Degradation of crude oil by a mixed population of bacteria isolated from sea-surface foams

A mixed bacteria population (EM₄) was isolated from foams formed on the surface of a zone chromically polluted by hydrocarbons (Gulf of Fos, French Mediterranean coast, October 1981). The population was able to degrade crude oil very effectively in the presence of sea water supplemented with nitrogen, phosphorus and iron. The percentage of hydrocarbon degradation was 81% at 30°C, pH 8, and partial oxygen pressure of 100%. After 12 d incubation, 92 and 83% of satured and aromatic compounds (mono-, di- and triaromatics) were degraded, respectively, as well as 63% of polar products and 48.5% of asphaltenes. Maximum degradation was attained at a sodium chloride concentration of between 400 and 800 mM with Population EM₄, which is constituted of 8 strains, four of which are weak halophiles. Bacterial growth on hydrocarbons induces the production in the culture medium of surface-active agents which are able to emulsify the substrate. There is high specificity between the nature of the growth substrate and such emulsifying activity, particularly as far as petroleum is concerned: only the culture medium from Population EM₄ is able to emulsify petroleum. These surface-active agents contain sugars and lipids (fatty acids, mono- and diglycerides). The foams, which always contain a high concentration of both hydrocarbons (100 to 180 mgl^-1) and bacteria that are able to grow on these types of substrates, have a strong emulsifying activity. Our results would seem to demonstrate the importance of biosurfactants in the elimination of hydrocarbons from polluted biotopes.     

Dehydrogenases as tools in the study of marine sediments

A method is outlined for the measurement of NADH₂-dependent dehydrogenase activity in whole samples as well as in fractions of carbonate sediments. The method is based on extraction of enzyme activity with phosphate buffer containing 0.12% Triton X-100, dialysis, and a photometric assay which permits initial velocity determinations of the enzyme reaction under controlled conditions of pH, temperature, and substrate concentration. The dehydrogenases extracted from carbonate sediment of a subtropical beach at Bermuda are characterized by low K _m values (0.03 to 0.07 mM NADH₂/1), pH maxima around 8.5, and temperature maxima between 35° and 40°C. Their vertical distribution in the sediment agrees with what is known about the distribution of biomass on beaches of this type, maximum activity occurring in the topmost centimetre. Approximately 80 to 90% of the total electron transport activity is contained in the grain fraction, the remainder in the interstitial fraction of a sediment sample. From this it can be inferred that the interstitial fauna contributes only little to the total energy budget of this type of sediment, which is dominated by the Aufwuchs on the sand grains. The electron transport activity (ETA) measured by means of the dehydrogenase assay is taken to represent the maximum transport capacity (ETC) of all the organisms living in the sand ecosystem. From published evidence it is inferred that oxygen consumption of sediment samples and of individual organisms measured in vivo usually accounts for 10 to 20% of the maximum electron transport capacity measured in vitro with saturating concentrations of substrates and with Triton X-100 as part of the enzyme assay. It is suggested further that the ratios of dehydrogenase activities in different fractions or zones of sediment, or of different organisms, may represent one of the best indicators, so far available, of energy relationships in marine sediments.Contribution No. 652 from the Bermuda Biological Station for Research.     

Hydrocarbons contamination and microbial degradation in mangrove sediments of the Niger Delta region (Nigeria)

The distribution of aliphatic hydrocarbons and polycyclic aromatic hydrocarbons (PAHs), and their degradability by bacteria in epipelic and benthic sediments from Qua Iboe Estuary mangrove ecosystem and associated creeks were investigated. The research findings revealed that total aliphatic hydrocarbons in sediments ranged from 16.82 mg·kg ^?1 to 210 mg kg ^?1, wheras total PAHs ranged from 6.30 to 35.55 mg·kg ^?1 dry weight of sediment. Low molecular mass (i.e. the 2–3-ring) PAHs were predominant in almost all the sampling points, whereas the higher molecular masses (4-, 5- and 6-ring PAHs) had the lowest concentrations. In general, the sediment samples ES ₂ (39.7%), ES ₃ (24.8%), BS ₁ (46.7%), BS ₂ (49.9%) and BS ₃ (44.2%) showed<50% contributions of Σ combustion–derived PAH (COMP-PAH) concentration to the Σ PAH concentrations, whereas ES ₁ (57%) contained>50% of COMP-PAHs. Our results have also shown that many mangrove bacteria have strong capacity to utilise Qua Iboe Light (QL) crude oil as the sole source of carbon and energy, while lower number of bacterial species including Bacillus sp., Micrococcus sp., Pseudomonas aeruginosa, Alcaligenes sp. and Flavobacterium sp. exhibited detectable PAHs degradability; and as such may serve as efficient degraders of QL crude oil contamination of mangrove ecosystem.     

