H2 production by mixed cultures

Production of H₂ from glucose by an anoxygenic phototrophic bacterium (Rhodobacter sphaeroides), a cyanobacterium (Synechococcus cedrorum) and a heterotrophic bacterium (Pseudomonas fluorescens) was tested individually and in mixed cultures of various combinations in light. H₂ production was maximal with a mixed culture of R. sphaeroides and P. fluorescens, which could be further enhanced by immobilization of the bacteria in alginate gel. Inhibition of H₂ photoproduction was observed in a mixture of S. cedrorum and P. fluorescens and a co-culture of all the three organisms.Ch. Sasikala and Ch. V. Ramana are and G. S. Prasad was with the Microbial Biotechnology Laboratory, Department of Botany, Osmania University, Hyderabad-500 007, India. G. S. Prasad is now with the Microbial Type Culture Collection Centre (MTCC), IMTECH, Chandigar, India.     

Growth and H2 production by Synechococcus spp. using organic/inorganic electron donors

Growth with simultaneous photoproduction of H₂ was obtained on various organic and inorganic compounds using axenic cultures of the oxygenic phototrophic bacteria Synechococcus sp. OU 103 and S. cedrorum. Highest H₂ production occurred with resting cells of S. cedrorum on malate (11.8 mmol H₂/vessel), whereas Synechococcus sp. OU 103 preferred sulphide (10.3 mmol H₂/vessel) as electron donor.The authors are with the Microbial Biotechnology Lab. Department of botany, Osmania University, Hyderabad 500 007, India.     

Relationship between unicellular cyanobacteria established by indirect immunofluorescence of intact cells

Antisera prepared against intact, viable cells were used to show the applicability of a serological approach to detect relationships between unicellular cyanobacteria. Antisera were raised against eight unicellular cyanobacteria and two chlorophycean unicellular organisms. The staining reactivity of each antiserum was tested by the fixed indirect immunofluorescence assay against the different organisms, and each organism was tested for its reactivity with all of the different antisera. Absorption of antisera with the appropriate heterologous antigens was used to further characterize the relationship betweenAnacystis nidulans andSynechococcus cedrorum, and also the relationship between two subcultures of an isolate distinguished by morphological features. Absorption of antiserum was also used for the removal of antibodies to contaminating bacteria. The approaches used are suggested as a useful tool for determining relationships between unicellular cyanobacteria.     

SIDEROCHROMES IN CYANOPHYCEAE: ISOLATION AND CHARACTERIZATION OF SCHIZOKINEN FROM ANABAENA SP.1

Schizokinen, a high affinity iron transport compound (siderochrome), previously described from Bacillus megaterium, has been isolated and characterized from low-iron cultures of a bluegreen alga, Anabaena sp. No conclusive evidence for production of an iron-repressible hydroxamate or catechol type siderochrome was observed in certain other bluegreen algae, such as Anabaena cylindrica Lemm., Coccochloris peniocystis (Kütz.) Drouet & Daily, Anacystis sp., Gloeocapsa alpicola (Lyng.) Born. or Chroococcus sp.     

Direct and indirect fluorescent antibody staining ofOphiobolus graminis Sacc. in culture and in the rhizosphere of cereal plants

Fluorescein isothiocyanate (FITC) conjugates were prepared to whole-cell and cell-wall fractions of four cultures ofOphiobolus graminis W1, W2, O1 and O₂. Homologous and heterologous direct and indirect FA staining of the four cultures ofO. graminis gave positive staining in all reactions. This indicated that the four cultures could not be differentiated by fluorescent antibody (FA) staining. Species specificity of the conjugates was shown by the staining ofO. graminis hyphae in the rhizosphere of wheat and oat roots. Plant tissues were not stained. Furthermore out of 52 rhizosphere isolates stained with whole-cell and cell-wall conjugates of the four cultures ofO. graminis, only 7 cross-reacted with the whole-cell conjugates whereas none cross-reacted with the cell-wall conjugates.These results indicate the potentiality of the FA staining technique as a serological tool in localizing, and identifyingO. graminis amongst mixed fungal populations in the rhizosphere of roots.     

