Distribution and Selection of Poly-3-Hydroxybutyrate Production Capacity in Methanotrophic Proteobacteria

Methanotrophs are known to produce poly-3-hydroxybutyrate (PHB), but there is conflicting evidence in the literature as to which genera produce the polymer. We screened type I and II proteobacterial methanotrophs that use the ribulose monophosphate and serine pathways for carbon assimilation, respectively, for both phaC, which encodes for PHB synthase, and the ability to produce PHB under nitrogen-limited conditions. Twelve strains from six different genera were evaluated. All type I strains tested negative for phaC and PHB production; all Type II strains tested positive for phaC and PHB production. In order to identify conditions that favor PHB production, we also evaluated a range of selection conditions using a diverse activated sludge inoculum. Use of medium typically recommended for methanotroph enrichment led to enrichments dominated by type I methanotrophs. Conditions that were selected for enrichments dominated by PHB-producing Type II methanotrophs were: (1) use of nitrogen gas as the sole nitrogen source in the absence of copper, (2) use of a dilute mineral salts media in the absence of copper, and (3) use of media prepared at pH values of 4–5.     

Intracellular PHB conversion in a Type II methanotroph studied by 13C NMR

Poly-β-hydroxybutyrate (PHB) formation under aerobic conditions via incorporation of [¹³C-2]acetate as a cosubstrate and its intracellular degradation under anaerobic conditions in a Type II methanotroph was studied by ¹³C NMR. During PHB synthesis in the presence of labelled acetate, low levels of β-hydroxybutyrate, butyrate, acetone, isopropanol, 2,3-butanediol and succinate were observed. Subsequent anaerobic PHB breakdown showed enhanced levels of these products at the expense of PHB. Fermentative metabolism occurring during anaerobic PHB degradation was confirmed in experiments with fully ¹³C-enriched cells, which were grown on ¹³C-labelled methane. β-hydroxybutyrate, butyrate, acetate, acetone, isopropanol, 2,3-butanediol and succinate were detected as multiple ¹³C-labelled compounds in the culture medium. Our results suggest that intracellular PHB degradation can be used as a reserve energy source by methanotrophs under anoxic conditions. Journal of Industrial Microbiology & Biotechnology (2001) 26, 15–21.     

Periodic change in DO concentration for efficient poly-β-hydroxy-butyrate production using temperature-inducible recombinant<Emphasis Type="Italic">Escherichia coli</Emphasis> with proteome analysis

RecombinantEscherichia coli strain harboring the λp _R-p _L promotor and heterologus poly-β-hydroxybutyrate (PHB) biosynthesis genes was used to investigate the effect of culture conditions on the efficient PHB production. The expression ofphb genes was induced by a temperature upshift from 33°C to 38°C. The protein expression levels were measured by using two-dimensional electrophoresis, and the enzyme activities were also measured to understand the effect of culture temperature, carbon sources, and the dissolved oxygen (DO) concentration on the metabolic regulations. AcetylCoA is an important branch point for PHB production. The decrease in DO concentration lowers the citrate synthase activity, thus limit the flux toward the TCA cycle, and increase the flux for PHB production. Since NADPH is required for PHB production, the PHB production does not continue leading the overproduction of acetate and lactate. Based on these observations, a new operation was considered where DO concentration was changed periodically, and it was verified its usefulness for the efficient PHB production by experiments.     

nifH Sequences and Nitrogen Fixation in Type I and Type II Methanotrophs

Some methane-oxidizing bacteria (methanotrophs) are known to be capable of expressing nitrogenase and utilizing N₂ as a nitrogen source. However, no sequences are available for nif genes in these strains, and the known nitrogen-fixing methanotrophs are confined mainly to a few genera. The purpose of this work was to assess the nitrogen-fixing capabilities of a variety of methanotroph strains. nifH gene fragments from four type I methanotrophs and seven type II methanotrophs were PCR amplified and sequenced. Nitrogenase activity was confirmed in selected type I and type II strains by acetylene reduction. Activities ranged from 0.4 to 3.3 nmol/min/mg of protein. Sequence analysis shows that the nifH sequences from the type I and type II strains cluster with nifH sequences from other gamma proteobacteria and alpha proteobacteria, respectively. The translated nifH sequences from three Methylomonas strains show high identity (95 to 99%) to several published translated environmental nifH sequences PCR amplified from rice roots and a freshwater lake. The translated nifH sequences from the type II strains show high identity (94 to 99%) to published translated nifH sequences from a variety of environments, including rice roots, a freshwater lake, an oligotrophic ocean, and forest soil. These results provide evidence for nitrogen fixation in a broad range of methanotrophs and suggest that nitrogen-fixing methanotrophs may be widespread and important in the nitrogen cycling of many environments.     

