himalaya, suggesting the vital role of PHA in bacterial adaptation and survival. Together, these findings had revealed the rich bacterial diversity and genetic machinery for PHA synthesis which does have potential for further utilization in the commercial applications.

Improved intracellular PHA determinations with novel spectrophotometric quantification methodologies based on Sudan black dye.

The presence of intracellular polyhydroxyalkanoates (PHAs) is usually studied using Sudan black dye solution (SB). In a previous work it was shown that the PHA could be directly quantified using the absorbance of SB fixed by PHA granules in wet cell samples. In the present paper, the optimum SB amount and the optimum conditions to be used for SB assays were determined following an experimental design by hybrid response surface methodology and desirability-function. In addition, a new methodology was developed in which it is shown that the amount of SB fixed by PHA granules can also be determined indirectly through the absorbance of the supernatant obtained from the stained cell samples. This alternative methodology allows a faster determination of the PHA content (involving 23 and 42 min for indirect and direct determinations, respectively), and can be undertaken by means of basic laboratory equipment and reagents. The correlation between PHA content in wet cell samples and the spectra of the SB stained supernatant was determined by means of multivariate and linear regression analysis. The best calibration adjustment (R2 = 0.91, RSE: 1.56%), and the good PHA prediction obtained (RSE = 1.81%), shows that the proposed methodology constitutes a reasonably precise way for PHA content determination. Thus, this methodology could anticipate the probable results of the above mentioned direct PHA determination. Compared with the most used techniques described in the scientific literature, the combined implementation of these two methodologies seems to be one of the most economical and environmentally friendly, suitable for rapid monitoring of the intracellular PHA content.

Overproduction of MCL-PHA with high 3-hydroxydecanoate Content.

Methods of producing medium-chain-length poly-3-hydroxyalkanoate (mcl-PHA) with high content of the dominant subunit, 3-hydroxydecanoate (HD), were examined with an emphasis on a high yield of polymer from decanoic acid. High HD content was achieved by using a ß-oxidation knockout mutant of Pseudomonas putida KT2440 (designated as P. putida DBA-F1) or by inhibiting ß-oxidation with addition of acrylic acid (Aa) to wild type P. putida KT2440 in carbon-limited, fed-batch fermentations. At a substrate feed ratio of decanoic acid and acetic acid to glucose (DAA:G) of 6:4 g/g, P. putida DBA-F1 accumulated significantly higher HD (97 mol%), but much lower biomass (8.5 g/L) and PHA (42% of dry biomass) than the wild type. Both biomass and PHA concentrations were improved by decreasing the ratio of DAA:G to 4:6. Moreover, when the substrate feed ratio was further decreased to 2:8, 18 g/L biomass containing 59% mcl-PHA consisting of 100 mol% HD was achieved. The yield of PHA from decanoic acid was 1.24 (g/g) indicating that de novo synthesis had contributed to production. Yeast extract and tryptone (YET) addition allowed the mutant strain to accumulate 74% mcl-PHA by weight with 97 mol% HD at a production rate of 0.41 g/L/hr, at least twice that of published data for any ß-oxidation knock-out mutant. Higher biomass concentration was achieved with Aa inhibition of ß-oxidation in the wild type but the HD content (84 mol%) was less than that of the mutant. A carbon balance showed a marked increase in supernantant organic carbon for the mutant indicating overflow metabolism. Increasing the dominant monomer content (HD) greatly increased melting point, crystallinity, and rate of crystallization.

Co-synthesis of medium-chain-length polyhydroxyalkanoates and CdS quantum dots nanoparticles in Pseudomonas putida KT2440.

Microbial polymers and nanomaterials production is a promising alternative for sustainable bioeconomics. To this end, we used Pseudomonas putida KT2440 as a cell factory in batch cultures to coproduce two important nanotechnology materials- medium-chain-length (MCL)-polyhydroxyalkanoates (PHAs) and CdS fluorescent nanoparticles (i.e. quantum dots [QDots]). Due to high cadmium resistance, biomass and PHA yields were almost unaffected by coproduction conditions. Fluorescent nanocrystal biosynthesis was possible only in presence of cysteine. Furthermore, this process took place exclusively in the cell, displaying the classical emission spectra of CdS QDots under UV-light exposure. Cell fluorescence, zeta potential values, and particles size of QDots depended on cadmium concentration and exposure time. Using standard PHA-extraction procedures, the biosynthesized QDots remained associated with the biomass, and the resulting PHAs presented no traces of CdS QDots. Transmission electron microscopy located the synthesized PHAs in the cell cytoplasm, whereas CdS nanocrystals were most likely located within the periplasmic space, exhibiting no apparent interaction. This is the first report presenting the microbial coproduction of MCL-PHAs and CdS QDots in P. putida KT2440, thus constituting a foundation for further bioprocess developments and strain engineering towards the efficient synthesis of these highly relevant bioproducts for nanotechnology.

Low frequency sonic waves assisted cloud point extraction of polyhydroxyalkanoate from Cupriavidus necator.

Low frequency sonic waves, less than 10kHz were introduced to assist cloud point extraction of polyhydroxyalkanoate from Cupriavidus necator present within the crude broth. Process parameters including surfactant system variables and sonication parameters were studied for their effect on extraction efficiency. Introduction of low frequency sonic waves assists in the dissolution of microbial cell wall by the surfactant micelles and release of cellular content, polyhydroxyalkanoate granules released were encapsulated by the micelle core which was confirmed by crotonic acid assay. In addition, sonic waves resulted in the separation of homogeneous surfactant and broth mixture into two distinct phases, top aqueous phase and polyhydroxyalkanoate enriched bottom surfactant rich phase. Mixed surfactant systems showed higher extraction efficiency compared to that of individual Triton X-100 concentrations, owing to increase in the hydrophobicity of the micellar core and its interaction with polyhydroxyalkanoate. Addition of salts to the mixed surfactant system induces screening of charged surfactant head groups and reduces inter-micellar repulsion, presence of ammonium ions lead to electrostatic repulsion and weaker cation sodium enhances the formation of micellar network. Addition of polyethylene glycol 8000 resulted in increasing interaction with the surfactant tails of the micelle core there by reducing the purity of polyhydroxyalkanoate.

Novel spectrophotometric technique for rapid determination of extractable PHA using Sudan black dye.

Classical techniques employed to determine the amount of extractable poly(hydroxyalkanoate)s (PHAs) from cells, are laborious and destructive. Sudan black staining is commonly used in the laboratory to investigate the presence of intracellular PHA. The aim of the present study was to develop a low-cost alternative technique to achieve a quick determination of extractable intracellular PHA. This methodology employs a basic laboratory spectroscopy equipment and Sudan black dye for spectra determination. The correlation between the content of PHA in cell samples taken directly from the culture flask and its spectra was determined using partial least square regression analysis and simple linear regression analysis. The best fit obtained for calibration correlation analysis (R2=0.944, RSE: 1.24%), together with the good extractable PHA predictions (RSE=0.51%) demonstrate that the proposed methodology constitutes a fast way with high potential for the determination of extractable PHA. Based on its simplicity and flexibility, its application would be suitable in routine monitoring and rapid quantification in large-scale processes involving PHA metabolism.