SELECTIVE INHIBITION OF BLUE-GREEN ALGAL GROWTH BY ETHIONINE AND OTHER AMINO ACID ANALOG1,2

Some metabolic analogs, including azaguanine, azathymine, azauracil, caffeine, 4-azaleucine, dl -ethionine, and dl -p-fluorophenylanlanine, were examined for their ability to repress the multiplication of algae from natural aquatic sources grown in defined or semidefined media. dl -ethionine, dl -p-fluorophenylalanine, and 4-azaleucine, in that order, inhibited the multiplication of blue-green but not other algal groups. The purine and pyrimidine analogs were not inhibitory. In chemically defined axenic media, dl -ethionine was about 100 times more inhibitory to the blue-green algae Synechococcus cedrorum and Anabaena cylindrica than to the eucaryotic algae Ochromonas danica and Euglena gracilis. The ciliate Tetrahymena pyriformis was at least 100-fold more resistant to ethionine than the algae. The unusual sensitivity of blue-green algae to ethionine and other amino acid analogs represents an exceptional phylelic character and may be useful in the control of these algae when they become a nuisance. Amino acid analogs such as ethionine may also serve to remove blue-green algae from cultures one may desire to render axenic.     

SODIUM: SOME EFFECTS ON BLUEGREEN ALGAL GROWTH1

The growth of heterocystous bluegreen algae in various concentrations of sodium, was examined in axenic culture as well as in situ studies. Anabaena cylindrica Lemm. with no Na⁺ added, suffered from decreased rates of acetylene reduction, ¹⁴C, assimilation, excretion of organic C as well as lower concentrations of chlorophyll a and particulate organic C compared to cultures supplied with 5, 10, and 50 mg Na⁺·l⁻¹ Sodium deficient algae released, extracellularly a higher percentage of previously fixed C as organic C. No differences in any parameter measured were demonstrable among cultures grown with 5, 10, and 50 mg Na⁺·l⁻¹ High nitrate concentrations (20 mg NO₃⁻·l⁻¹) resulted in decreased rates of acetylene reduction and heterocyst numbers in. Na sufficient, and Na deficient cultures: however, decreased, cellular Na content at high NO₃⁻ levels occurred only in N deficient, cultures. Higher percentages of excreted organic C occurred with increasing NO₃⁻ concentrations in Na deficient cultures. Sodium enrichment of natural bluegreen populations with the addition of 50, 100, and 200 mg Na⁺·l⁻¹ elicited neither a stimulatory nor an inhibitory response in photosynthetic C fixation. In contrast, the addition of small amounts of Na⁺ (5 mg·l⁻) resulted in increased C fixation. However, since the Na. concentration of the lake water, at ca. 5 mg Na⁺·l⁻¹, was sufficient for growth of the bluegreens present, sodium, is not assumed to be limiting under most natural conditions. No increase in in situ acetylene reduction rates occurred with additions of sodium.     

ACUTE TOXICITY OF SOME BLUEGREEN ALGAE TO THE PROTOZOAN PARAMECIUM CAUDATUM1

Four species of bluegreen algae were tested for possible effect on the protozoan Paramecium caudatum Ehrenberg. Toxicity was demonstrated using lyophilized cells of Fischerella epiphytica Ghose and Gloeotrichia echinulata (Smith) Richter. Nostoc linckia (Roth) Bornet & Thuret failed to show any effects when lyophilized but became toxic when sonified. Anabaena flos-aquae (Lyngb.) Bréb. was nontoxic in all tests. G. echinulata was lethal at 0.1 mg·ml^?1 which is comparable to the toxic concentration of Aphanizomenon flos-aquae (L.) Ralfs reported for microcrustaceans.     

