Single Carbon Substrates Research Articles

Phenotypic plasticity in fungi: a review with observations on Aureobasidium pullulans

Phenotypic plasticity in fungi is reviewed in the context of observations on phenotypic changes in the colony morphology of the fungus Aureobasidium pullulans. The variation in colony form is shown to depend on (i) the types of single carbon substrates (sugars and sugar alcohols) used in the growth medium, (ii) colony age, (iii) incubation temperature, (iv) light cycle and (v) substrate type. Expanding colonies grow in a developmental sequence that show synchronize growth phase shifts as well as unusual transitions from homogeneous to sectored, yeast to mycelial and giant to microcolonial growth forms. Epigenetic influences on phenotypic switches are suggested to be potential causes of form changes. The desirable properties of a model organism for studying phenotypic plasticity are discussed and past work on the yeast-mycelial transition of fungi is reviewed.

A novel Bacillus sp. accumulating poly (3-hydroxybutyrate-co-3-hydroxyvalerate) from a single carbon substrate

The objective of this paper was to report a bacterium designated as 88D, capable of producing poly (3-hydroxybutyrate-co-3-hydroxyvalerate) [P (3HB-co-3HV)] copolymer from a single carbon source, which was isolated from a municipal sewage treatment plant in Hyderabad, India. This microorganism, based on the phenotypical features and genotypic investigations, was identified as Bacillus sp. The optimal growth of Bacillus sp. 88D occurred between 28 and 30 degrees C and at pH 7. The strain yielded a maximum of 64.62% dry cell weight (DCW) polymer in the medium containing glucose as carbon source, which was followed by 60.46% DCW polymer in glycerol containing medium. Bacillus sp. 88D produced P (3HB-co-3HV) from glucose or glycerol, when they were used as a single carbon substrate. This bacterium produced polyhydrxybutyrate (PHB) when sodium acetate was used as sole carbon substrate. The viscosity average molecular mass (Mv) of the copolymers ranged from 523 to 627 kDa. The physical, chemical and mechanical properties of the biopolymers were characterized.

Production of PHB and P (3HB-co-3HV) biopolymers by Bacillus megaterium strain OU303A isolated from municipal sewage sludge

Bacillus megaterium strain OU303A isolated from municipal sewage sludge was selected for the study of biosynthesis of polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-hydroxyvalerate P (HB-co-HV) copolymer. The strain yielded a maximum of 62.43% DCW polymer in the medium containing glycerol as carbon source, which was followed by 58.63% DCW polymer in glucose containing medium. We found that this strain was capable of producing 2.5% hydroxyvalerate copolymer from a single carbon substrate, glucose. The strain showed an increase in the amount of HV monomer content, when the precursor for the copolymer was included in the fermentation medium. The characterization of the biopolymers was carried out using FTIR, GC-MS, H1 NMR and DSC. This is the first report of B. megaterium strain producing HV copolymer, without the addition of any precursor in the fermentation medium.

The effect of glycerol as a sole and secondary substrate on the growth and fatty acid composition of Rhodotorula glutinis

Rhodotorula glutinis is a yeast that produces copious quantities of lipids in the form of triacylglycerols (TAG) and can be used to make biodiesel via a transesterification process. The ester bonds in the TAG are broken leaving behind two products: fatty acid methyl esters and glycerol that could provide an inexpensive carbon source to grow oleaginous yeast R. glutinis. Described here are the effects of different growth substrates on TAG accumulation and fatty acids produced by R. glutinis. Yeast cultured 24 h on medium containing dextrose, xylose, glycerol, dextrose and xylose, xylose and glycerol, or dextrose and glycerol accumulated 16, 12, 25, 10, 21, and 34% TAG on a dry weight basis, respectively. Lipids were extracted from R. glutinis culture and transesterified to form fatty acid methyl esters. The results show a difference in the degree of saturation for the carbon sources tested. Cells cultivated on glycerol alone had the highest degree of unsaturated fatty acids at 53% while xylose had the lowest at 25%. R. glutinis can be cultivated on all sugars tested as single carbon substrates or in mixtures. Glycerol may be used as secondary or primary carbon substrate.

