Accumulation of Poly[(R)-3-Hydroxyalkanoates] in Pseudomonas oleovorans during Growth with Octanoate in Continuous Culture at Different Dilution Rates

Pseudomonas oleovorans ATCC 29347 was grown in chemostat culture at different dilution rates with mineral media varying in their ratios of octanoate to ammonia (C₀/N₀ ratio). At all dilution rates tested, three distinct growth regimes were observed: (i) carbon limitation with NH₄⁺ in excess at low C₀/N₀ ratios, (ii) purely nitrogen-limited growth conditions at high C₀/N₀ ratios with residual octanoate in the culture supernatant, and (iii) an intermediate zone of dual-nutrient-limited growth conditions where both the concentration of octanoate and that of ammonia were very low. The dual-nutrient-limited growth zone shifted to higher C₀/N₀ ratios with decreasing dilution rates, and the extension of the dual-nutrient-limited growth zone was inversely proportional to the growth rate. The cells accumulated the storage compound medium-chain-length poly[(R)-3-hydroxyalkanoate] (mcl-PHA) during dual (C and N)-nutrient-limited and N-limited growth conditions. Within the dual-nutrient-limited growth zone, the cellular mcl-PHA contents increased when the C₀/N₀ ratio in the feed was increased, whereas the cellular mcl-PHA level was independent from the feed C₀/N₀ ratio during N-limited growth. The monomeric composition of the accumulated mcl-PHA was independent of both the dilution rate and the feed C₀/N₀ ratio and consisted of 12 mol% 3-hydroxyhexanoic acid and 88 mol% 3-hydroxyoctanoic acid. Accumulation of mcl-PHA led to an increase in the cellular C/N ratio and to changes in elemental growth yields for nitrogen and carbon.     

Accumulation of poly[(R)-3-hydroxyalkanoates] in Pseudomonas 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.     

Tailor-made olefinic medium-chain-length poly[(R)-3-hydroxyalkanoates] by Pseudomonas putida GPo1: batch versus chemostat production

Functionalized medium-chain-length polyhydroxyalkanoates (mclPHAs) have gained much interest in research on biopolymers because of their ease of chemical modification. Tailored olefinic mclPHA production from mixtures of octanoic acid and 10-undecenoic acid was investigated in batch and dual (C,N) nutrient limited chemostat cultures of Pseudomonas putida GPo1 (ATCC 29347). In a batch culture, where P. putida GPo1 was grown on a mixture of octanoic acid (58 mol%) and 10-undecenoic acid (42 mol%), it was found that the fraction of aliphatic monomers was slightly lower in mclPHA produced during exponential growth than during late stationary phase. Thus, the total monomeric composition changed over time indicating different kinetics for the two carbon substrates. Chemostat experiments showed that the dual (C,N) nutrient limited growth regime (DNLGR) for 10-undecenoic acid coincided with the one for octanoic acid. Five different chemostats on equimolar mixtures of octanoic acid and 10-undecenoic acid within the DNLGR revealed that the monomeric composition of mclPHA was not a function of the carbon to nitrogen (C(0)/N(0)) ratio in the feed medium but rather of the dilution rate. The fraction of aliphatic monomers in the accumulated mclPHA was slightly lower at high dilution rates and increased towards low dilution rates, again indicating different kinetics for the two carbon substrates in P. putida GPo1.     

Continuous Production of Long-Side-Chain Poly-beta-Hydroxyalkanoates by Pseudomonas oleovorans