EFFECT OF CYSTEINE AND METHIONINE ON SULFATE UPTAKE AND PRIMARY PRODUCTIVITY BY AXENIC ALGAL CULTURES AND LAKE MICROPLANKTON COMMUNITIES1

The presence of up to 500 μg sulfur·l^?1 of an equimolar mixture of cysteine and methionine had virtually no effect on the SO₄^2- uptake rate of Navicula pelliculosa, (Bréb.) Hilse whereas the rate of Ankistrodesmus falcatus (Corda) Ralfs was decreased by the presence of 500 μg S· l^?1 and Anabaena flos-aquae (Lyngbye) Bréb. by 50 μg S·l^?1. Primary productivity in these axenic cultures was affected (decreased) only in A. falcatus. The C:S uptake ratio was lowest in N. pelliculosa and highest in A. falcatus. Considering these species as representative of groups of naturally occurring algae, patterns of SO₄^2- uptake and primary productivity in a eutrophic and a moderately oligotrophic lake reflected the results of the algal culturing experiments: SO₄^2- uptake rates, relative to primary productivity, were higher in the presence of diatoms and bluegreen algae and lower when green algae were present; the addition of the cysteine I methionine mixture to the lake waters decreased the rate of microplankton SO₄^2- uptake in correlation with the makeup of the algal community; primary productivity decreased upon the addition of cysteine I methionine when green algae were relatively abundant. It is concluded that, in most fresh water systems, the effects of organic sulfur pollution on algal SO₄^2- uptake and primary productivity are insignificant as compared to other ecological changes that occur due to that pollution.     

Influence of rhizobia,azotobacter and blue-green algae on N content and yield of rice

Summary Inoculation with root-nodule bacteria had favourable influence on N-uptake and yield of wheat. Since waterlogged root region of rice permits higher nitrogenase activity a pot culture experiment was conducted using same nine strains of rhizobia,Azotobacter chroococcum and bluegreen algae as inoculants.R. leguminosarum in combination with 50 kg N ha⁻¹;R. japonicum and a strain of rhizobium isolated from moong bean increased the yield of paddy cv. Pusa-33. On the other hand an adverse effect of bacterial inoculation and of applied N was observed in case of Azotobacter, and rhizobia isolated from green gram, cicer, soyabean and clover. The importance of plant type, growth conditions and application of inorganic N in determining the success of plant-rhizobial associations is emphasised.     

INTERACTION OF PLECTONEMA BORYANUM (CYANOPHYCEAE) AND THE LPP-CYANOPHAGES IN CONTINUOUS CULTURE1

Continuous culture techniques are used to study long-term population interactions between Plectonema boryanum Gomont, a filamentous bluegreen alga, and the LPP-viruses which infect it. After LPP-I (virulent cyanophage) infection of sensitive algae, 3 oscillations occur in cell density with concomitant oscillations in virus titer before final stabilization of both algal and viral concentrations. After LPP-ID and LPP-2 (temperate viruses) infection, oscillation in cell density occurred with burst of virus particles. Resistant algae always repopulated the chemostat; lysogeny was not established. The interaction between Plectonema that was resistant to virus infection and the 3 LPP-cyanophages resulted in rapid elimination of the viruses from the chemostat in the effluent. When lysogenic P. boryanum was tested, a law population of virus was present in the chemostat throughout the incubation period indicative of spontancous induction. Clones of lysogenic algae were isolated.     

CELL WALL CONSTITUENTS OF APHANOTHECE HALOPHYTICA (CYANOPHYTA)1

Some aspects of the cell wall and extracellular polysaccharide (ECPS) of the obligate halophile Aphanothece halophytica Frémy (Chroococcales) have been investigated. Extracellular polysaccharide concentration was found to remain constant on a per cell basis in medium containing from 1–3 M NaCl. The rate of ECPS production remained constant during mid-log growth phase and increased substantially as the culture reached stationary phase. The lipopolysaccharide of this organism was found to possess a low and unusual fatty acid content when compared to other chroococcalean forms. The cell wall appears to contain a typical gram-negative peptidoglycan. The covalently attached protein resembles the envelope protein of extremely halophilic bacteria in its possession of a similar molar percentage of amino acids with lipophilic R-groups and a high acidic amino acid fraction. The ECPS and cell wall fractions of A. halophytica were found to chemically more closely resemble those from other non-halophilic, chroococcalean bluegreen algae than those from the obligately halophilic bacteria.     

