Synthesis of <Emphasis Type="Italic">scl</Emphasis>-poly (3-hydroxyalkanoates) by <Emphasis Type="Italic">Bacillus cereus</Emphasis> found in freshwater,from monosaccharides and disaccharides

Background

Polyhydroxyalkanoates are a good substitute for synthetic plastic because they are highly biocompatible, ecofriendly, and biodegradable. Bacteria in freshwater bodies such as rivers, tube wells, and canals are exposed to alternating high and low concentrations of substrates that induce PHA production.

Methods

Fresh water samples were collected for isolation of bacterial strains. Screening of PHA in bacterial cells was performed with Sudan and Nile Red staining. Extracted PHA was characterized by FTIR.

Results

In this study, nine bacterial isolates were selected for PHA production on the basis of phenotypic screening. Their ability to accumulate PHAs was determined using different monosaccharides and disaccharides. Two bacterial isolates Bacillus cereus T1 (KY746353) and Bacillus cereus R3 (KY746354) produced PHAs. Optimal growth of the bacterial strain (T1) was observed in the presence of glucose, followed by maximum production of PHAs (63% PHAs) during the logarithmic phase of growth. B. cereus R3 (KY746354) accumulated 60% PHAs by dry cell weight.

Conclusion

PHA accumulation was relatively less with fructose, but both strains showed increased production (up to 50%) with sucrose. The polymer produced was characterized by Fourier-transform infrared spectroscopy (FTIR), which showed that the compound contains short-chain PHAs.

     

Simultaneous production of polyhydroxyalkanoates and rhamnolipids by Pseudomonas aeruginosa

The feasibility of the simultaneous production of polyhydroxyalkanoates (PHAs) and rhamnolipids, as a novel approach to reduce their production costs, was demonstrated by the cultivation of Pseudomonas aeruginosa IFO3924. Fairly large amounts of PHAs and rhamnolipids were obtained from the bacterial cells and the culture supernatant, respectively. Decanoate was a more suitable carbon source than ethanol and glucose for the simultaneous production, although glucose was suitable for cell growth without an induction period under pH control. The kind of carbon source affected PHA monomer composition markedly and PHA molecular weight slightly. Monorhamnolipids and dirhamnolipids were included in the rhamnolipids extracted from the culture supernatant using decanoate, glucose, or ethanol as the carbon source. Both PHAs and rhamnolipids were synthesized after the growth phase. PHA content in the cell reached a maximum when the carbon source was exhausted. After exhaustion of the carbon source, PHA content decreased rapidly, but rhamnolipid synthesis, which followed PHA synthesis, continued. This resulted in a time lag for the attainment of maximum levels of PHAs and rhamnolipids. The reusability of the cells used in rhamnolipid production was evaluated in the repeated batch culture of P. aeruginosa IFO3924 for the simultaneous production of PHAs and rhamnolipids. High concentrations of rhamnolipids in the culture supernatant were attained at the end of both the first and second batch cultures. High PHA content was achieved in the resting cells that were finally harvested after the second batch. Simultaneous production of PHAs and rhamnolipids will enhance the availability of valuable biocatalysts of bacterial cells, and dispel the common belief that the production cost of PHAs accumulated intracellularly is almost impossible to become lower than that of cells themselves.     

Rhamnolipid biosurfactant production by strains of Pseudomonas aeruginosa using low-cost raw materials

This study was aimed at the development of economical methods for higher yields of biosurfactant by suggesting the use of low-cost raw materials. Two oil-degrading strains, Pseudomonas aeruginosa GS9-119 and DS10-129, were used to optimize a substrate for maximum rhamnolipid production. Among the two strains, the latter produced maxima of 4.31, 2.98, and 1.77 g/L rhamnolipid biosurfactant using soybean oil, safflower oil, and glycerol, respectively. The yield of biosurfactant steadily increased even after the bacterial cultures reached the stationary phase of growth. Characterization of rhamnolipids using mass spectrometry revealed the presence of dirhamnolipids (Rha-Rha-C(10)-C(10)). Emulsification activity of the rhamnolipid biosurfactant produced by P. aeruginosa DS10-129 was greater than 70% using all the hydrocarbons tested, including xylene, benzene, hexane, crude oil, kerosene, gasoline, and diesel. P. aeruginosa GS9-119 emulsified only hexane and kerosene to that level.     