Mixed carbon sources for nitrate reduction in activated sludge-identification of bacteria and process activity studies

Mixtures of methanol and acetate as carbon source were investigated in order to determine their capacity to enhance denitrification and for analysis of the microbial composition and carbon degradation activity in activated sludge from wastewater treatment plants. Laboratory batch reactors at 20 °C were used for nitrate uptake rate (NUR) measurements in order to investigate the anoxic activity, while single and mixed carbon substrates were added to activated sludge. Microautoradiography (MAR) in combination with fluorescence in situ hybridisation (FISH) were applied for microbial analysis during exposure to different carbon sources. The NUR increased with additions of a mixture of acetate and methanol compared with additions of a single carbon source. MAR–FISH measurements demonstrated that the probe-defined group of Azoarcus was the main group of bacteria utilising acetate and the only active group utilising methanol under anoxic conditions. The present study indicated an improved denitrification potential by additions of a mixed carbon source compared with commonly used single-carbon additions. It is also established that Azoarcus bacteria are involved in the degradation of both acetate and methanol in the anoxic activated sludge.

A novel mcl PHA-producing bacterium, Pseudomonas guezennei sp. nov., isolated from a ‘kopara’ mat located in Rangiroa, an atoll of French Polynesia

The aim of the present study was to describe an aerobic, mesophilic and heterotrophic bacterium, designated RA26, able to produce a medium-chain-length polyhydroxyalkanoate (PHA). It was isolated from a French Polynesian bacterial mat located in the atoll of Rangiroa. This micro-organism, on the basis of the phenotypical features and genotypic investigations can be clearly assigned to the Pseudomonas genus and the name of Pseudomonas guezennei is proposed. Optimal growth occurs between 33 and 37 degrees C, at a pH between 6.4 and 7.1 and at ionic strength of 15 g l(-1) of sea salts. The G+C content of DNA is 63.2%. Under laboratory conditions, this bacterium produced a novel, medium-chain-length PHA, mainly composed of 3-hydroxydecanaote (64 mol.%) and 3-hydroxyoctanoate (24 mol.%) (GC-MS, NMR) from a single nonrelated carbon substrate, i.e. glucose. The bacterium P. guezennei produces a novel PHA mcl with elastomeric properties. PHAs share physical and material properties that recommend them for application in various areas, and are considered as an alternative to nonbiodegradable plastics produced from fossil oils. In this study, we describe a new bacteria with the capability to synthesize a novel PHA with promising biotechnological applications.

Carbon substrate utilization, antibiotic sensitivity, and numerical taxonomy of bacterial isolates from the Great Salt Plains of Oklahoma

The current work extends the phenotypic characterization of a bacterial culture collection from the Great Salt Plains of Oklahoma. This barren expanse of mud flats is typically crusted with thalassohaline salt evaporites. The initial account of the aerobic heterotrophic bacteria from the Great Salt Plains described 105 halotolerant isolates that represented 47 phylotypes. Extensive phenotypic analyses were performed on 76 isolates representing 37 unique phylotypes. The current report extends these observations for 60 of the isolates by measuring a wider set of phenotypic characteristics. Utilization patterns for 45 carbon substrates were used to assign the isolates into seven coherent phenons, along with several singletons and a group of isolates that did not grow on single carbon substrates. Most of the isolates were able to utilize nearly all of the nitrogen sources tested, with nitrate being the least utilized. Little antibiotic resistance was seen in the collection as a whole; however, certain phenons were enriched for antibiotic-resistant organisms. A total of 81 phenotypic characteristics were used to generate dendrograms. The numerical taxonomy trees essentially agreed with those generated using 16S rRNA gene sequences. The pattern of carbon substrate utilization showed substantial changes at different salinities that may have relevance to the variable salinities microbes experience at the Salt Plains over time.