Effects of long-term acclimation to environmental hypercapnia on extracellular acid–base status and metabolic capacity in Mediterranean fish <Emphasis Type="Italic">Sparus aurata</Emphasis>

In the context of future scenarios of anthropogenic CO₂ accumulation in marine surface waters, the present study addresses the effects of long-term hypercapnia on a Mediterranean fish, Sparus aurata. By equilibration with elevated CO₂ levels seawater pH was lowered to a value of 7.3, close to the maximum pH drop expected in marine surface waters from atmospheric CO₂ accumulation. Intra- and extracellular acid–base parameters as well as changes in enzyme profiles were studied in red and white muscles and the heart under both normocapnia and hypercapnia. The activities of pyruvate kinase (PK), lactate dehydrogenase (L-LDH), citrate synthase (CS), malate dehydrogenase and and 3-hydroxyacyl CoA dehydrogenase (HOAD) reflect the pathways and capacity of oxidative processes in metabolism. Long-term hypercapnia caused a transient reduction in blood plasma pH (pH_e) as well as in intracellular pH (pH_i). Compensation of the acidosis occurred through increased plasma and cellular bicarbonate levels. Changes in enzymatic activities, especially the increase in the activity of L-LDH, paralleled by a drop in CS activity in white and red muscles reflect a shift from aerobic to anaerobic pathways of substrate oxidation during long-term acclimation under hypercapnia. The present results suggest that moderate environmental hypercapnia changes the metabolic profile in tissues of S. aurata. Consequences for slow processes like growth and reproduction potential as well as potential harm at population, species and ecosystem levels require further investigation.     

Contrasting effects of sulfide and thiosulfate on symbiotic CO2-assimilation of Phallodrilus leukodermatus (Annelida)

Experiments were conducted in order to specify the reductants responsible for the carbon dioxide fixation of the symbiotic sulfur bacteria in the gutless marine oligochaete Phallodrilus leukodermatus (Annelida) from shallow calcareous sediments in Bermuda. Carbon dioxide-uptake rates were suppressed by S⁼ and stimulated by S₂O₃ ⁼. Individuals which hosted bacteria containing reserve energy substances maintained a high short-term CO₂-uptake activity, while bacteria in worm homogenates and in worms treated with an antibiotic (Baypen) did not show any significant metabolic activity. Absolute uptake rates in P. leukodermatus were usually considerably higher than those reported for other animals harbouring prokaryotic sulfuroxidizing symbionts. Utilization of thiosulfate rather than sulfide is compatible with the preferred occurrence of the worms around the redox discontinuity layer and has been confirmed in other thiobiotic animals. Sulfur stored in the symbiotic bacteria appears to be oxidized to sulfate and be excreted when the worms are held under energy-limited conditions. The data emphasize the complexity of the possible metabolic pathways involved in the oxidation of reduced-sulfur compounds by bacterial symbionts in marine invertebrates.     