Shake flask experiments showed that Pseudomonas oleovorans began to be growth inhibited at 4.65 g of sodium octanoate liter, with total inhibition at 6 g liter. In chemostat studies with 2 g of ammonium sulfate and 8 g of octanoate liter in the feed, the maximum specific growth rate was 0.51 h, and the maximum specific rate of poly-beta-hydroxyalkanoate (PHA) production was 0.074 g of PHA g of cellular protein h at a dilution rate (D) of 0.25 h. When the specific growth rate (mu) was <0.3 h, the PHA composition was relatively constant with a C(4)/C(6)/C(8)/C(10) ratio of 0.1:1.7:20.7:1.0. At mu > 0.3 h, a decrease in the percentage of C(8) with a concomitant increase in C(10) monomers as mu increased was probably due to the effects of higher concentrations of unmetabolized octanoate in the fermentor. At D = 0.24 h and an increasing carbon/nitrogen ratio, the percentage of PHA in the biomass was constant at 13% (wt/wt), indicating that nitrogen limitation did not affect PHA accumulation. Under carbon-limited conditions, the yield of biomass from substrate was 0.76 g of biomass g of octanoate consumed, the yield of PHA was 0.085 g of PHA g of octanoate used, and 7.9 g of octanoate was consumed for each gram of NH(4) supplied. The maintenance coefficient was 0.046 g of octanoate g of biomass h. Replacement of sodium octanoate with octanoic acid appeared to result in transport-limited growth due to the water insolubility of the acid.     

Diel variation of the cellular carbon to nitrogen ratio of Chlorella autotrophica (Chlorophyta) growing in phosphorus‐ and nitrogen‐limited continuous cultures

We investigated the relationship between daily growth rates and diel variation of carbon (C) metabolism and C to nitrogen (N) ratio under P‐ and N‐limitation in the green algae Chlorella autotrophica. To do this, continuous cultures of C. autotrophica were maintained in a cyclostat culture system under 14:10 light:dark cycle over a series of P‐ and N‐limited growth rates. Cell abundance, together with cell size, as reflected by side scatter signal from flow cytometric analysis demonstrated a synchronized diel pattern with cell division occurring at night. Under either type of nutrient limitation, the cellular C:N ratio increased through the light period and decreased through the dark period over all growth rates, indicating a higher diel variation of C metabolism than that of N. Daily average cellular C:N ratios were higher at lower dilution rates under both types of nutrient limitation but cell enlargement was only observed at lower dilution rates under P‐limitation. Carbon specific growth rates during the dark period positively correlated with cellular daily growth rates (dilution rates), with net loss of C during night at the lowest growth rates under N‐limitation. Under P‐limitation, dark C specific growth rates were close to zero at low dilution rates but also exhibited an increasing trend at high dilution rates. In general, diel variations of cellular C:N were low when dark C specific growth rates were high. This result indicated that the fast growing cells performed dark C assimilation at high rates, hence diminished the uncoupling of C and N metabolism at night.     

Co-utilization of polymerized carbon sources by Bacteroides ovatus grown in a two-stage continuous culture system

Bacteroides ovatus NCTC 11153 was grown in a two-stage continuous culture system at various growth rates (vessel 1, D = 0.06 to 0.19 h-1; vessel 2, D = 0.03 to 0.09 h-1) on media containing mixtures of starch and arabinogalactan as carbon sources. The cell-associated enzyme activities needed to hydrolyze both substrates (amylase, arabinogalactanase, alpha-glucosidase, beta-galactosidase, and alpha-arabinofuranosidase) were variously influenced by growth rate and polysaccharide availability but were detected under all growth conditions tested. Measurements of residual carbohydrate in spent culture media showed that both polysaccharides were co-utilized during growth under putative C-limited conditions. The arabinogalactan was partly depolymerized in N-limited chemostats, and significant amounts of arabinose- and galactose-containing oligosaccharides accumulated in the cultures, indicating that starch was being preferentially utilized. Acetate, propionate, and succinate were the major fermentation products formed by C-limited bacteria, but under N limitation, lactate was also produced. Molar ratios of succinate increased concomitantly with the dilution rate in C-limited chemostats, whereas molar ratios of propionate decreased. During N-limited growth, however, decarboxylation of succinate to propionate was relatively independent of growth rate. Cell viability was higher in C-limited cultures compared with those grown under N limitation and was greatest at high dilution rates, irrespective of nutrient limitation.     

Co-utilization of polymerized carbon sources by Bacteroides ovatus grown in a two-stage continuous culture system.