Poly-beta-hydroxybutyrate biosynthesis in the facultative methylotroph methylobacterium extorquens AM1: identification and mutation of gap11, gap20, and phaR.

Methylobacterium extorquens AM1, a serine cycle facultative methylotroph, accumulates poly-beta-hydroxybutyrate (PHB) as a carbon and energy reserve material during growth on both multicarbon- and single-carbon substrates. Recently, the identification and mutation of the genes involved in the biosynthesis and degradation of PHB have been described for this bacterium, demonstrating that two of the genes of the PHB cycle (phaA and phaB) are also involved in C(1) and C(2) metabolism, as part of a novel pathway for glyoxylate regeneration in the serine cycle (N. Korotkova and M. E. Lidstrom, J. Bacteriol. 183:1038-1046, 2001; N. Korotkova, L. Chistoserdova, V. Kuksa, and M. E. Lidstrom, J. Bacteriol. 184:1750-1758, 2002). In this work, three new genes involved in PHB biosynthesis in this bacterium have been investigated via mutation and phenotypic analysis: gap11, gap20, and phaR. We demonstrate that gap11 and gap20 encode two major granule-associated proteins (phasins) and that mutants with mutations in these genes are defective in PHB production and also in growth on C(2) compounds, while they show wild-type growth characteristics on C(1) or multicarbon compounds. The phaR mutant shows defects in both PHB accumulation and growth characteristics when grown on C(1) compounds and has defects in PHB accumulation but grows normally on C(3) and C(4) compounds, while both PHB accumulation and growth rate are at wild-type levels during growth on C(2) compounds. Our results suggest that this phenotype is due to altered fluxes of acetyl coenzyme A (CoA), a major intermediate in C(1), C(2), and heterotrophic metabolism in M. extorquens AM1, as well as the entry metabolite for the PHB cycle. Therefore, it seems likely that PhaR acts to control acetyl-CoA flux to PHB in this methylotrophic bacterium.

Genetic variability of the postharvest pathogen Gilbertella persicaria: identification of randomly amplified polymorphic DNA (RAPD) markers correlating with (+) and (-) mating types.

Random amplified polymorphic DNA (RAPD) and isoenzyme polymorphisms among 16 isolates of the postharvest pathogen Gilbertella persicaria were examined. Six different 10-bp primers were used to determine the extent of intraspecific genetic variability. Nine composite amplification types were identified. RAPD markers were obtained which correlated with the mating types of the G. persicaria isolates. The variability of the isoenzyme patterns was very low and no correlation was found between the isoenzyme markers and the mating abilities. When 80 single carbon substrates were tested in utilization assays, most of them were utilized uniformly by the 16 G. persicaria strains. However, some compounds elicited differences between the isolates representing the two mating types. Beta-alanine (0.2%) has little effect on the germination of the sporangiospores of the (+) isolates, but inhibited the germination of (-) sporangiospores. Glycerol-1-monoacetate supported the growth of both mating types, but at concentrations higher than 4% this was accompanied with a compact (colonial) growth for plus mating type isolates only.

Accumulation of poly[(R)-3-hydroxyalkanoates] inPseudomonas oleovorans during growth in batch and chemostat culture with different carbon sources