Polyhydroxyalkanoate (PHA) accumulating bacteria from the gut of higher termite <Emphasis Type="Italic">Macrotermes carbonarius</Emphasis> (Blattodea: Termitidae)

The continuous quest for bacterial strains capable of accumulating polyhydroxyalkanoate (PHA) utilizing cheaper and renewable carbon source prompted us to explore newer and diverse environments like the gut of termites. Among the bacterial strains isolated from the gut of higher termite Macrotermes carbonarius, three strains were found to accumulate PHA, as observed by microscopic studies and PHA production experiments. Among them, strain MC1 with rapid growth and higher PHA accumulation was selected for further studies. API kit-50 CHB and 16S rRNA gene sequence analysis results indicated the strain to have 99% homology with Bacillus megaterium and Bacillus flexus. Bacillus sp. MC1 was able to accumulate PHA during the growth phase utilizing different carbon sources like glucose, fructose, sodium acetate, sodium valerate and 1,4-butanediol. Gas chromatography analysis of the polymer has shown it to be basically composed of poly (3-hydroxybutyrate) (PHB). Growth associated PHB biosynthesis was best in the presence of sodium acetate with 39 wt% after 16 h of cultivation. Though previous studies provided evidence confirming the presence of PHA producing bacteria in termite gut, isolation and characterization of these strains in pure culture has not been documented yet. Presence of other morphotypes in the termite gut with PHA like granular inclusions was evident from the transmission electron microscopy studies. This is a novel report and shows the feasibility of using potent strains capable of utilizing lignocellulosic degradation products as a renewable carbon source for the production of PHA in the future.     

Effects of Granule-Associated Protein PhaP on Glycerol-Dependent Growth and Polymer Production in Poly(3-Hydroxybutyrate)-Producing Escherichia coli

Polyhydroxyalkanoates (PHAs) are accumulated as intracellular granules by many bacteria under unfavorable conditions, enhancing their fitness and stress resistance. Poly(3-hydroxybutyrate) (PHB) is the most widespread and best-known PHA. Apart from the genes that catalyze polymer biosynthesis, natural PHA producers have several genes for proteins involved in granule formation and/or with regulatory functions, such as phasins, that have been shown to affect polymer synthesis. This study evaluates the effect of PhaP, a phasin, on bacterial growth and PHB accumulation from glycerol in bioreactor cultures of recombinant Escherichia coli carrying phaBAC from Azotobacter sp. strain FA8. Cells expressing phaP grew more, and accumulated more PHB, both using glucose and using glycerol as carbon sources. When cultures were grown in a bioreactor using glycerol, PhaP-bearing cells produced more polymer (2.6 times) and more biomass (1.9 times) than did those without the phasin. The effect of this protein on growth promotion and polymer accumulation is expected to be even greater in high-density cultures, such as those used in the industrial production of the polymer. The recombinant strain presented in this work has been successfully used for the production of PHB from glycerol in bioreactor studies, allowing the production of 7.9 g/liter of the polymer in a semisynthetic medium in 48-h batch cultures. The development of bacterial strains that can efficiently use this substrate can help to make the industrial production of PHAs economically feasible.     

Pseudomonas aeruginosa biosurfactant production in continuous culture with glucose as carbon source

Rsan-ver, a strain of Pseudomonas aeruginosa isolated at this department, was used for the development of a continuous process for biosurfactant production. The active compounds were identified as rhamnolipids. A final medium for production was designed in continuous culture by means of medium shifts, since the formation of surface-active compounds was decisively influenced by the composition and concentration of the medium components. In the presence of yeast extract, biosurfactant production was poor. For the nitrogen-source nitrate, which was superior to ammonium, an optimum carbon-to-nitrogen ratio of ca. 18 existed. The iron concentration needed to be minimized to 27.5 micrograms of FeSO4 X 7H2O per g of glucose. A carbon-to-phosphate ratio below 16 yielded the maximum production of rhamnolipids. The final productivity dilution rate diagram indicated that biosurfactant production was correlated to low growth rates (dilution rate below 0.15 h-1). With a medium containing 18.2 g of glucose liter-1, a biosurfactant concentration (expressed as rhamnolipids) of up to 1.5 g liter-1 was obtained in the cell-free culture liquid.     