Ecology and characteristics of bdellovibrios from three tropical marine habitats

Marine bdellovibrios have not previously been reported from the southern hemisphere, and knowledge of their occurrence in marine ecosystems is rudimentary. This study examined quantitative and qualitative aspects of bdellovibrios parasitic to the bacterium Vibrio alginolyticus at each of three representative tropical marine habitats of the Great Barrier Reef. Bdellovibrios were found in the water column throughout a 12 mo period from May 1992 at a sandy beach, a mangrove and a fringing coral reef. Their abundance was correlated with water temperature (P<0.001) and was highest in summer, lowest in winter and intermediate in spring and autumn. Over the sampling period, bdellovibrios were most abundant at the mangrove habitat (36.6 ml^-1) and least abundant at the reef (9.5 ml^-1), but there was substantial variability in numbers at all habitats among seasons and months of the year. On some occasions no bdellovibrios were found in replicate samples from the beach and reef habitats, while on others the maximum detectable by the method used (180 ml^-1) was sometimes found at the beach and mangrove habitats. Bdellovibrios within each habitat were uniformly distributed among sampling sites (P>0.05). They were more abundant in sub-surface than bottom waters in summer, but the reverse occurred in winter. Midwater samples usually had least bdellovibrios. Bdellovibrio numbers were significantly correlated with those of potential host bacteria—colony-forming bacteria at all habitats and total bacteria at the beach and reef habitats. Strain characteristics, primarily based on host range, indicated qualitative differences in bdellovibrio populations among habitats. Pseudomonas atlantica, P. aeruginosa, P. marina, Cytophaga marinoflava, Vibrio gazogenes, V. mimicus and a Spirillum-like bacterium were not parasitised by bdellovibrios from any habitat. Of the other 25 Vibrio spp. tested, most were parasitised by the majority of bdellovibrio strains from each habitat. Strain differences were principally with respect to parasitism of non-Vibrio bacteria. All strains required Na⁺ and grew at 35°C, but some failed to grow at 15°C.     

Distribution de certaines bactéries hétérotrophes aérobies dans les sédiments profonds de Méditerranée Orientale

Ninety five sediment samples were extracted from 16 cores collected at depths from 283 to 4,350 m in the Eastern Mediterranean Sea. Generally, the microbial population of superficial layers is lower than 10⁵ bacteria/g sediment. At depths below 1,000 m this population is lower than 10⁴. Bacterial counts at different levels of the sediment show that the microbial population is only important in the superficial layer. Below the surface, bacterial counts are generally between 10² to 10³ cells/ml. Some levels appear to be sterile; none of the 12 culture media gave rise to development with the sediment samples. Studies of some physiological groups of bacteria show scarcity of chitinoclastic and agarolytic bacteria and absence of cellulolytic and sulphate reducers. On the other hand, amylolytic, gelatinolytic, lypolytic and nitrate-reducing bacteria are widely distributed among the microbial population of marine sediments. This population of deep sediments includes a few auxotrophic bacteria.     

Influence of the mineralogical composition on microbial activities in marine sediments: an experimental approach

We investigated the influence of the mineralogical composition of marine sediments on bacterial activity in experimental microcosms. Calcite and quartz were added to natural marine sediments and microbial response in terms of total bacterial abundance and biomass, β-D-glucosidase exo-enzymatic activity and bacterial incorporation of a radio-labelled (³H-leucine) substrate were investigated for a period of one month. We report here that after 15 days the mineralogical composition of the sediment (calcite vs. quartz) had an impact on bacterial abundance and activity (reduced for ca 15% and 56%, respectively). However, such impact was mitigated or even disappeared in high organic nutrient conditions.     

Interrelationships between sulfate reducing and methane producing bacteria in coastal sediments with intense sulfide production

Coastal sediments receiving different amounts of organic carbon through sedimentation were investigated with respect to sulfate reduction and methanogenic activity. Sampling was carried out at sediment temperatures of 7° and 15°C. Sulfate-reducing and methanogenic bacteria were found at all depths. Sulfate reduction decreased with depth and the highest sulfide concentrations were found a few centimeters below the sediment surface, up to 15 mM at 15°C and pH 7.1. In the same segments a maximum in the methane concentration was also found, 0.91 mM. The high sulfide concentration inhibited the methane formation from acetate but not from carbon dioxide. In the organic rich sediment sulfate reduction was limited by the diffusion of SO ₄ ⁺ into the sediment and methane production from acetate by sulfide diffusion out of the sediment. When electron acceptor concentration limits sulfate reduction, thermodynamic calculations show that the utilization of electron donors more reduced than acetate is favored. In the sediment with the high carbon-input, acetate predominated at 15°C whereas in the low carbon-input sediment hardly any short chain organic acids were detected. The possibility of a shift in sulfate reduction from acetate oxidation to acetate production is discussed.     

