Poly-beta-Hydroxybutyrate Accumulation as a Measure of Unbalanced Growth of the Estuarine Detrital Microbiota

The procaryotic endogenous storage material poly-beta-hydroxybutyrate (PHB) can be induced to accumulate in the estuarine detrital microbiota under conditions which suggest unbalanced growth, such as limitation of a critical factor(s) in the presence of carbon and energy sources. Changes in PHB-to-lipid phosphate ratios detected in field samples can be mimicked in the laboratory with common estuarine stresses. Acute anoxia or low pH induces conditions of no growth with depression of both the synthesis and catabolism of PHB without change in the lipid phosphate. Balanced growth induced by nutrients increases the lipid phosphate, depresses PHB synthesis, and stimulates PHB catabolism, resulting in a low ratio of PHB to lipid phosphate. Unbalanced growth induced to a small extent by high salinity or much more readily by dark upland runoff water results in rapid accumulation of PHB and slowing of PHB catabolism with little change in lipid phospate. Unbalanced growth conditions result in high PHB-to-lipid phosphate ratios in the detrital microbiota.     

Recovery of Poly-β-Hydroxybutyrate from Estuarine Microflora

Poly-β-hydroxybutyrate (PHB) is a uniquely procaryotic endogenous storage polymer whose metabolism has been shown to reflect environmental perturbations in laboratory monocultures. When hydrolyzed for 45 min in 5% sodium hypochlorite, PHB can be isolated from estuarine detrital microflora in high yield and purified free from non-PHB microbial components. Lyophilization of frozen estuarine samples shortens the exposure time to NaOCl necessary for maximal recovery. Lyophilized samples of hardwood leaves, Vallisneria, and the aerobic upper millimeter of estuarine muds yielded PHB. The efficiency of incorporation of sodium [1-¹⁴C]acetate into PHB is very high and is stimulated by aeration. PHB was not recovered from the anaerobic portions of sediments unless they were aerated for a short time. Levels of PHB in the detrital microbial community do not correlate with the microbial biomass as measured by the extractible lipid phosphate, suggesting that PHB-like eucaryotic endogenous storage materials may more accurately reflect the metabolic status of the population than its biomass.     

Recovery of Poly-beta-Hydroxybutyrate from Estuarine Microflora

Poly-beta-hydroxybutyrate (PHB) is a uniquely procaryotic endogenous storage polymer whose metabolism has been shown to reflect environmental perturbations in laboratory monocultures. When hydrolyzed for 45 min in 5% sodium hypochlorite, PHB can be isolated from estuarine detrital microflora in high yield and purified free from non-PHB microbial components. Lyophilization of frozen estuarine samples shortens the exposure time to NaOCl necessary for maximal recovery. Lyophilized samples of hardwood leaves, Vallisneria, and the aerobic upper millimeter of estuarine muds yielded PHB. The efficiency of incorporation of sodium [1-C]acetate into PHB is very high and is stimulated by aeration. PHB was not recovered from the anaerobic portions of sediments unless they were aerated for a short time. Levels of PHB in the detrital microbial community do not correlate with the microbial biomass as measured by the extractible lipid phosphate, suggesting that PHB-like eucaryotic endogenous storage materials may more accurately reflect the metabolic status of the population than its biomass.     

Effect of Light on Biomass and Community Structure of Estuarine Detrital Microbiota

Comparison of estuarine detrital microbiota grown with and without light in the absence of macroscopic grazing showed shifts in the community structure that enabled correlation between various biochemical measures. Analysis of these biochemical measures showed that growth in light induces the smallest increases in procaryotic attributes such as muramic acid; wall glucosamine; lipid phosphate; total extractable adenosine nucleotides; short-branched, cyclopropane, and cis-vaccenic fatty acids; lipid glucose and mannose; the incorporation of acetate into lipid; and the formation of deoxyribonucleic acid from thymidine. Measures of the microfauna such as lipid inositol and the γ-linolenic series of polyenoic fatty acids also increased minimally in the light-grown microbiota. Measures of sulfo-lipid synthesis, lipid glycerol, total extractable palmitate, 18-carbon polyenoic fatty acids, and total polyenoic fatty acids longer than 20 carbons increased 10- to 15-fold in algae and fungi. Chlorophyll a, lipid galactose, and the 16- and 20- carbon polyenoic fatty acids characteristic of diatoms increased maximally in the light. This increase of diatom measure correlated with the sheets of diatoms detected by scanning electron microscopy.     