Bacteroides ovatus NCTC 11153 was grown in a two-stage continuous culture system at various growth rates (vessel 1, D = 0.06 to 0.19 h-1; vessel 2, D = 0.03 to 0.09 h-1) on media containing mixtures of starch and arabinogalactan as carbon sources. The cell-associated enzyme activities needed to hydrolyze both substrates (amylase, arabinogalactanase, alpha-glucosidase, beta-galactosidase, and alpha-arabinofuranosidase) were variously influenced by growth rate and polysaccharide availability but were detected under all growth conditions tested. Measurements of residual carbohydrate in spent culture media showed that both polysaccharides were co-utilized during growth under putative C-limited conditions. The arabinogalactan was partly depolymerized in N-limited chemostats, and significant amounts of arabinose- and galactose-containing oligosaccharides accumulated in the cultures, indicating that starch was being preferentially utilized. Acetate, propionate, and succinate were the major fermentation products formed by C-limited bacteria, but under N limitation, lactate was also produced. Molar ratios of succinate increased concomitantly with the dilution rate in C-limited chemostats, whereas molar ratios of propionate decreased. During N-limited growth, however, decarboxylation of succinate to propionate was relatively independent of growth rate. Cell viability was higher in C-limited cultures compared with those grown under N limitation and was greatest at high dilution rates, irrespective of nutrient limitation.     

The effect of growth in continuous culture at various input C:N ratios on the expression of proteins involved in the transport and metabolism of methanol and short-chain amides by Methylophilus methylotrophus

Physiological regulation of proteins involved in the transport and metabolism of methanol and short-chain amides by Methylophilus methylotrophus was investigated following growth in continuous culture at various input C:N ratios. The concentrations of the methanol porin and methanol dehydrogenase were highest during C-limited growth (C:N<4.6), but declined gradually as a function of the increasing C:N ratio and were lowest during N-limited growth (C:N>16.3). In contrast, the concentrations of the amide-urea porin, the amide-urea binding protein, formamidase and acetamidase (together with formamidase and acetamidase activities) were lowest during C-limited growth, but increased sharply as a function of the C:N ratio and were highest during dual CN-limited and N-limited growth (C:N 4.6–16.3). The results are discussed in terms of the physiological and biochemical requirements of growth at varying C:N ratios.     

Growth and accumulation dynamics of poly(3-hydroxyalkanoate) (PHA) in Pseudomonas putida GPo1 cultivated in continuous culture under transient feed conditions

It has been shown that Pseudomonas putida GPo1 is able to grow in continuous culture simultaneously limited by ammonium (N source) and octanoate (C source), and concomitantly accumulate poly([R]-3-hydroxyalkanoate) (PHA). Under such growth conditions the material properties of PHA can be fine-tuned if a second PHA precursor substrate is supplied. To determine the range of dual carbon and nitrogen (C, N)-limited growth conditions, tedious chemostat experiments need to be carried out for each carbon source separately. To determine the growth regime, the C/N ratio of the feed (f) to a chemostat was changed in a stepwise manner at a constant dilution rate of 0.3/h. Dual-(C, N)-limited growth was observed between C(f) /N(f) ≤ 6.4 g/g and C(f) /N(f) >9.5 g/g. In the following, we analyzed alternative approaches, using continuous medium gradients at the same dilution rate, that do not require time consuming establishments of steady states. Different dynamic approaches were selected in which the C(f) /N(f) ratio was changed continuously through a convex increase of C(f) , a convex increase of N(f) , or a linear decrease of C(f) (gradients 1, 2, and 3, respectively). In these experiments, the dual-(C, N)-limited growth regime was between 7.2 and 11.0 g/g for gradient 1, 4.3 and 6.9 g/g for gradient 2, and 5.1 and 8.9 g/g for gradient 3. A mathematical equation was developed that compensated a time delay of the gradient that was caused by the wash-in/wash-out effects of the medium feed.     