Pseudomonas aeruginosa biosurfactant production in continuous culture with glucose as carbon source.

Rsan-ver, a strain of Pseudomonas aeruginosa isolated at this department, was used for the development of a continuous process for biosurfactant production. The active compounds were identified as rhamnolipids. A final medium for production was designed in continuous culture by means of medium shifts, since the formation of surface-active compounds was decisively influenced by the composition and concentration of the medium components. In the presence of yeast extract, biosurfactant production was poor. For the nitrogen-source nitrate, which was superior to ammonium, an optimum carbon-to-nitrogen ratio of ca. 18 existed. The iron concentration needed to be minimized to 27.5 micrograms of FeSO4 X 7H2O per g of glucose. A carbon-to-phosphate ratio below 16 yielded the maximum production of rhamnolipids. The final productivity dilution rate diagram indicated that biosurfactant production was correlated to low growth rates (dilution rate below 0.15 h-1). With a medium containing 18.2 g of glucose liter-1, a biosurfactant concentration (expressed as rhamnolipids) of up to 1.5 g liter-1 was obtained in the cell-free culture liquid.     

Accumulation of polyhydroxyalkanoates by halophilic archaea isolated from traditional solar salterns of India

Extremely halophilic archaeal isolates obtained from brine and sediment samples of solar salterns of Goa and Tamil Nadu, India were screened for accumulation of polyhydroxyalkanoates (PHA). Seven polymer accumulating haloarchaeal strains (TN4, TN5, TN6, TN7, TN9, TN10 and BBK2) were selected based on their growth and intensity of fluorescence when grown on 20 % NaCl synthetic medium supplemented with 2 % glucose and incorporated with Nile red dye. The polymer was quantified by conversion of PHA to crotonic acid which gave a characteristic absorption maxima at 235 nm. On the basis of phenotypic and genotypic characterization the cultures TN4, TN5, TN6, TN7, TN10 and BBK2 were grouped under genus Haloferax whereas isolate TN9 was grouped under the genus Halogeometricum. Growth kinetics and polymer accumulation studies revealed that the culture Halogeometricum borinquense strain TN9 accumulates PHA maximally at the mid-log phase, i.e. 5th day of growth (approx. 14 wt% PHA of CDW). Analysis of the polymer by IR, ¹H NMR and ¹³C NMR confirmed it to be a homopolymer of 3-hydroxybutyrate.     

Medium chain length polyhydroxyalkanoate (mcl-PHA) production from volatile fatty acids derived from the anaerobic digestion of grass

A two step biological process for the conversion of grass biomass to the biodegradable polymer medium chain length polyhydroxyalkanoate (mcl-PHA) was achieved through the use of anaerobic and aerobic microbial processes. Anaerobic digestion (mixed culture) of ensiled grass was achieved with a recirculated leach bed bioreactor resulting in the production of a leachate, containing 15.3 g/l of volatile fatty acids (VFAs) ranging from acetic to valeric acid with butyric acid predominating (12.8 g/l). The VFA mixture was concentrated to 732.5 g/l with a 93.3 % yield of butyric acid (643.9 g/l). Three individual Pseudomonas putida strains, KT2440, CA-3 and GO16 (single pure cultures), differed in their ability to grow and accumulate PHA from VFAs. P. putida CA-3 achieved the highest biomass and PHA on average with individual fatty acids, exhibited the greatest tolerance to higher concentrations of butyric acid (up to 40 mM) compared to the other strains and exhibited a maximum growth rate (μ_MAX?=?0.45 h^?1). Based on these observations P. putida CA-3 was chosen as the test strain with the concentrated VFA mixture derived from the AD leachate. P. putida CA-3 achieved 1.56 g of biomass/l and accumulated 39 % of the cell dry weight as PHA (nitrogen limitation) in shake flasks. The PHA was composed predominantly of 3-hydroxydecanoic acid (>65 mol%).     