海水中藻菌共培养体系对碳氮磷的吸收转化

海洋环境中,细菌和微藻之间的物质交换是生源要素在自然界中迁移转化的重要方式。为进一步了解生源要素的生物地球化学循环,在实验室模拟条件下,研究了共培养体系中营养盐和有机物在细菌和微藻之间的转换。通过纯培养中肋骨条藻(Skeletonema costatum)、东海原甲藻(Prorocentrum donghaiense)、天然海水中的细菌以及藻菌混合培养,分析了营养盐和有机物随藻菌生物量的变化情况,并计算了溶解有机碳(DOC)和溶解有机氮(DON)的浓度比值[(DOC/DON)a]。结果发现,在共培养体系中,细菌对中肋骨条藻的生长有抑制作用,对东海原甲藻影响不明显;中肋骨条藻有利于细菌生长,东海原甲藻抑制细菌生长,这种不同可能与微藻的粒径有关。海洋细菌在2种藻的指数生长均期均会促进微藻吸收氨氮(NH_4-N),但在生长末期NH_4-N以释放为主。硝氮(NO_3-N)的浓度与藻的生长呈负相关,但在衰亡期NO_3-N略有增加,表明NO_3-N再生所需时间较长。细菌对硝氮的吸收量较少,但对其再生有贡献。细菌和中肋骨条藻对磷酸盐(PO_4-P)的吸收存在竞争,但与东海原甲藻的竞争关系不明显。不同培养体系中DOC浓度变化不同,在藻菌共培养体系中增加较快,纯藻培养体系中增加缓慢,在纯菌培养体系中缓慢减少。通过对DOC与DON浓度比值的分析,发现用判断颗粒有机碳(POC)来源的方法可以分析DOC的来源。     

SPECIES‐SPECIFIC VARIATION IN FATTY ACID CONCENTRATIONS OF FOUR PHYTOPLANKTON SPECIES: DOES PHOSPHORUS SUPPLY INFLUENCE THE EFFECT OF LIGHT INTENSITY OR TEMPERATURE?1

We tested, in the laboratory, the influence of light intensity, temperature, and phosphorus (P) supply on fatty acid (FA) concentrations of four freshwater algae: the green algae Scenedesmus quadricauda (Turpin) Bréb. and Chlamydomonas globosa J. Snow, the cryptophyte Cryptomonas ovata Ehrenb., and the diatom Cyclotella meneghiniana Kütz. We investigated the main and interactive effects of two variables on algal FA concentrations (i.e., light intensity and P supply or temperature and P supply). Interactive effects of light intensity and P supply were most pronounced in C. meneghiniana, but were also found in S. quadricauda and C. ovata. Changes in several saturated and unsaturated FA concentrations with light were more distinct in the low‐P treatments than in the high‐P treatments. Interactive effects of temperature and P supply on various FA concentrations were observed in all four species, but there was no consistent pattern. In lake ecosystems, P limitation often coincides with high light intensities and temperatures in summer. Therefore, it is important to examine how combinations of these environmental conditions affect FA concentrations of primary producers that are important sources of FAs for higher trophic levels.     

Metronidazole and the isolation of temperature-sensitive photosynthetic mutants in cyanobacteria

A procedure has been developed for use of metronidazole (2-methyl-5-nitroimidazole-1-ethanol) as an enrichment agent during the isolation of temperature-sensitive, photosynthetic mutants in the cyanobacteriumSynechococcus cedrorum. The protocol includes incubation with this drug following mutagenesis withN-methyl-N-nitro-N-nitrosoguanidine. Incubation of photosynthetically activeS. cedrorum cells with 1 mM metronidazole causes a light-dependent reduction of cell viability. Maximum reduction in cell viability occurred following 6 h of incubation. Cessation of electron transport reduced the impact of the drug by five orders of magnitude. Yet during the time of incubation, metronidazole did not influence the electron transport capacities of theS. cedrorum cells, suggesting that the thylakoid membrane was not the target of the toxic effects of this drug. In addition, this drug was found to be an effective electron acceptor to photosystem I although high concentrations were required to observe maximum rates of electron transfer. Metronidazole interacted in a noncompetitive manner with methyl viologen, which suggested that those two acceptors to photosystem I have unique reduction sites on theS. cedrorum thylakoid membrane. The temperature-sensitive strains that were isolated using the procedure presented here were assessed for photosynthetic electron transport and chlorophyll fluorescence (induction kinetics and low-temperature emission spectra) characteristics. Approximately one-half of the temperature-sensitive mutants isolated possessed abnormal photosynthetic properties when shifted to the restrictive temperature (40°C). A total of 31 strains have been characterized and initially classified, showing abnormalities throughout the photosynthetic electron-transport chain.     