Lysosomes and sulfide-oxidizing bodies in the bacteriocytes of Lucina pectinata, a cytochemical and microanalysis approach

Lucina pectinata is a large tropical clam living deeply burrowed in the black, reducing mud of mangrove swamps. It is known to possess hemoglobin in the cytoplasmic areas of its bacteriocytes, which harbor sulfide-oxidizing bacteria. The bacteriocytes also possess lysosome-like microbodies containing either membrane whorls or electron-dense granules in which free heme compounds have been identified. The cytochemical detection of acid phosphatase and arylsulphatase through EDX (energy-dispersive X-ray) microanalysis strongly suggests that the bacteriocytes of L. pectinata contain, in fact, two different types of microbodies. Some of these (devoid of dense granules) possess a variable amount of lysosomal enzymes and occasionally a limited quantity of iron, which may result from a recycling process of hemoglobin. Their main function seems to be the digestion of a limited proportion of symbiotic bacteria. They represent genuine secondary lysosomes with a functionally acidic pH. The second type of microbodies is characterized by dense granules containing sulfur and iron hemes but no lysosomal enzymes. Their sulfide-oxidizing activity was substantiated by benzyl viologen assay, with Na₂S as a substrate. These microbodies appear to be similar to the sulfide-oxidizing bodies (SOBs) described in the bacteriocytes of other bivalve species with symbiotic thioautotrophic bacteria; however, their sulfide-oxidizing activity appears to be non-enzymatic. They are discrete organelles, characterized by a functionally basic pH and pseudoperoxidasic activity, and have been termed SOBs. Therefore, the bacteriocytes of L. pectinata possess at the same time functional lysosomes and functional SOBs. Received: 17 August 2000 / Accepted: 20 December 2000     

Purification and some properties of β-N-acetyl-D-glucosaminidase from prawn (<Emphasis Type="Italic">Penaeus vannamei</Emphasis>)

The -N-acetyl-D-glucosaminidase (NAGase, EC 3.2.1.52) from prawn (Penaeus vannamei) was purified by extraction with 30% ethanol solution and ammonium sulfate fractionation, then chromatographed on Sephadex G-100 followed by DEAE-cellulose (DE-32) columns. The purified enzyme determined to be homogeneous by polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE. The specific activity of the purified enzyme was 1,560 U mg^–1. Enzyme molecular weight was determined to be 105,000 Da; it contained two subunits of the same mass (45,000 Da). The pI value was calculated to be 4.8 by isoelectric focusing. The optimum pH and optimum temperature of the enzyme for the hydrolysis of pNP--D-GlcNAc (enzyme substrate) were determined to be pH 5.2 and 45°C, respectively. The behavior of the enzyme during hydrolysis of pNP--D-GlcNAc followed Michaelis–Menten kinetics, with K_m=0.254 mM and V_m=9.438 M min^–1, at pH 5.2 and 37°C. The stability of the enzyme was investigated, and the results showed that the enzyme was stable in a pH range from 4.2 to 10.0 and at temperatures <40°C. The effects of metal ions on the enzyme were also studied. Li⁺, Na⁺ and K⁺ had no influence on enzyme activity. Mg²⁺, Ca²⁺ and Mn²⁺ activated the enzyme, while Ba²⁺, Zn²⁺, Co²⁺, Cd²⁺, Hg²⁺, Pb²⁺ Cu²⁺, Fe³⁺ and Al³⁺ showed various degrees of inhibitory effects on the enzyme.Communicated by O. Kinne, Oldendorf/Luhe     

Characterization of alkaline phosphatase and organic phosphorous utilization in the oceanic dinoflagellate Pyrocystis noctiluca