Effect of heterologous expression of phaG [(R)-3-hydroxyacyl-ACP-CoA transferase] on polyhydroxyalkanoate accumulation from the aromatic hydrocarbon phenylacetic acid in Pseudomonas species

Five Pseudomonas strains capable of growth with the aromatic carboxylic acid phenylacetic acid were investigated with a view to improving PHA accumulation. The overexpression of (R)-3-hydroxyacyl-ACP-CoA transferase (PhaG) from Pseudomonas putida CA-3 increased PHA accumulation in only one of the five strains tested, namely Pseudomonas jessenii C8. Recombinant P. jessenii C8 harbouring the phaG gene showed a 4.1-fold increase (9.6-39% cell dry weight) in PHA accumulation when grown on phenylacetic acid (15 mM) compared with the wild-type strain. This is the highest reported level of PHA accumulation from phenylacetic acid. This is also the first time the heterologous expression of phaG has resulted in improved PHA accumulation from an aromatic carbon source. The growth patterns of the wild type and recombinant strains were very similar, with no significant differences observed in carbon and nitrogen utilization.     

Polymeric Beta-Hydroxyalkanoates from Environmental Samples and Bacillus megaterium

The procaryotic endogenous storage polymer known as poly-beta-hydroxybutyrate is actually a mixed polymer of short-chain beta-hydroxy fatty acids. A method for the quantitative recovery of this mixed polymer, called poly-beta-hydroxyalkanoate (PHA), with analysis by capillary gas-liquid chromatography, showed the presence of at least 11 short-chain beta-hydroxy acids in polymers extracted from marine sediments. Polymers extracted from Bacillus megaterium monocultures were also a complex mixture of beta-hydroxy acids with chain lengths between four and eight carbons. Lyophilized sediments were extracted in a modified Soxhlet extractor, and the polymer was purified with ethanol and diethyl ether washes. The purified polymer was treated with ethanol-chloroform-hydrochloric acid (8.5:2.5:1) for 4 h at 100°C, a treatment which resulted in the formation of the ethyl esters of the constituent beta-hydroxy acids. Subsequent assay of the products by gas-liquid chromatography indicated excellent reproducibility and sensitivity (detection limit, 100 fmol). Disturbing sediments mechanically or adding natural chelators increased all major PHA components relative to the bacterial biomass. Gardening of sedimentary microbes by Clymenella sp., an annelid worm, induced decreases in PHA, with changes in the relative proportion of component beta-hydroxy acids. The concentration of PHA relative to the bacterial biomass can reflect the recent metabolic status of the microbiota.     

Development of a bioprocess to convert PET derived terephthalic acid and biodiesel derived glycerol to medium chain length polyhydroxyalkanoate

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.     

Production of polyhydroxyalkanoates by mixed culture: recent trends and biotechnological importance

Polyhydroxyalkanoates (PHAs) are the polymers of hydroxyalkanoates that accumulate as carbon/energy or reducing-power storage material in various microorganisms. PHAs have been attracting considerable attention as biodegradable substitutes for conventional polymers. To reduce their production cost, a great deal of effort has been devoted to developing better bacterial strains and more efficient fermentation/recovery processes. The use of mixed cultures and cheap substrates can reduce the production cost of PHA. Accumulation of PHA by mixed cultures occurs under transient conditions mainly caused by intermittent feeding and variation in the electron donor/acceptor presence. The maximum capacity for PHA storage and the PHA production rate are dependent on the substrate and the operating conditions used. This work reviews the development of PHA research. Aspects discussed include metabolism and various mechanisms for PHA production by mixed cultures; kinetics of PHA accumulation and conversion; effects of carbon source and temperature on PHA production using mixed cultures; PHA production process design; and characteristics of PHA produced by mixed cultures.     

The Effect of Rhamnolipid Biosurfactant Produced by <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> on Model Bacterial Strains and Isolates from Industrial Wastewater

In this study, the effect of rhamnolipid biosurfactant produced by Pseudomonas fluorescens on bacterial strains, laboratory strains, and isolates from industrial wastewater was investigated. It was shown that biosurfactant, depending on the concentration, has a neutral or detrimental effect on the growth and protein release of model Gram (+) strain Bacillus subtilis 168. The growth and protein release of model Gram (−) strain Pseudomonas aeruginosa 1390 was not influenced by the presence of biosurfactant in the medium. Rhamnolipid biosurfactant at the used concentrations supported the growth of some slow growing on hexadecane bacterial isolates, members of the microbial community. Changes in cell surface hydrophobicity and permeability of some Gram (+) and Gram (−) isolates in the presence of rhamnolipid biosurfactant were followed in experiments in vitro. It was found that bacterial cells treated with biosurfactant became more or less hydrophobic than untreated cells depending on individual characteristics and abilities of the strains. For all treated strains, an increase in the amount of released protein was observed with increasing the amount of biosurfactant, probably due to increased cell permeability as a result of changes in the organization of cell surface structures. The results obtained could contribute to clarify the relationships between members of the microbial community as well as suggest the efficiency of surface properties of rhamnolipid biosurfactant from Pseudomonas fluorescens making it potentially applicable in bioremediation of hydrocarbon-polluted environments.     