Mass production of poly-β-hydroxybutyric acid by fully automatic fed-batch culture of methylotroph

Summary Fifty-one methylotrophs were checked with respect to their ability of poly--hydroxybutyric acid (PHB) production from methanol. One of them, Pseudomonas sp. K, was chosen from its good growth on a minimum synthetic medium. Optimal temperature and pH for its growth were 30° C and 7.0, respectively. Concentrations of PO ₄ ^3- and NH ₄ ⁺ in the medium should be kept at low levels. PHB formation was stimulated by deficiency of nutrient such as NH ₄ ⁺ , SO ₄ ^2- , Mg²⁺, Fe²⁺ or Mn²⁺. Among them, nitrogen deficiency was chosen from its effectiveness and easiness for PHB accumulation.The microorganism was cultivated to produce a large amount of poly--hydroxybutyric acid (PHB) from methanol by means of microcomputer-aided fully automatic fed-batch culture technique. During the cultivation, temperature, dissolved oxygen concentration (DO), and methanol concentration in the culture broth were maintained at 30° C 2.5±0.5 ppm and 0.5±0.2 g/l, respectively. Other nutrients, nitrogen source and mineral ions, were also controlled to maintain their initial concentrations in the medium during cell growth phase. When the high cell concentration was achieved (160 g/l), feedings of ammonia and minerals were stopped and only methanol was supplied successively to accumulate PHB. At 175 h, high concentration of PHB (136 g/l) was obtained and total cell concentration became 206 g/l. DO must be maintained above the critical level during the PHB formation phase, too. PHB yield from methanol (g PHB/g methanol) was 0.18 and the maximum PHB content reached 66% of dry weight. Solid PHB produced by the strain had the melting point of 176° C and the average molecular weight of 3.0x10⁵.     

Growth characteristics of non-heterocystous cyanobacterium Plectonema boryanum with N2 as nitrogen source

Strains of filamentous, non-heterocystous cyanobacteria from the Pasteur Culture Collection (PCC), able to synthesize nitrogenase under anaerobic test conditions, were tested for growth with N₂ as sole nitrogen source at low O₂ partial pressure (less than 0.05%). Plectonema boryanum (PCC 73110) exhibited exponential growth under these conditions. This capacity was restricted to light intensities not exceeding 500 lux. Growth rates were 0.014/h at 200 and 0.023 at 500 lux and similar to those of anaerobic and aerobic control cultures with nitrate as N-source. For N₂-fixing cultures incubated at 200 and 500 lux, acetylene reduction rates were 4–8 and 5–14 nmol C₂H₄ per mg protein per min, respectively. The ratio of phycocyanine to chlorophyll was higher (200 lux) or slightly reduced (500 lux) in N₂-fixing cultures as compared to control cultures with nitrate as N-source. On the basis of epifluorescence microscopy and microfluorimetry, no differences in pigment contents were found between individual cells or filaments of N₂-fixing cultures. Also no noteworthy differences were observed between the pycobiliprotein composition of individual cells in N₂ fixing cultures as compared to nitrate-grown controls. Thus the observed exponential growth of P. boryanum at low light intensities implies simultaneous nitrogen fixation and oxygenic photosynthesis. Additional continuous culture experiments showed that N₂-fixing exponential growth was dependent on O₂ partial pressures lower than 0.2–0.4%.The other strains tested (PCC 6412, 6602, 7403, 7104) did not grow under such conditions.Abbreviations Chl chlorophyll - PBP phycobiliproteins - PC phycocyanin - PCC Pasteur Culture Collection - OD optical density     

Identification and physiological characterization of the nitrogen fixing bacterium Corynebacterium autotrophicum GZ 29