Increased Bioplastic Production with an RNA Polymerase Sigma Factor SigE during Nitrogen Starvation in Synechocystis sp. PCC 6803

Because cyanobacteria directly harvest CO₂ and light energy, their carbon metabolism is important for both basic and applied sciences. Here, we show that overexpression of the sigma factor sigE in Synechocystis sp. PCC 6803 widely changes sugar catabolism and increases production of the biodegradable polyester polyhydroxybutyrate (PHB) during nitrogen starvation. sigE overexpression elevates the levels of proteins implicated in glycogen catabolism, the oxidative pentose phosphate pathway, and polyhydroxyalkanoate biosynthesis. PHB accumulation is enhanced by sigE overexpression under nitrogen-limited conditions, yet the molecular weights of PHBs synthesized by the parental glucose-tolerant and sigE overexpression strain are similar. Although gene expression induced by nitrogen starvation is changed and other metabolites (such as GDP-mannose and citrate) accumulate under sigE overexpression, genetic engineering of this sigma factor altered the metabolic pathway from glycogen to PHB during nitrogen starvation.     

Effect of phosphate supply and aeration on poly-β-hydroxybutyrate production in Azotobacter chroococcum

The effects of phosphate supply and aeration on cell growth and PHB accumulation were investigated in Azotobacter chroococcum 23 with the aim of increasing PHB production. Phosphate limitation favoured PHB formation in Azotobacter chroococcum 23, but inhibited growth. Azotobacter chroococcum 23 cells demonstrated intensive uptake of orthophosphate during exponential growth. At the highest phosphate concentration (1·5 g/litre) and low aeration the amount of intracellular orthophosphate/g residual biomass was highest. Under conditions of fed-batch fermentation the possibility of controlling the PHB production process by the phosphate level in the cultivation medium was demonstrated. A 36 h fed-batch fermentation resulted in a biomass yield of 110 g/litre with a PHB cellular concentration of 75% dry weight, PHB content 82·5 g/litre, PHB yield Y_P/S = 0·24 g/g and process productivity 2·29 g/litre·h.     

New insight into the role of the PhaP phasin of Ralstonia eutropha in promoting synthesis of polyhydroxybutyrate

Phasins are proteins that are proposed to play important roles in polyhydroxyalkanoate synthesis and granule formation. Here the phasin PhaP of Ralstonia eutropha has been analyzed with regard to its role in the synthesis of polyhydroxybutyrate (PHB). Purified recombinant PhaP, antibodies against PhaP, and an R. eutropha phaP deletion strain have been generated for this analysis. Studies with the phaP deletion strain show that PhaP must accumulate to high levels in order to play its normal role in PHB synthesis and that the accumulation of PhaP to low levels is functionally equivalent to the absence of PhaP. PhaP positively affects PHB synthesis under growth conditions which promote production of PHB to low, intermediate, or high levels. The levels of PhaP generally parallel levels of PHB in cells. The results are consistent with models whereby PhaP promotes PHB synthesis by regulating the surface/volume ratio of PHB granules or by interacting with polyhydroxyalkanoate synthase and indicate that PhaP plays an important role in PHB synthesis from the early stages in PHB production and across a range of growth conditions.     

Use of lipid composition and metabolism to examine structure and activity of estuarine detrital microflora

Earlier studies have shown that the activity of the estuarine detrital microflora measured by various enzyme activities, muramic acid and adenosine 5'-triphosphate (ATP) content, heterotrophic potentials, and respiratory activities correlates with the incorporation of C and P into the microbial lipids. In this study, these lipids were reproducibly fractionated into neutral lipid, glycolipid, and phospholipid classes. Distinct differences between the active microflora of oak leaves, sweet gum leaves, and pine needles were evidenced both in the rate of lipid synthesis and in the proportions of neutral lipids, glycolipids, and phospholipids. Successional changes in the microflora of leaves incubated in a semitropical estuary, previously suggested by ATP-to-muramic acid ratios and scanning electron micrography, were reflected in changes in the proportions of C in major lipid classes when analyzed from the same type of detritus. Short incubation times with C gave lipid compositions rich in phospholipids that are typical for the faster-growing bacterial populations; longer incubation with C gave lipid compositions richer in neutral and glycolipids, more characteristic of slower-growing eukaryotes or morphologically more complex prokaryotes. The metabolism of the lipids of the estuarine detrital microflora was examined by a pulse-chase experiment with C. Glycolipids lost C at a rate equal to the loss of C of the slow component of muramic acid. Individual phospholipids lost C from their backbone glycerol esters at different rates.     