Differences and dynamic changes in the cell surface properties of three Pseudomonas aeruginosa strains isolated from petroleum-polluted soil as a response to various carbon sources and the external addition of rhamnolipids

Three Pseudomonas aeruginosa strains isolated from petroleum-polluted soil were the subject of studies concerning changes in cell surface properties. Fundamentally different reactions could be observed for each of the studied strains after a cultivation on various carbon sources. The experiments carried out during the logarithmic growth phase showed, that the changes in the cell surface hydrophobocity values were dynamic and substrate dependant. An external addition of rhamnolipids to the tested systems resulted in further shifts in the CSH values. All of the strains displayed miscellaneous phenotypic properties during MATH, sedimentation profile, Zeta potential and surface tension measurements. The obtained results lead to a conclusion, that the presence of rhamnolipids seems to be the key factor to this phenomenon, as all of the studied strains exhibited the ability to produce this biosurfactant in a different degree.     

Production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from plant oil by engineered Ralstonia eutropha strains

The polyhydroxyalkanoate (PHA) copolymer poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(HB-co-HHx)] has been shown to have potential to serve as a commercial bioplastic. Synthesis of P(HB-co-HHx) from plant oil has been demonstrated with recombinant Ralstonia eutropha strains expressing heterologous PHA synthases capable of incorporating HB and HHx into the polymer. With these strains, however, short-chain-length fatty acids had to be included in the medium to generate PHA with high HHx content. Our group has engineered two R. eutropha strains that accumulate high levels of P(HB-co-HHx) with significant HHx content directly from palm oil, one of the world's most abundant plant oils. The strains express a newly characterized PHA synthase gene from the bacterium Rhodococcus aetherivorans I24. Expression of an enoyl coenzyme A (enoyl-CoA) hydratase gene (phaJ) from Pseudomonas aeruginosa was shown to increase PHA accumulation. Furthermore, varying the activity of acetoacetyl-CoA reductase (encoded by phaB) altered the level of HHx in the polymer. The strains with the highest PHA titers utilized plasmids for recombinant gene expression, so an R. eutropha plasmid stability system was developed. In this system, the essential pyrroline-5-carboxylate reductase gene proC was deleted from strain genomes and expressed from a plasmid, making the plasmid necessary for growth in minimal media. This study resulted in two engineered strains for production of P(HB-co-HHx) from palm oil. In palm oil fermentations, one strain accumulated 71% of its cell dry weight as PHA with 17 mol% HHx, while the other strain accumulated 66% of its cell dry weight as PHA with 30 mol% HHx.     

The conversion of BTEX compounds by single and defined mixed cultures to medium-chain-length polyhydroxyalkanoate

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.     

Polyhydroxyalkanoate Production and Degradation Patterns in <Emphasis Type="Italic">Bacillus</Emphasis> Species

Bacteria under stress conditions of excess of carbon (C) and limitations of nutrients divert its metabolism towards C storage as energy reservoir—polyhydroxyalkanoate (PHA). Different Bacillus species—B. cereus and B. thuringiensis, were monitored to produce PHA from different C sources—glucose, crude glycerol and their combination at 37 °C for period up to 192 h. PHA production and its composition was found to vary with feed and bacterial strains. PHA production on crude glycerol continued to increase up to 120 h, reaching a maximum of 2725 mg/L with an effective yield of 71% of the dry cell mass. Depolymerization of PHA was observe to initiate after 96 h of incubation up to 192 h. PHA degradation products have been envisaged to be applied in medical field: tissue engineering, drug carriers, memory enhancers, antiosteoporosis, biodegradable implants. The PHA production and degradation cycle for 192 h has not been reported previously in literature.