The coryneform hydrogen bacterium strain GZ 29, assigned to Corynebacterium autotrophicum fixed molecular nitrogen under autotrophic (H₂, CO₂) as well as under heterotrophic (sucrose) conditions. Physiological parameters of nitrogen fixation were measured under heterotrophic conditions. The optimal dissolved oxygen concentration for cells grown in a fermenter with N₂ was rather low (0.14 mg O₂/l) compared with cells grown in the presence of NH ₄ ⁺ (4.45 mg O₂/l). C. autotrophicum GZ 29 had a doubling time of 3.7 h at 30°C with N₂ as N-source and sucrose as carbon source and at optimal pO₂. Acetylene reduction reached values of 12 nmoles of ethylene produced/minxmg protein. Although the oxygen concentration in the growing culture was kept constant, the optimal dissolved oxygen tension for the acetylene reduction assay shifted to higher pO₂-values. The overall efficiency of nitrogen fixation amounted to 22 mg N fixed/g sucrose consumed; it reached a maximal value of 65 mg N fixed/g sucrose consumed at the beginning of the exponential growth phase. Intact cells reduced acetylene even under anaerobic test conditions; further anaerobic metabolic activity could not be ascertained so far.     

Nitrate removal and DO levels in batch wetland mesocosms: Cattail (Typha spp.) versus bulrush (Scirpus spp.)

Nitrate removal rates and dissolved oxygen (DO) levels were evaluated in small batch-mode wetland mesocosms with two different plant species, cattail (Typha spp.) and bulrush (Scirpus spp.), and associated mineral-dominated sediment collected from a mature treatment wetland. Nitrate loss in both cattail and bulrush mesocosms was first-order in nature. First-order volumetric rate constants (k_V) were 0.30 d⁻¹ for cattail and 0.21 d⁻¹ for bulrush and rates of nitrate loss were significantly different between plant treatments (p < 0.005). On an areal basis, maximum rates of nitrate removal were around 500 mg N/(m² d) early in the experiment when nitrate levels were high (> 15 mg N/L). Areal removal rates were on average 25% higher in cattail versus bulrush mesocosms. DO in mesocosm water was significantly higher in bulrush versus cattail (p < 0.001). DO in bulrush generally ranged between 0.5 and 2 mg/L, while DO in cattail mesocosms was consistently below 0.3 mg/L. Based on cumulative frequency analysis, DO exceeded 1 mg/L around 50% of the time in bulrush, but only 2% of the time in cattail. DO in bulrush exhibited a statistically significant diel cycle with DO peaks in the late afternoon and DO minimums in the early morning hours. Difference in nitrate removal rates between wetland plant treatments may have been due to differing plant carbon quality. Cattail litter, which has been shown in other studies to exhibit superior biodegradability, may have enhanced biological denitrification by fueling heterotrophic microbial activity, which in turn may have depressed DO levels, a prerequisite for denitrification. Our results show that the cattail is more effective than bulrush for treating nitrate-dominant wastewaters.     

Nitrogen metabolism in a new obligate methanotroph, 'Methylosinus' strain 6

A new obligate methanotroph was isolated and characterized. It was classified as a 'Methylosinus' species and named 'Methylosinus' sp. strain 6. Nitrogen metabolism in 'Methylosinus' 6 was found to be similar to other Type II methanotrophs, including the assimilation of nitrogen exclusively by the glutamine synthetase/glutamate synthase system. However, unlike other Type II methanotrophs, it appeared that glutamine synthetase activity was regulated by adenylylation in this organism. 'Methylosinus' 6 was grown in continuous culture with either dinitrogen or nitrate as sole nitrogen source under various dissolved oxygen tensions. Higher rates of methane oxidation and a more developed intracytoplasmic membrane system were found at lower oxygen tensions with nitrate as the nitrogen source but at higher oxygen tensions with dinitrogen as the nitrogen source. This suggested that carbon metabolism was influenced by nitrogen metabolism in this organism.     