Formation and utilisation of carbon reserves by Rhizobium

The accumulation and utilisation of poly-β-hydroxybutyrate (PHB) and extracellular polysaccharide (ECPS) have been studied in Rhizobium NZP 2037 and it was found that PHB synthesis was slow during growth whereas ECPS formation was not affected. PHB synthesis only was dependent on the pH of the medium. PHB and ECPS were both used as carbon sources in the absence of other exogenous carbon. ECPS when given to starved cells as sole carbon source in the absence of nitrogen was used for PHB synthesis by the organism. In the presence of nitrogen ECPS was found to support growth. The extracellular breakdown of ECPS was demonstrated and was accompanied by the release of its constituent glucose. This was favoured by low pH.     

Poly(3-Hydroxybutyrate) Synthesis by Recombinant Escherichia coli arcA Mutants in Microaerobiosis

We assessed the effects of different arcA mutations on poly(3-hydroxybutyrate) (PHB) synthesis in recombinant Escherichia coli strains carrying the pha synthesis genes from Azotobacter sp. strain FA8. The arcA mutations used were an internal deletion and the arcA2 allele, a leaky mutation for some of the characteristics of the Arc phenotype which confers high respiratory capacity. PHB synthesis was not detected in the wild-type strain in shaken flask cultures under low-oxygen conditions, while ArcA mutants gave rise to polymer accumulation of up to 24% of their cell dry weight. When grown under microaerobic conditions in a bioreactor, the arcA deletion mutant reached a PHB content of 27% ± 2%. Under the same conditions, higher biomass and PHB concentrations were observed for the strain bearing the arcA2 allele, resulting in a PHB content of 35% ± 3%. This strain grew in a simple medium at a specific growth rate of 0.69 ± 0.07 h⁻¹, whereas the deletion mutant needed several nutritional additives and showed a specific growth rate of 0.56 ± 0.06 h⁻¹. The results presented here suggest that arcA mutations could play a role in heterologous PHB synthesis in microaerobiosis.     

Effects of Grazing by Estuarine Gammaridean Amphipods on the Microbiota of Allochthonous Detritus

Estuarine gammaridean amphipods grazing at natural population density on detrital microbiota affected the microbial community composition, biomass, and metabolic activity without affecting the physical structure of the leaves. Total microbial biomass estimated by adenosine triphosphate and lipid phosphate or observed by scanning electron microscopy was greater on grazed than on ungrazed detritus. The rates of oxygen consumption, poly-β-hydroxybutyrate synthesis, total lipid biosynthesis, and release of ¹⁴CO₂ from radioactively prelabeled microbiota were higher on grazed than on ungrazed leaves, indicating stimulation of the metabolic activity of grazed detrital microbes. This was true with rates based either on the dry leaf weight or microbial biomass. Alkaline phosphatase activity was lower in the grazed system, consistent with enhanced inorganic phosphate cycling. The loss of ¹⁴C from both total lipid and poly-β-hydroxybutyrate of microorganisms prelabeled with ¹⁴C was greater from grazed than ungrazed microbes. There was a faster decrease in the ¹⁴C-glycolipid than in the ¹⁴C-neutral lipid or ¹⁴C-phospholipid fractions. Analysis of specific phospholipids showed losses of the metabolically stable [¹⁴C]glycerolphosphorylcholine derived from phosphatidylcholine and much more rapid metabolism of the bacterial lipid phosphatidylglycerol measured as [¹⁴C]glycerolphosphorylglycerol with amphipod grazing. The biochemical data supported scanning electron microscopy observations of a shift as the grazing proceeded from a bacterial/fungal community to one dominated by bacteria.     