Phosphate depleted Pyrocystis noctiluca (Murray) Schuett 1895 has at least one phosphomonoesterase (EC 3:1:3:1) which is triphasic between 0.1 and 222 mol P. The enzyme has a broad temperature range with maximum activity at 50 °C and a Q₁₀ of 1.4 to 1.5. A break in the Arrhenius plot at 35 °C implies the enzyme is membrane-bound. Cytological staining of whole cells and cell fractionation studies (showing 26 times higher specific activity in the particulate compared with the cytoplasmic fraction) suggest the enzyme is plasmalemma-bound. The enzyme has an absolute metal requirement which would be satisfied by Mg⁺⁺ but not Mn⁺⁺, Zn⁺⁺, Fe⁺⁺, or Co⁺⁺ at seawater concentrations. Alkaline phosphatase is a stable enzyme whose activity is not altered by inhibitors of protein synthesis. Orthophosphate inhibition of enzyme activity was largely eliminated in the presence of these inhibitors. Apparently, a protein induced by PO₄ ^3-, rather than PO₄ ^3- itself, inhibits alkaline phosphatase. Cell-free alkaline phosphatase can hydrolyze a variety of phosphate esters and linear polymers of inorganic phosphorus as well as disolved organic phosphorus from tropical oceanic waters. These same hydrolysable organic and inorganic phosphorus compounds support the axenic culture growth of P. noctiluca, suggesting that naturally occurring hydrolysable organic phosphorus compounds may also support the growth of this alga.     

Nitrogen fixation in the rhizosphere of marine angiosperms

High rates of acetylene reduction were observed in systems containing excised rhizomes of the Caribbean marine angiosperms Thalassia testudinum, Syringodium filiforme and Diplanthera wrightii, and the temperate marine angiosperm Zostera marina. For 4 plant and plant-sediment systems the ratio of acetylene reduced/N₂ fixed varied from 2.6 to 4.6. For T. testudinum the estimated rates of nitrogen fixation are in agreement with estimated requirements of the plant for nitrogen. For a typical T. testudinum stand, N₂ fixation is estimated to be 100 to 500 kg N/hectare per year. Numbers of N₂-fixing bacteria in the rhizosphere sediments were roughly 50 to 300 times more abundant than those in the nonrhizosphere sediments, and in both types of sediments were of the same orders as the estimated numbers of heterotrophic aerobes.Canadian IBP Contribution No. 137.     

Ammonia production and kinetic properties of glutamate dehydrogenase in the sipinculid Phascolosoma arcuatum exposed to anoxia

The amounts of total NH ₄ ⁺ detected in the external media in which Phascolosoma arcuatum had been exposed to various periods of anoxia were significantly greater than those in which the worms were exposed to normoxia for a similar period. The increased NH ₄ ⁺ production by P. arcuatum during anoxic exposure was unlikely to be due to an increased catabolism of adenine nucleotides or urea. In contrast, there were significant decreases in the concentrations of several free amino acids in the coelomic plasma and body tissues of individuals during the 48 h of anoxic exposure. The amount of NH ₄ ⁺ produced by the anoxic P. arcuatum could be accounted for by the decreases in the concentrations of aspartate or glycine. Increases in the catabolism of free amino acids (FAA), leading to the increased production of NH ₄ ⁺ , in P. arcuatum during anoxia were supported by the detection of significant changes in the kinetic properties of glutamate dehydrogenase (GDH), in the deaminating direction, from worms exposed to anoxia for 48 h. The apparent increase in the affinity of GDH from the anoxic worm to glutamate would bring about a greater deaminating activity at physiological concentrations of ths substrate. P. arcuatum used in these experiments were collected from the mangrove swamp at Mandai, Singapore between 1990 and 1993.     