Microcallus growth from maize protoplasts

Maize (Zea mays L.) protoplasts obtained from Type I and Type II calli from several genotypes were shown to be capable of synthesizing cell walls and forming small clusters of cells. The medium used also supported cluster formation from protoplasts obtained from root tips. The effects of various additions to the medium (such as casein hydrolysate, coconut water, amino acids, sugars, phytohormones, nitrate, calcium, and dimethylsulfoxide as well as pH variations on cellcluster formation were determined. The method of culture (protoplasts plated in agarose or supported in alginate beads in liquid medium) as well as several components of the medium were found to be critical for microcallus formation. Protoplasts obtained from embryogenic Type I callus and cultured in the medium of C. Nitsch and J.P. Nitsch (1967, Planta 72, 355–370) modified by various additions (NN 67-mod medium) were affected most by various sugars, casein hydrolysate, coconut water, and a combination of the auxins napthalene-1-acetic acid (2 mg/l) and 2,4-dichlorophenoxyacetic acid (0.1 mg/l), and the cytokinin N⁶-benzylaminopurine (0.5 mg/l). Cluster size in the agarose culture system was from 0.1 to 0.5 mm diameter and in the alginate culture system, up to 2.0 mm diameter.     

Rice roots select for type I methanotrophs in rice field soil

Methanotrophs are an important regulator for reducing methane (CH₄) emissions from rice field soils. The type I group of the proteobacterial methanotrophs are generally favored at low CH₄ concentration and high O₂ availability, while the type II group lives better under high CH₄ and limiting O₂ conditions. Such physiological differences are possibly reflected in their ecological preferences. In the present study, methanotrophic compositions were compared between rice-planted soil and non-planted soil and between the rhizosphere and rice roots by using terminal restriction fragment length polymorphism (T-RFLP) analysis of particulate methane monooxygenase (pmoA) genes. In addition, the effects of rice variety and nitrogen fertilizer were evaluated. The results showed that the terminal restriction fragments (T-RFs), which were characteristic for type I methanotrophs, substantially increased in the rhizosphere and on the roots compared with non-planted soils. Furthermore, the relative abundances of the type I methanotroph T-RFs were greater on roots than in the rhizosphere. Of type I methanotrophs, the 79 bp T-RF, which was characteristic for an unknown group or Methylococcus/Methylocaldum, markedly increased in field samples, while the 437 bp, which possibly represented Methylomonas, dominated in microcosm samples. These results suggested that type I methanotrophs were enriched or selected for by rice roots compared to type II methanotrophs. However, the members of type I methanotrophs are dynamic and sensitive to environmental change. Rice planting appeared to increase the copy number of pmoA genes relative to the non-planted soils. However, neither the rice variety nor the N fertilizer significantly influenced the dynamics of the methanotrophic community.     

Diversity of methanotrophs in urea-fertilized tropical rice agroecosystem

Laboratory experiments were conducted to study the population size, diversity and methane oxidation potential of methanotrophs in tropical rice agroecosystem under the influence of N-fertilizer. Results indicate that the diversity of methane oxidizing bacteria (MOB) is altered in fertilizer treated soils compared to untreated control. Nevertheless, Type I MOB still dominated in the fertilized soils whereas the diversity of Type II methanotrophs decreases. Control soils have higher MOB population and CH₄ oxidation capacity than fertilized soils. Rhizospheric soil is more populated than non-rhizospheric soil in both unfertilized and fertilized conditions. Variation in K_m and V_max of methane oxidation in soils appears to be due to variation in methanotrophic community. Experimental results indicate that methanotrophic community differs both quantitatively and qualitatively in unfertilized and fertilized soils.     

Field application of nitrogen and phenylacetylene to mitigate greenhouse gas emissions from landfill cover soils: effects on microbial community structure

Landfills are large sources of CH₄, but a considerable amount of CH₄ can be removed in situ by methanotrophs if their activity can be stimulated through the addition of nitrogen. Nitrogen can, however, lead to increased N₂O production. To examine the effects of nitrogen and a selective inhibitor on CH₄ oxidation and N₂O production in situ, 0.5 M of NH₄Cl and 0.25 M of KNO₃, with and without 0.01% (w/v) phenylacetylene, were applied to test plots at a landfill in Kalamazoo, MI from 2007 November to 2009 July. Nitrogen amendments stimulated N₂O production but had no effect on CH₄ oxidation. The addition of phenylacetylene stimulated CH₄ oxidation while reducing N₂O production. Methanotrophs possessing particulate methane monooxygenase and archaeal ammonia-oxidizers (AOAs) were abundant. The addition of nitrogen reduced methanotrophic diversity, particularly for type I methanotrophs. The simultaneous addition of phenylacetylene increased methanotrophic diversity and the presence of type I methanotrophs. Clone libraries of the archaeal amoA gene showed that the addition of nitrogen increased AOAs affiliated with Crenarchaeal group 1.1b, while they decreased with the simultaneous addition of phenylacetylene. These results suggest that the addition of phenylacetylene with nitrogen reduces N₂O production by selectively inhibiting AOAs and/or type II methanotrophs.     