Effects of Temperature and Carbon-Nitrogen (C/N) Ratio on the Performance of Anaerobic Co-Digestion of Dairy Manure,Chicken Manure and Rice Straw: Focusing on Ammonia Inhibition

Anaerobic digestion is a promising alternative to disposal organic waste and co-digestion of mixed organic wastes has recently attracted more interest. This study investigated the effects of temperature and carbon-nitrogen (C/N) ratio on the performance of anaerobic co-digestion of dairy manure (DM), chicken manure (CM) and rice straw (RS). We found that increased temperature improved the methane potential, but the rate was reduced from mesophilic (30∼40°C) to thermophilic conditions (50∼60°C), due to the accumulation of ammonium nitrogen and free ammonia and the occurrence of ammonia inhibition. Significant ammonia inhibition was observed with a C/N ratio of 15 at 35°C and at a C/N ratio of 20 at 55°C. The increase of C/N ratios reduced the negative effects of ammonia and maximum methane potentials were achieved with C/N ratios of 25 and 30 at 35°C and 55°C, respectively. When temperature increased, an increase was required in the feed C/N ratio, in order to reduce the risk of ammonia inhibition. Our results revealed an interactive effect between temperature and C/N on digestion performance.     

INTERACTING EFFECTS OF LIGHT AND NUTRIENT LIMITATION ON THE GROWTH RATE OF SYNECHOCOCCUS LINEARIS (CYANOPHYCEAE)1

The blue-green alga Synechococcus linearis (Naeg.) Kom. was grown in P- and N-limited chemostats over a range of potentially limiting irradiances in order to determine the combined effects of light and nutrient limitation on some aspects of the composition and metabolism of this alga. Over a narrow range of low irradiances, simultaneous limitation of growth rate by light and either N or P was shown. This simultaneous limitation of growth rate by a nutrient and a physical factor can be explained by the ability of an increased supply of one to compensate in part for a decreased supply of the other. At all irradiances, the internal concentration of the limiting nutrient increased with increasing dilution rate, and the results could be fitted to the Droop relationship. With decreasing irradiance, the internal concentration of the limiting nutrient increased. There appeared to be little or no effect of light on the minimum internal concentration of P but that of N increased with decreasing light. Both chlorophyll a and biliprotein per unit particulate C increased with increasing dilution rate and decreasing irradiance. The critical N/P ratio increased with decreasing light as the N requirement of N-limited cells increased faster than did the P requirement of P-limited cells. The composition of exponentially growing cells in complete medium varied much less with light. Neither dilution rate nor irradiance during growth had a great effect on saturated rates of P or N uptake or alkaline phosphatase activity. Calculated assimilation ratios increased with light and dilution rate. The role of the flexibility of nutrient composition in adaptation to adverse conditions and the implications of the results for the use of physiological indicators of nutrient status are discussed.     

Nitrogen supply effects on productivity and potential leaf litter decay of Carex species from peatlands differing in nutrient limitation

We investigated the effect of increased N-supply on productivity and potential litter decay rates of Carex species, which are the dominant vascular plant species in peatlands in the Netherlands. We hypothesized that: (1) under conditions of N-limited plant growth, increased N-supply will lead to increased productivity but will not affect C:N ratios of plant litter and potential decay rates of that litter; and (2) under conditions of P-limited plant growth, increased N-supply will not affect productivity but it will lead to lower C:N ratios in plant litter and thereby to a higher potential decay rate of that litter. These hypotheses were tested by fertilization experiments (addition of 10 g N m^-2 year^-1) in peatlands in which plant growth was N-limited and P-limited, respectively. We investigated the effects of fertilization on net C-fixation by plant biomass, N uptake, leaf litter chemistry and potential leaf litter decay. In a P-limited peatland, dominated by Carex lasiocarpa, there was no significant increase of net C-fixation by plant biomass upon enhanced N-supply, although N-uptake had increased significantly compared with the unfertilized control. Due to the N-fertilization the C:N ratio in the plant biomass decreased significantly. Similarly, the C:N ratio of leaf litter produced at the end of the experiment showed a significant decrease upon enhanced N-supply. The potential decay rate of that litter, measured as CO₂-evolution from the litter under aerobic conditions, was significantly increase upon enhanced N-supply. In a N-limited peatland, dominated by C. acutiformis, the net C-fixation by plant biomass increased with increasing N-supply, whereas the increase in N-uptake was not significant. The C:N ratio of both living plant material and of dead leaves did not change in response to N-fertilization. The potential decay rate of the leaf litter was not affected by N-supply. The results agree with our hypotheses. This implies that atmospheric N-deposition may affect the CO₂-sink function of peatlands, but the effect is dependent on the nature of nutrient limitation. In peatlands where plant growth is N-limited, increased N-supply leads to an increase in the net accumulation of C. Under conditions of P-limited plant growth, however, the net C-accumulation will decrease, because productivity is not further increased, whereas the amount of C lost through decomposition of dead organic matter is increased. As plant growth in most terrestrial ecosystems is N-limited, increased N-supply will in most peatlands lead to an increase of net C-accumulation.     