环草隆对城市绿地重金属污染土壤有机氮矿化、基础呼吸及相关酶活性的影响

城市土壤重金属和有机污染物复合污染广泛存在,而城市草坪除草剂的应用使城市绿地土壤的农药污染问题成为了新的关注点。为了准确评价城市绿地重金属污染土壤的农药污染生态风险,选择不同重金属污染程度的土壤为研究对象,以土壤有机氮矿化量、基础呼吸以及土壤酶活性为指标,采用室内模拟试验方法,探讨了草坪除草剂环草隆污染对土壤微生物的生态毒理效应。结果表明:(1)土壤有机氮矿化、基础呼吸、芳基硫酸酯酶和碱性磷酸酶对重金属和环草隆污染响应较为敏感,脲酶和蔗糖酶对重金属和环草隆污染不敏感。(2)环草隆浓度为0~1 000 mg·kg~(-1)范围内,和污染较轻的样点N土壤的碱性磷酸酶活性抑制(激活)率的线性相关关系显著,和污染较为严重的样点D和G土壤的芳基硫酸酯酶活性抑制(激活)率的线性关系显著。(3)土壤中环草隆对样点D和G土壤芳香硫酸酯酶活性、对样点N土壤碱性磷酸酶活性抑制(激活)率的EC10分别为568 mg·kg~(-1)、1 306 mg·kg~(-1)(抑制值)和56 mg·kg~(-1)(激活值)、99 mg·kg~(-1),EC50分别为1 901 mg·kg~(-1)、3 806 mg·kg~(-1)、2 321 mg·kg~(-1)。以上研究结果能够为城市土壤重金属和农药复合污染生态风险评价提供基础数据和技术方法。     

Microbial control of organic carbon in marine sediments: Coupled chemoautotrophy and heterotrophy

The chemoautotrophic fixation of carbon dioxide is responsible for an appreciable component of the organic matter apparent as a carbon-rick peak at 40 cm sub-bottom in the marine muds of Halifax Harbour, Nova Scotia, Canada. Dissolved oxygen and sulfide profiles show that the 40 cm horizon represents a transitional environment from aerobic to underlying anaerobic conditions. A dissolved sulfate maximum and pH minimum at 40 cm indicate that sulfur-oxidizing chemoautotrophs are associated with the organic carbon production at this horizon. The stimulation of CO₂-fixation by thiosulfate and inhibition by anaerobic conditions, ammonia, nitrate and nitrite further support the contention that sulfur-oxidizers are primary producers at this horizon. Heterotrophic activity data show that both aerobic and anaerobic heterotrophs are active in the sediments. These data, in conjunction with the measurement of CO₂-fixation and the calculation of organic carbon accumulation over time, show that the peak of organic carbon observed is residual carbon that is not heterotrophically recycled to CO₂ at the 40 cm horizon.     

A comparative study of the glutamate dehydrogenase activity in several species of marine phytoplankton

Kinetic studies on the NADP-linked glutamate dehydrogenase (GDH) activity in 8 species of marine phytoplankton have been carried out. The pH optima, linearity of reactions, apparent energies of activation, and apparent K _m values for NADPH, NH ₄ ^>+ and -ketoglutarate were determined. Based on these data, an accurate, sensitive enzyme assay procedure is presented which was successfully tested on shipboard during a recent cruise in an upwelling region. Preliminary studies on inhibition and activation of the NADP-linked GDH activity have also been carried out. In addition to NADP-linked GDH activity, NAD-linked GDH activity was discovered in certain species of phytoplankton. The possible physiological roles of NAD-and NADP-linked GDH enzymes are discussed.Contribution No. 911 from the Department of Oceanography, University of Washington. This research was supported by the National Science Foundation Grants GA-34165 and GX-33502.     

Molecular mechanisms of adaptation to low temperature in marine poikilotherms. Some regulatory properties of dehydrogenases from two arctic species