Identity of active methanotrophs in landfill cover soil as revealed by DNA-stable isotope probing

A considerable amount of methane produced during decomposition of landfill waste can be oxidized in landfill cover soil by methane-oxidizing bacteria (methanotrophs) thus reducing greenhouse gas emissions to the atmosphere. The identity of active methanotrophs in Roscommon landfill cover soil, a slightly acidic peat soil, was assessed by DNA-stable isotope probing (SIP). Landfill cover soil slurries were incubated with (13)C-labelled methane and under either nutrient-rich nitrate mineral salt medium or water. The identity of active methanotrophs was revealed by analysis of (13)C-labelled DNA fractions. The diversity of functional genes (pmoA and mmoX) and 16S rRNA genes was analyzed using clone libraries, microarrays and denaturing gradient gel electrophoresis. 16S rRNA gene analysis revealed that the cover soil was mainly dominated by Type II methanotrophs closely related to the genera Methylocella and Methylocapsa and to Methylocystis species. These results were supported by analysis of mmoX genes in (13)C-DNA. Analysis of pmoA gene diversity indicated that a significant proportion of active bacteria were also closely related to the Type I methanotrophs, Methylobacter and Methylomonas species. Environmental conditions in the slightly acidic peat soil from Roscommon landfill cover allow establishment of both Type I and Type II methanotrophs.     

The Effects of Age, Darkness and Nitrate on Poly-β-hydroxybutyrate Levels and Nitrogen-Fixing Ability of Rhizobium in Lupinus angustifolius

The effects of age, darkness and nitrate on nitrogenase activity, poly-β-hydroxybutyrate (PHB) and β-hydroxybutyrate dehydrogenase (BOHB DH) activity in Rhizobium lupini while in symbiosis with Lupinus angustifolius were assessed. It was concluded that PHB functions as a reserve material in root nodules and is involved in the maintenance of nitrogen fixation when the availability of photosynthates is restricted. PHB accumulation and the development of BOHB-DH activity did not take place until the peak of nitrogenase activity had passed. All three parameters reached a steady level when the host plant was approximately 24 days old. Deprivation of photosynthates due to continuous darkness resulted in major losses of PHB and BOHB-DH activity until N₂-fixation ceased. Nitrate addition without restriction of photosynthates resulted in diminished nitrogenase activity. Apart from a slight delay in accumulation. PHB levels were not affected. It was found important to standardize sampling since N₂-fixation was affected by plant culture and diurnal cycles.     

Influence of electron acceptor,carbon, nitrogen,and phosphorus on polyhydroxyalkanoate (PHA) production by <Emphasis Type="Italic">Brachymonas</Emphasis> sp. P12

Polyhydroxyalkanoates (PHAs), intracellular carbon and energy reserve compounds in many bacteria, have been used extensively in biodegradable plastics. PHA formation is influenced by nutrient limitations and growth conditions. To characterize the PHA accumulation in a new denitrifying phosphorus-removing bacterium Brachymonas sp. P12, batch experiments were conducted in which the electron acceptor (oxygen or nitrate) was varied and different concentrations of carbon (acetate), nitrogen (NH₄Cl), and phosphorus (KH₂PO₄) were used. Polyhydroxybutyrate (PHB) was the dominant product during PHA formation when acetate was the sole carbon source. The PHB content of aerobically growing cells increased from 431 to 636 mg PHB g⁻¹ biomass, but the PHB concentration of an anoxic culture decreased (−218 mg PHB g⁻¹ biomass), when PHB was utilized simultaneously with acetate as an electron donor for anoxic denitrification. The specific PHB production rate of the carbon-limited batch, 158.2 mg PHB g⁻¹ biomass h⁻¹, was much greater than that of batches with normal or excess carbon. The effects of phosphorus and nitrogen concentrations on PHB accumulation were clearly less than the effect of carbon concentration. According to the correlation between the specific PHB production rate and the specific cell growth rate, PHB accumulation by Brachymonas sp. P12 is enhanced by nutrient limitation, is growth-associated, and provides additional energy for the biosynthesis of non-PHB cell constituents to increase the cell growth rate beyond the usual level.     