Production of poly-3-hydroxybutyrate by Bacillus sp. JMa5 cultivated in molasses media

A strain of Bacillus sp. coded JMa5 was isolated from molasses contaminated soil. The strain was able to grow at a temperature as high as 45°C and in 250 g/l molasses although the optimal growth temperature was 35–37°C. Cell density reached 30 g/l 8 h after inoculation in a batch culture with an initial concentration of 210 g/l molasses. Under fed-batch conditions, the cells grew to a dry weight of 70 g/l after 30 h of fermentation. The strain accumulated 25–35%, (w/w) polyhydroxybutyrate (PHB) during fermentation. PHB accumulation was a growth-associated process. Factors that normally promote PHB production include high ratios of carbon to nitrogen, and carbon to phosphorus in growth media. Low dissolved oxygen supply resulted in sporulation, which reduced PHB contents and dry weights of the cells. It seems that sporulation induced by reduced supply of nutrients is the reason that PHB content is generally low in the Bacillus strain.     

Kinetic Studies and Biochemical Pathway Analysis of Anaerobic Poly-(R)-3-Hydroxybutyric Acid Synthesis in Escherichia coli

Poly-(R)-3-hydroxybutyric acid (PHB) was synthesized anaerobically in recombinant Escherichia coli. The host anaerobically accumulated PHB to more than 50% of its cell dry weight during cultivation in either growth or nongrowth medium. The maximum specific PHB production rate during growth-associated synthesis was approximately 2.3 ± 0.2 mmol of PHB/g of residual cell dry weight/h. The by-product secretion profiles differed significantly between the PHB-synthesizing strain and the control strain. PHB production decreased acetate accumulation for both growth and nongrowth-associated PHB synthesis. For instance under nongrowth cultivation, the PHB-synthesizing culture produced approximately 66% less acetate on a glucose yield basis as compared to a control culture. A theoretical biochemical network model was used to provide a rational basis to interpret the experimental results like the fermentation product secretion profiles and to study E. coli network capabilities under anaerobic conditions. For example, the maximum theoretical carbon yield for anaerobic PHB synthesis in E. coli is 0.8. The presented study is expected to be generally useful for analyzing, interpreting, and engineering cellular metabolisms.     

Nuclear Magnetic Resonance Studies of Poly(3-Hydroxybutyrate) and Polyphosphate Metabolism in Alcaligenes eutrophus

The metabolic pathways of poly(3-hydroxybutyrate) (PHB) and polyphosphate in the microorganism Alcaligenes eutrophus H16 were studied by ¹H, ¹³C, and ³¹P nuclear magnetic resonance (NMR) spectroscopy and by conventional analytical techniques. A. eutrophus cells accumulated two storage polymers of PHB and polyphosphate in the presence of carbon and phosphate sources under aerobic conditions after exhaustion of nitrogen sources. The solid-state cross-polarization/magic-angle spinning ¹³C NMR spectroscopy was used to study the biosynthetic pathways of PHB and other cellular biomass components from ¹³C-labeled acetate. The solid-state ¹³C NMR analysis of lyophilized intact cells grown on [1-¹³C]acetate indicated that the carbonyl carbon of acetate was selectively incorporated both into the carbonyl and methine carbons of PHB and into the carbonyl carbons of proteins. The ³¹P NMR analysis of A. eutrophus cells in suspension showed that the synthesis of intracellular polyphosphate was closely related to the synthesis of PHB. The roles of PHB and polyphosphate in the cells were studied under conditions of carbon, phosphorus, and nitrogen source starvation. Under both aerobic and anaerobic conditions PHB was degraded, whereas little polyphosphate was degraded. The rate of PHB degradation under anaerobic conditions was faster than that under aerobic conditions. Under anaerobic conditions, acetate and 3-hydroxybutyrate were produced as the major extracellular metabolites. The implications of this observation are discussed in connection with the regulation of PHB and polyphosphate metabolism in A. eutrophus.     