Pseudomonas oleovorans (ATCC 29347) was grown in batch and chemostat cultures with citrate, hexanoate, heptanoate, octanoate, and nonanoate as single carbon substrates. The growth medium for batch cultures was adjusted such that nitrogen (NH(4)(+)) limitation terminated the exponential-growth phase. During batch cultivation with octanoate or nonanoate the biomass continued to increase after depletion of ammonium due to the accumulation of medium-chain-length poly[(R)-3-hydroxyalkanoates] (mcl-PHAs). Additionally, a significant rate of mcl-PHA accumulation was also observed in the exponential-growth phase of batch cultures. It is well known that the accumulation of reserve materials is strongly dependent on the ratio of nutrients (here of carbon, C, and of nitrogen, N) and that in a batch culture the ratio of C:N is continuously changing. Therefore, we have also investigated the effect of defined ratios of C:N under constant cultivation conditions, namely at a fixed dilution rate (D) in a chemostat fed with different medium C:N ratios. These experiments were performed at a constant D of 0.2 h(-1). The concentration of the nitrogen source in the inflowing medium (N()) was kept constant, while its carbon concentration (C()) was increased stepwise, resulting in an increase of the medium carbon to nitrogen ratio (C()/N() ratio). The culture parameters and the cell composition of steady-state cultures were determined as a function of the C()/N() ratio in the feed medium. Mcl-PHA accumulation was detected during growth with the fatty acids, and three distinct regimes of growth limitation were discovered: In addition to carbon limitation at low, and nitrogen limitation at high C()/N() ratios, an intermediate growth regime of simultaneous limitation by carbon and nitrogen was detected where both substrates were used to completion. The width of this dual-nutrient-limited growth regime was dependent on the change in the yield factors for carbon and nitrogen (Y(X/C), Y(X/N)) measured during single-nutrient-limited growth.

Evaluation of cell activity and of methods for the cultivation of bacteria from a natural lake community

The viability and culturability of chemoheterotrophic bacteria in Zwischenahner Meer, a shallow eutrophic lake in Northern Germany, were assessed by different techniques. The fraction of metabolically active cells was measured by the CTC reduction method. At three sampling times, numbers of CTC-reducing cells represented only a minor fraction (≤8%) of the total cell count. Only a slight stimulation of CTC reduction was observed upon the addition of single carbon substrates or substrate mixtures. A medium mimicking the low natural ion concentrations in freshwater was devised and used for the cultivation of bacteria in most probable number (MPN) series and on membrane filters. Similar to the number of CTC-reducing cells, the number of culturable cells as determined in MPN series never exceeded 7% of the total cell count with any of the carbon substrates tested. This indicated that the type of carbon substrate was not the major determinant of the rather low culturability. However, we observed a significant inhibition of bacterial growth in MPN series in which the typical R2A medium was used, or when phosphate buffer instead of HEPES was employed. In contrast to the low numbers of CTC-reducing cells and the low MPN values, up to 58% of the bacterial cells could divide on membrane filters incubated on top of the same type of medium that had been used in the MPN series. It is concluded that most of the bacteria in Zwischenahner Meer are able to grow and divide, but do not reach high cell densities even in improved culture media. Therefore, the majority of bacteria may be classified as viable but nonculturable, but would not be detected by the CTC reduction method.

Evaluation of cell activity and of methods for the cultivation of bacteria from a natural lake community

The viability and culturability of chemoheterotrophic bacteria in Zwischenahner Meer, a shallow eutrophic lake in Northern Germany, were assessed by different techniques. The fraction of metabolically active cells was measured by the CTC reduction method. At three sampling times, numbers of CTC-reducing cells represented only a minor fraction (≤8%) of the total cell count. Only a slight stimulation of CTC reduction was observed upon the addition of single carbon substrates or substrate mixtures. A medium mimicking the low natural ion concentrations in freshwater was devised and used for the cultivation of bacteria in most probable number (MPN) series and on membrane filters. Similar to the number of CTC-reducing cells, the number of culturable cells as determined in MPN series never exceeded 7% of the total cell count with any of the carbon substrates tested. This indicated that the type of carbon substrate was not the major determinant of the rather low culturability. However, we observed a significant inhibition of bacterial growth in MPN series in which the typical R2A medium was used, or when phosphate buffer instead of HEPES was employed. In contrast to the low numbers of CTC-reducing cells and the low MPN values, up to 58% of the bacterial cells could divide on membrane filters incubated on top of the same type of medium that had been used in the MPN series. It is concluded that most of the bacteria in Zwischenahner Meer are able to grow and divide, but do not reach high cell densities even in improved culture media. Therefore, the majority of bacteria may be classified as viable but nonculturable, but would not be detected by the CTC reduction method.