Evaluation of 16s rRNA and cellular fatty acid profiles as markers of human intestinal bacterial growth in the chemostat

Chemostats were used to study the effects of carbon and nitrogen limitation and specific growth rate on 16S rRNA synthesis and cellular fatty acid (CFA) profiles in four human intestinal bacteria (Bacteroides thetaiotaomicron, Bifidobacterium adolescentis, Clostridium bifermentans and Cl. difficile). Cellular fatty acid synthesis varied with dilution rate and nutrient availability in different species, but these cellular constituents were relatively stable phenotypic characteristics in Bact. thetaiotaomicron, where branched chain and hydroxy CFA were good taxonomic markers. Conversely, CFA in the Gram-positive bacteria varied markedly with changes in growth environment. For example, in chemostats, cyclopropane CFA were only synthesized in Cl. bifermentans and Cl. difficile under N-limited conditions. Similarly, Dimethyl acetal (DMA) fatty acids in Bif. adolescentis were primarily produced during N-limited growth, and this was inversely related to dilution rate. At low growth rates, 16S rRNA concentrations (microg rRNA per ml culture) correlated with viable bacterial counts, but were more closely related to specific growth rate when expressed as a function of cell mass (microg rRNA per mg dry weight bacteria). However, this did not reveal differences in bacterial population size and rRNA concentration in C-limited cultures. Thus, at low dilution rates, C limitation strongly reduced rRNA synthesis in Cl. bifermentans, despite viable cell counts being similar to those in N-limited cultures. These results indicate that, while 16S rRNA is a useful indicator of microbial activity, cell growth rate does not necessarily relate to rRNA concentration under all nutritional conditions. Consequently, bowel habit and diet will affect both CFA and rRNA content in bacteria isolated from intestinal samples, and this should be taken into consideration when interpreting such data measurements.     

In vivo blending of medium chain length polyhydroxy-alkanoates and polyhydroxybutyrate using recombinant Pseudomonas putida harboring phbCAB operon of Ralstonia eutropha

The in vivo blending of medium chain length polyhydroxyalkanoates (mcl-PHA) and polyhydroxybutyrate (PHB) was carried out using recombinant Pseudomonas putida after transforming the phbCAB operon of Ralstonia eutropha. The most suitable carbon sources for the production of mcl-PHA and PHB blends were identified to be octanoate and gluconate. The molar fractions of 3-hydroxyoctanoate and 3-hydroxybutyrate in the polymer blends were effectively modulated by controlling the mixing ratio of octanoate and gluconate, thereby producing a composition ranging from 95% mcl-PHA to 78% PHB.     

Plant effects on soil N mineralization are mediated by the composition of multiple soil organic fractions

Despite the topic of soil nitrogen (N) mineralization being well-studied, very few studies have addressed the relative contribution of different plant and soil variables in influencing soil N mineralization rates, and thus the supply of inorganic N to plants. Here, we used data from a well-studied N-limited grassland to address the relative effects of six plant and soil variables on net and on gross rates of soil N mineralization. We also addressed whether plant effects on soil N mineralization were mediated by changes in C and N concentrations of multiple soil organic matter (SOM) fractions. Regression analyses show that key plant traits (i.e., plant C:N ratios and total root mass) were more important than total C and N concentrations of bulk soil in influencing N mineralization. This was mainly because plant traits influenced the C and N concentration (and C:N ratios) of different SOM fractions, which in turn were significantly associated with changes in net and gross N mineralization. In particular, C:N ratios of a labile soil fraction were negatively related to net soil N mineralization rates, whereas total soil C and N concentrations of more recalcitrant fractions were positively related to gross N mineralization. Our study suggests that changes in belowground N-cycling can be better predicted by simultaneously addressing how plant C:N ratios and root mass affect the composition and distribution of different SOM pools in N-limited grassland systems.     