The ntrB and ntrC Genes Are Involved in the Regulation of Poly-3-Hydroxybutyrate Biosynthesis by Ammonia in Azospirillum brasilense Sp7

Azospirillum brasilense Sp7 and its ntrA (rpoN), ntrBC, and ntrC mutants have been evaluated for their capabilities of poly-3-hydroxybutyrate (PHB) accumulation in media with high and low ammonia concentrations. It was observed that the ntrBC and ntrC mutants can produce PHB in both low- and high-C/N-ratio media, while no significant PHB production was observed for the wild type or the ntrA mutant in low-C/N-ratio media. Further investigation by fermentation analysis indicated that the ntrBC and ntrC mutants were able to grow and accumulate PHB simultaneously in the presence of a high concentration of ammonia in the medium, while little PHB was produced in the wild type and ntrA (rpoN) mutant during active growth phase. These results provide the first genetic evidence that the ntrB and ntrC genes are involved in the regulation of PHB synthesis by ammonia in A. brasilense Sp7.     

Inorganic phosphate effect on alternate peripheral pathways of glucose catabolism in Pseudomonas cepacia

Abstract The effect of the inorganic phosphate concentration on the activity of the enzyme of alternate peripheral pathways of glucose catabolism was studied in Pseudomonas cepacia ATCC 17759. Growth with low glucose concentration (0.5% w/v) and 20 mM phosphate resulted in induced levels of the phosphorylative pathway enzymes when compared with the levels of these same enzymes in high glucose concentration (2% w/v). However, an expansion of the oxidative pathway was detected during growth with 0.5% (w/v) of glucose and high phosphate concentration (160 mM). Moreover, under high phosphate (160 mM) and high glucose (2% w/v) growth conditions, glucokinase activity was increased preferentially relative to levels of direct oxidative pathway enzymes.     

Effects of substrate biodegradability on the mass and activity of the associated estuarine microbiota

Multiple biochemical assays of microbial mass and activities were applied to the estuarine detrital microbiota colonizing morphologically similar polyvinyl chloride needles and needles from slash pine (Pinus elliottii). Biodegradable pine needles consistently showed 2- to 10-fold higher values of extractable adenosine 5'-triphosphate, rates of oxygen utilization, activities of alkaline phosphatase and phosphodiesterase, and the mucopeptide cell wall component muramic acid than did the polyvinyl chloride needles, during a 14-week incubation in a semitropical estuary. The higher activities by the microbiota of the biodegradable substrate correlated with estimates of the microbial density from scanning electron microscopy. The microbial community associated with the nondegradable substrate showed minimal activity of beta-d-galactosidase, beta-d-glucosidase, and alpha-d-mannosidase in contrast to the biota of the degradable substrate, which showed 10- to 100-fold higher activities of these glycoesterases. These enzymes logically could be involved in catabolism of the carbohydrate polymers of the detritus. Assuming equivalent rates of predation, a surface that is also a utilizable substrate supports a three- to fivefold more active microbial population.     

Overexpression of NAD kinase in recombinant <Emphasis Type="BoldItalic">Escherichia coli</Emphasis> harboring the <Emphasis Type="BoldItalic">phbCAB</Emphasis> operon improves poly(3-hydroxybutyrate) production

NAD kinase was overexpressed to enhance the accumulation of poly(3-hydroxybutyrate) (PHB) in recombinant Escherichia coli harboring PHB synthesis pathway via an accelerated supply of NADPH, which is one of the most crucial factors influencing PHB production. A high copy number expression plasmid pE76 led to a stronger NAD kinase activity than that brought about by the low copy number plasmid pELRY. Overexpressing NAD kinase in recombinant E. coli was found not to have a negative effect on cell growth in the absence of PHB synthesis. Shake flask experiments demonstrated that excess NAD kinase in E. coli harboring the PHB synthesis operon could increase the accumulation of PHB to 16–35 wt.% compared with the controls; meanwhile, NADP concentration was enhanced threefold to sixfold. Although the two NAD kinase overexpression recombinants exhibited large disparity on NAD kinase activity, their influence on cell growth and PHB accumulation was not proportional. Under the same growth conditions without process optimization, the NAD kinase-overexpressing recombinant produced 14 g/L PHB compared with 7 g/L produced by the control in a 28-h fermentor study. In addition, substrate to PHB yield Y _PHB/glucose showed an increase from 0.08 g PHB/g glucose for the control to 0.15 g PHB/g glucose for the NAD kinase-overexpressing strain, a 76% increase for the Y _PHB/glucose. These results clearly showed that the overexpression of NAD kinase could be used to enhance the PHB synthesis.