The properties of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase from gill tissue of the tanner crab Chionocetes bairdi, and lactate dehydrogenase (LDH) and glyceraldehyde dehydrogenase from skeletal muscle of C. bairdi and the yellowfin sole Limanda aspera were examined over the physiological temperature range of the animals. Both animals were obtained in the Bering Sea in winter, and their enzymes appear to be remarkably cold-adapted. Affinity of sole LDH for substrate appears to increase with decreasing temperature, thus keeping reaction rate essentially independent of temperature at physiological concentrations of the substrate. Calculated values of activation energy are low, in keeping with the argument that organisms from cold environments have enzymes with a reduced energy of activation. In addition, Hill plots of the substrate saturation curves for lactate dehydrogenase from muscle of sole indicate that there is a facilitation of allosteric behaviour at low temperatures. Maximum affinity of sole LDH for substrate in the absence of univalent cations occurs at 3°C, while in the presence of 150 mN K⁺, it occurs between 0° to-2°C. The effects of Mg²⁺ on enzyme activity appear to be determined by concentration of substrate and temperature. Thus, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase are stimulated more effectively by Mg²⁺ at low temperature and at low substrate levels whereas, at high concentrations of substrate, they are relatively independent of the bivalent cation. All four dehydrogenases are affected by the univalent cations Na⁺, K⁺ and NH₄ ⁺ in a manner which appears to be determined, in part at least, by concentration of substrate and by temperature. These findings suggest mechanisms for the maintenance and regulation of enzyme activity in poikilothermic tissues at low and changing temperatures.     

Effects of methionine sulfoximine on growth and nitrogen assimilation of the marine microalga Chlorella autotrophica

Chlorella autotrophica Shihira and Krauss (clone 580), a euryhaline microalga from the marine coastal environment is subject to large fluctuations in external salinity and nitrogen supply. The alga exhibits maximum growth at salinities lower than 100% ASW (artificial seawater). Cells divide faster and show higher cell yields when the supply of either NH ₄ ⁺ or NO ₃ ^- is increased above 0.2 mM. Cells growing on NH ₄ ⁺ show high levels of NADPH-glutamate dehydrogenase (GDH) activity, and the levels of glutamine synthetase (GS) are decreased to very low levels under these conditions. Methionine sulfoximine (MSX), an inhibitor of GS, has little effect on cell division and nitrogen assimilation of cells growing on NH ₄ ⁺ . Cells growing on NO ₃ ^- , however, show marked inhibition (65%) in nitrogen assimilation in the presence of 5 mM MSX. This MSX concentration also causes growth retardation and a progressive decrease in cell protein and nitrogen content. GS is almost completely inhibited by 5 mM MSX in both NH ₄ ⁺ and NO ₃ ^- -grown cells. Cells growing on NH ₄ ⁺ maintain high levels of NADPH-GDH activity in the presence of MSX. NADPH-GDH activity in MSX-treated NO ₃ ^- -grown cells increases, and, in the presence of 5 mM MSX, reaches 40% of the level found in NH ₄ ⁺ -grown cells. These results are consistent with NADPH-GDH providing an alternate pathway for NH ₄ ⁺ assimilation by this marine Chlorella species.     

Photosynthetic carbon acquisition in the red alga Gracilaria conferta

In this continuing study on photosynthesis of the marine red alga Gracilaria conferta, it was found that ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in crude extracts had a K _m (CO₂) of 85 M. Since seawater contains only ca. 10 M CO₂, it appears that this alga must possess a CO₂ concetrating system in order to supply sufficient CO₂ to the vicinity of the enzyme. Because this species is a C₃ plant (and thus lacks the C₄ system for concentrating CO₂), but can utilize HCO₃ ^- as an exogenous carbon source, we examined whether HCO₃ ^- uptake could be the initial step of such a CO₂ concetrating system. The surface pH of G. conferta thalli was 9.4 during photosynthesis. At this pH, estimated maximal uncatalyzed HCO₃ ^- dehydration (CO₂ formation) within the unstirred layer was too slow to account for measured phostosynthetic rates, even in the presence of an external carbonic anhydrase inhibitor. This observation, and the marked pH increase in the unstirred layer following the onset of light, suggests that a HCO₃ ^- transport system (probably coupled to transmembrane H⁺/OH^- fluxes) operates at the plasmalemma level. The involvement of surface-bound carbonic anhydrase in such a system remains, however, obscure. The apparent need of marine macroalgae such as G. conferta for CO₂ concentrating mechanisms is discussed with regard to their low affinity of Rubisco to CO₂ and the low rate of CO₂ supply in water. The close similarity between rates of Rubisco carboxylation and measured photosynthesis further suggests that the carboxylase activity, rather than inorganic carbon transport and intercoversion events, could be an internal limiting factor for photosynthetic rates of G. conferta.