Production of polyhydroxyalkanoates by Pseudomonas nitroreducens

A strain coded AS 1.2343 was isolated from oil-contaminated soil in an oil-field in North China Tianjian City and it was identified as Pseudomonas nitroreducens. The strain demonstrated some unusual ability to synthesize polyhydroxybutyrate (PHB) homopolymer from medium-chain-length (mcl) fatty acids including hexanoate and octanoate. While polyhydroxyalkanoates (PHA) consisting of mcl hydroxyalkanoate (HA) monomers such as hydroxyoctanoate (HO) and hydroxydecanoate (HD) were the major compositions when butyrate, decanoate, lauric acid and tetradecanoic acid were used as substrates for the cell growth, respectively. PHA was accumulated up to 77% of the cell dry weight when growth was conducted in lauric acid, it appeared that the HA contents in the PHA would not be much affected by the changing of the lauric acid concentration. Varying the concentration ratio of butyrate to octanoate could change the composition of PHA accumulated by the strain. Yet PHB homopolymer was always the only polyester synthesized by the strain, regardless of the octanoate concentration change. Additionally, the ratio of carbon to nitrogen (C/N) in butyrate media was found to have effects on the PHA monomer content, as C/N increased from 2 to 100, content of HB decreased from 100% to 7%. PHA polyester synthesized by cells of Pseudomonas nitroreducens AS 1.2343 was a blend polymers consisting of acetone-insoluble HB and acetone-soluble mcl HA monomers.     

Maximum production strategy for biodegradable copolymer P(HB-co-HV) in fed-batch culture of Alcaligenes eutrophus

A novel strategy for the maximum production of a biodegradable copolymer, poly(3-hydroxybutyric-co-hydroxyvaleric) acid, P(HB-co-HV), was developed, based on the kinetic parameters obtained from fed-batch culture experiments of Alcaligenes eutrophus. The effects of various culture conditions such as mole ratio of carbon:nitrogen in feed medium (C/N); total fatty acids concentrations; and addition ratio of fatty acids on cultivation properties such as the specific rates of cell formation, mu (h-1), P(HB-co-HV) production, rho[g.P(HB-co-HV)/g.cell/h], production yield from fatty acids [g.P(HB-co-HV)/g.fatty acid], and mole fraction of monomeric units in the copolymer [mol.(HV)/{mol.(HB) + mol.(HV)}], were investigated. When nitrogen supply was sufficient for cell growth; that is, C/N (mol.nitrogen atom/mol.carbon atom) was low, mu was high, but rho and the production yield were low, because fatty acids were used mainly for energy formation and anabolic reactions in the cells. On the other hand, when nitrogen supply was limited for cell growth-that is, C/N was high-rho was high. The highest value of rho was obtained when C/N was 75. As the mole ratio of valeric acid (VA) to butyric acid (BA) in the feed medium was increased, the mole fraction of HV units in P(HB-co-HV) increased linearly. When the ratio of BA to VA in the feed medium was kept at a constant value, but C/N was increased, the mole fraction of HV units decreased. In particular, when C/N was >12, the mole fraction of HV units decreased linearly as C/N increased. When VA was utilized as the sole carbon source and C/N was fixed at 4, P(HB-co-HV) with the highest mole fraction of HV units (67 mol%) was achieved. From these results, it was shown that both C/N and the mole ratio of BA to VA in the feed medium should be well controlled for an optimal production of P(HB-co-HV) with the desired value of the mole fraction of HV units. When the addition ratio of butyric acid was 50 wt% of total fatty acids, a maximum production strategy for P(HB-co-HV) was developed and realized experimentally, which was based on a model of the relationship between mu and rho.     