Metabolic activity of bacterioplankton communities assessed by flow cytometry and single carbon substrate utilization

Metabolic activity of bacterioplankton communities assessed by flow cytometry and single carbon substrate utilization

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by newly isolated Agrobacterium sp. SH-1 and GW-014 from structurally unrelated single carbon substrates

Various isolates from activated sludge and soil samples were investigated for their ability to synthesize poly(3-hydroxybutyrate-co-3-hydroxyvalerate), P(3HB-co-3HV), from structurally unrelated single carbon substrates. Isolated Gram-negative bacteria, Agrobacterium sp. SH-1 and GW-014, could synthesize P(3HB-co-3HV) with 3HV monomer mole percent in the range from 1.3 to 11.4 from single carbon substrate, such as glucose, xylose or sucrose. Terpolyesters of 3HB, 3HV and 4-hydroxybutyrate (4HB) were also accumulated by Agrobacterium sp. SH-1 and GW-014 in a nitrogen-free production stage with various hydroxyalkanoate substrates. The structure of P(3HB-co-3HV) synthesized from glucose was confirmed by gas chromatographicmass spectrometric (GC-MS) analysis, and its molecular weight, polydispersity index and melting temperature were determined to be 620000, 1.2 and 178°C, respectively. Total polymer content up to 78% (w/w) of dry cell weight and 3HV mole percent up to 50% could be achieved by fed-batch production from glucose and propionate.

Biodegradation of chlorophenol mixtures by Pseudomonas putida

The dynamic growth behavior of Pseudomonas putida has been studied when resting calls were inoculated into a growth medium containing inhibitory concentrations of mixtures of phenol and monochlorophenols. Resting cells inoculated into single carbon substrate media did not demonstrate enhanced cell lysis by any of the phenol substrates. The apprarent death rate was reduced as the concentrations of phenol or chlorophenols were increased. This behavior was modeled by employing a constant specific death rate (k(d) = 0.0075 h(-1)) and assuming all organic species result in a lag-phase, specific growth rate which may be larger or smaller than k(d). Logarithmic biomass growth on pure monochlorophenols did not occur within 2 weeks after inoculation. Logarithmic growth phases were only observed when the monochlorophenols were cometabolized with phenol. The delay time over which the lag phase exists increased exponentially with phenol concentration and linearly with monochlorophenol concentration. The log growth yield coefficient decreased linearly with monochlorophenol concentration.The lag-phase, specific growth rate was found to decrease exponentially with the concentration of monochlorophenols. This resulted in a 50% lag growth rate inhibition for both 3- and 4-chlorophenol of 9 ppm and for 2-chlorophenol of only 2 ppm. The new, empirical correlations are shown to closely model the complete lag and log growth behavior ot P. putida on phenol and chlorophenol mixtures.

Catabolic enzymes of the acetogen Butyribacterium methylotrophicum grown on single-carbon substrates.

When grown on formate, formate-CO, and methanol-CO, Butyribacterium methylotrophicum contained high levels of tetrahydrofolate (H4folate) and required enzymes, carbon monoxide dehydrogenase, formate dehydrogenase, and hydrogenase. The activities of methylene-H4folate reductase were comparable to other H4 folate activities (which ranged from 0.55 to 9.28 mumol/min per mg of protein) when measured by an improved procedure. The H4folate activities in formate-grown cells were twice those found in formate-CO-grown cells. This result correlated with a growth yield on formate that was one-half that on formate-CO. The stoichiometry of the formyl-H4folate synthetase reaction was 1 mol of ATP per 1 mol of formate. The methylene-H4folate dehydrogenase was NAD+ dependent. We conclude that B. methylotrophicum utilizes these enzymes in homoacetogenic catabolism.