Modeling of the mixed culture and periodic control for PHB production

The unstructured mathematical model was developed in the present investigation for the mixed culture, where the metabolites produced by one microorganism is assimilated by the other microorganism. For this, we specifically employed such model system in which sugars such as glucose were converted to lactate by Lactobacillus delbrueckii and the lactate was converted in turn to poly-β-hydroxybutyrate (PHB) by Ralstonia eutropha in one fermentor. Several batch and fed-batch culture experiments were conducted using each microorganism at different dissolved oxygen (DO) concentrations. Those experimental data were then fitted to the mathematical model, which can describe the dynamics of a mixed culture. Some of the model parameters were expressed as functions of DO concentrations, and some of the other model parameters were tuned based on the mixed culture experiments. The model developed describes the effects of such concentrations of glucose, lactate, DO, and NH₃ on the dynamic behavior of such concentrations as both microorganisms, glucose, lactate, and PHB. Optimal operating condition was then investigated using the model developed. It was found that the periodic change in DO concentration improved such performance as PHB yield, and it was verified by experiments. The optimal NH₃ concentration profile was also obtained for the efficient PHB production by the application of the maximum principle.     

Control of glucose feeding using exit gas data and its application to the production of PHB from tapioca hydrolysate byAlcaligenes eutrophus

Summary A method to estimate the glucose concentration in the culture broth using CO₂ evolution rate (CER) data from a mass spectrometer was developed.Alcaligenes eutrophus was cultivated to produce poly(3-hydroxybutyric acid) (PHB) from tapioca hydrolysate using this method. Thek value (g glucose/mol CO₂), defined as the glucose consumption per CO₂ evolution, decreased with culture time and was automatically changed using CER data. The glucose concentration in the culture broth could be controlled at 10 to 20 g/L. A final cell concentration of 106 g/L, PHB concentration of 61 g/L. and PHB content of 58 % of dry cell weight were obtained after 59 h of cultivation.     

Production and Consumption of Nitric Oxide by Three Methanotrophic Bacteria

We studied nitrogen oxide production and consumption by methanotrophs Methylobacter luteus (group I), Methylosinus trichosporium OB3b (group II), and an isolate from a hardwood swamp soil, here identified by 16S ribosomal DNA sequencing as Methylobacter sp. strain T20 (group I). All could consume nitric oxide (nitrogen monoxide, NO), and produce small amounts of nitrous oxide (N₂O). Only Methylobacter strain T20 produced large amounts of NO (>250 parts per million by volume [ppmv] in the headspace) at specific activities of up to 2.0 × 10⁻¹⁷ mol of NO cell⁻¹ day⁻¹, mostly after a culture became O₂ limited. Production of NO by strain T20 occurred mostly in nitrate-containing medium under anaerobic or nearly anaerobic conditions, was inhibited by chlorate, tungstate, and O₂, and required CH₄. Denitrification (methanol-supported N₂O production from nitrate in the presence of acetylene) could not be detected and thus did not appear to be involved in the production of NO. Furthermore, cd₁ and Cu nitrite reductases, NO reductase, and N₂O reductase could not be detected by PCR amplification of the nirS, nirK, norB, and nosZ genes, respectively. M. luteus and M. trichosporium produced some NO in ammonium-containing medium under aerobic conditions, likely as a result of methanotrophic nitrification and chemical decomposition of nitrite. For Methylobacter strain T20, arginine did not stimulate NO production under aerobiosis, suggesting that NO synthase was not involved. We conclude that strain T20 causes assimilatory reduction of nitrate to nitrite, which then decomposes chemically to NO. The production of NO by methanotrophs such as Methylobacter strain T20 could be of ecological significance in habitats near aerobic-anaerobic interfaces where fluctuating O₂ and nitrate availability occur.