Regulation of ammonia assimilation in an obligate methylotroph Methylobacillus flagellatum under steady-state and transient growth conditions

The obligate methylotroph Methylobacillus flagellatum was grown in the presence of different ammonium concentrations and the regulation of the enzymes associated with ammonium assimilation was investigated in steady-state and transient growth regimes. As the medium changed from C-limitation to dual C/N- and finally to N-limitation, the culture passed through three definite growth phases. The NADP⁺-dependent glutamate dehydrogenase (GDH) was present under ammonium limitation of the culture growth (at 2 mmol l^-1 of ammonium in the growth medium) and increased in response to an increase in nitrogen availability. Glutamine synthetase (GS) and glutamate synthase (GOGAT) activities were negligible during C- and C/N-limitation. In N-limited cells the GOGAT activity increased as the dilution rate increased up to 0.35 h^-1, and then sharply dropped. In the N-sufficient cultures both NAD⁺- and NADP⁺-dependent isocitrate dehydrogenase (NAD-ICDH and NADP-ICDH) activities were up-regulated as dilution rate increased, but in the N-limited culture the NAD-ICDH activity was up-regulated whereas NADP-ICDH one was down-regulated. Pulse additions of ammonium and methanol demonstrated the coordinate regulation of the GDH and ICDHs activities. When pulses were added to the C/N-limited cultures, there was an immediate utilization of the nutrients, resulting in an increase in biomass; at the same time the GDH and ICDH activities increased and the GS and GOGAT activities decreased. When the same ammonium/methanol pulse was added into the N-limited culture, there was a 3-hours delay in the culture response, after which the substrates were utilized at rates close to the ones shown by the C/N-limited culture after the analogous pulse.     

Lipid production by an unsaturated fatty acid auxotroph of the oleaginous yeast Apiotrichum curvatum grown in single-stage continuous culture

An unsaturated fatty acid auxotroph of the oleaginous yeast Apiotrichum curvatum, named UfaM3, blocked in the conversion of stearic to oleic acid was cultivated in single-stage continuous culture. The influence of consumed carbon to nitrogen ratios (C/N ratios, g g^–1) obtained at various dilution rates (D) on fatty acid (FA) accumulation and its profiles were studied. In continuous culture in N-limited medium a maximum FA accumulation of 45.6% (g g^–1 of dry biomass) was obtained at an optimal D of 0.049 h^–1, recording an efficiency of substrate conversion of 0.48 g g^–1 and 0.22 g g^–1 for biomass and lipids, respectively. The quality of lipid approached cocoa butter at an optimal C/N ratio of between 20 and 30. The C/N ratio in the incoming medium was 38.5 g g^–1 with 30 g l^–1 of glucose and both C and N sources were completely consumed at a critical D of 0.07 h^–1. The stability of the mutant was demonstrated in the steady-state conditions of the chemostat with regard to the FA composition of its lipids. Correspondence to: P. J. Blanc     

What are the effects of nitrogen deficiency on growth components of lettuce?

Relationships between nitrogen (N) content and growth are routinely measured in plants. This study determined the effects of N on the separate morphological and physiological components of plant growth, to assess how N-limited growth is effected through these components. Lettuce ( Lactuca sativa ) plants were grown hydroponically under contrasting N-supply regimes, with the external N supply either maintained continuously throughout the period of study, or withdrawn for up to 14 d. Richards' growth functions, selected using an objective curve-fitting technique, accounted for 99.0 and 99.1% of the variation in plant dry weight for control and N-limited plants respectively. Sublinear relationships occurred between N and relative growth rates under restricted N-supply conditions, consistent with previous observations. There were effects of treatment on morphological and physiological components of growth. Leaf weight ratio increased over time in control plants and decreased in N- limited plants. Shoot:root ratio followed a similar pattern. On a whole-plant basis, assimilation of carbon decreased in N-limited plants, a response paralleled by differences in stomatal conductance between treatments. Changes in C assimilation, expressed as a function of stomatal conductance to water vapour, suggest that the effects of N limitation on growth did not result directly from a lack of photosynthetic enzymes. Relationships between plant N content and components of growth will depend on the availability of different N pools for remobilization and use within the plant.