Simultaneous production of inulase and lactase in batch and continuous cultures of Kluyveromyces fragilis

Under different induction conditions, the industrial yeast Kluyveromyces fragilis is an excellent producer of the enzymes inulase (β- d-fructofuranoside fructohydrolase, EC 3.2.1.26) and lactase (β- d-galactoside galactohydrolase, EC 3.2.1.23), producing 27 and 1.6 U mg −1 dry cell weight, respectively. In order to improve overall enzyme yields, conditions for the simultaneous production of both enzymes in a one-stage fermentation have been examined. Techniques employed include carbon-limited batch and continuous culture, single and mixed carbon substrates, and the use of a mutant semi-constitutive for inulase production. Synthesis of both enzymes suffered strongly from carbon catabolite repression in batch cultures grown on single and mixed inducing substrates. Only glycerol and dl-malate did not repress either enzyme. The non-metabolizable analogues of lactose, isopropyl-β- d-thiogalactoside and methyl-β- d-thiogalactoside induced lactase in glycerol grown batch cultures, but were ineffective in sucrose grown continuous cultures. They also depressed the normally high levels of inulase in such continuous cultures. The highest simultaneous inulase and lactase activities in the wild-type yeast were obtained in continuous culture on an equal mixture of d-fructose and d-galactose; 25 and 0.78 U mg −1 dry cell weight, respectively. In this fermentation the combined yield per unit carbon substrate of the two enzymes was 141%, compared to a reference value of 100% for the highest yield of each enzyme in separate fermentations. On the same mixture of d-fructose and d-galactose, the mutant produced ∼60 and 0.70 U mg −1 dry cell weight, respectively. The combined enzyme yield per unit of carbon substrate was 172%.

Single-carbon catabolism in acetogens: analysis of carbon flow in Acetobacterium woodii and Butyribacterium methylotrophicum by fermentation and 13C nuclear magnetic resonance measurement

The catabolism of methanol, formate, or carbon monoxide to acetate or butyrate or both was examined in two acetogenic bacteria. Butyribacterium methylotrophicum simultaneously transformed methanol and formate mainly to butyrate with concomitant H2 and CO2 production and consumption. In contrast, methanol plus CO was primarily converted to acetate, and only slight amounts of CO2 were produced. In vivo 13C nuclear magnetic resonance analysis of [13C]methanol transformation by B. methylotrophicum indicated that methanol was predominantly incorporated into the methyl of acetate. 13CO2 was produced and then consumed, and butyrate was formed from the condensation of two acetate precursors. The analysis of the position of acetate labeled by a given 13C single-carbon substrate when B. methylotrophicum or Acetobacterium woodii was grown in the presence of a second one-carbon substrate indicated two trends: when methanol was consumed, CO, CO2, or formate predominantly labeled the acetate carboxyl; when CO was consumed, CO2 and formate were principally funneled into the acetate methyl group, and CO remained a better carboxyl precursor. These data suggest a model of acetate synthesis via the combined operation of two readily reversible single-carbon pathways which are linked by CO2.

Nutritional growth requirements forButyribacterium methylotrophicum on single carbon substrates and glucose

Butyribacterium methylotrophicum, an anaerobic acetogen, obligately required pantothenate for growth on either glucose, CH3OH−CO2, H2−CO2, or carbon monoxide. Growth on glucose but not single carbon substrates was stimulated by lipoate and biotin. Sulfide but not sulfate served as the sole sulfur source for growth. This study established thatB. methylotrophicum was both a true autotroph when grown on H2−CO2 and a unicarbonotroph on CO as the sole carbon and energy source. In addition, the vitamin requirements of this species further suggest its distinctiveness fromEubacterium limosum (Butyribacterium rettgeri).