Formation of N-Carboxymethyl Amino Acid from the Reaction of α-Amino Acid with Glyoxal

Enrichment cultures in a medium containing 0.1% methanol and 0.1% bicarbonate at pH 7.0 under anaerobic conditions in the light became mainly green in color. Forty-four enrichment cultures, which showed abundant growth, were obtained from 46 different sources and found to contain cells of methanol-utilizing bacteria and green algae as predominant members. From these enrichment cultures, two strains of bacteria and two strains of algae were isolated. The microorganisms isolated were designated as bacterium No. 7, bacterium No. 8, Chlorella sp. A-1 and Chlorella sp. B-1, respectively. Stable mixed cultures were easily formed by mixing the isolated cultures of bacteria and algae. Both methanol and bicarbonate were necessary for the growth of the mixed cultures under anaerobic-light conditions. Growth behavior of the mixed cultures was examined on a medium containing 0.1% methanol and 0.1 % bicarbonate at 30°C in the light (about 6000 lx). The maximum specific growth rate for the cultures, µ_max, was 0.092 hr^?1 (doubling time, 7.5 hr). The maximum cell yield was 0.87 g dry-cell weight per g of methanol used. The protein content of the biomass was 65%.     

The production of clonal and axenic cultures of microalgae using fluorescence-activated cell sorting

Unialgal cultures of the flagellate algae Cyanophora paradoxa, Haematococcus lacustris, Monomastix sp., Scherffelia dubia and Spermatozopsis similis which contained bacteria were sorted by flow cytometry to obtain axenic clonal cultures. The variables used for fluorescence-activated cell sorting (FACS) were chlorophyll autofluorescence, forward scatter and side scatter of the laser beam. To produce clonal cultures, a single cell was sorted into each culture flask. Depending on the species, about 20–30% of the sorted cultures grew successfully and at least 20% of these were axenic even if the numerical ratio betweeen bacteria and algae in the original cultures was as high as 300:1. FACS represents an effective and rapid method for the preparation of clonal and axenic cultures of microalgae.     

Natural diet of herbivorous zooplankton in Lake Xolotlán (Managua)

The natural diet of herbivorous zooplankton was analyzed for a better understanding of the trophic relationships in Lake Xolotlán (Managua). Gut content of the most common zooplankton species (Arctodiaptomus dorsalis, Moina micrura andDiaphanosoma fluviatile) were analyzed. The quality and the relative abundance of ingested algae was studied. The most consumed algae during the dry season (February) wereCyclotella, Oocystis andLyngbya, and during the rainy season (November)Cyclotella, Oocystis andChroococcus. The kind of food ingested byA. dorsalis andM. micrura was similar in both periods. There was no similarity with the diet ofD. fluviatile. During the dry season, when algal biomass in the lake was high,A. dorsalis andM. micrura ingested a higher proportion of diatoms. During the rainy season, when food was scarce, the diet was more diverse and bluegreen algae (Chroococcus) were ingested in a larger proportion.Diaphanosoma fluviatile ingested predominantly tiny particles (bacteria and detritus) and algal food consisted mainly of green algae (Oocystis) and probably (not studied) nanoplanktonic algae. The results were consistent with those obtained in other lakes, indicating the importance of diatoms and green algae for zooplankton nutrition.     

CYTOCHROME c548 IN NOSTOC SP. (CYANOPHYCEAE): AN ELECTRON ACCEPTOR FROM REDUCED NADP IN THE DARK1

A c-type, carbon monoxide-binding cytochrome was isolated and studied from an endophytic Nostoc sp. Enzymatic transfer of electrons from NADPH to this cytochrome was demonstrated in cell-free preparations under dark, anaerobic conditions, probably mediated by NADPH-cytochrome reductase or ferredoxin-NADP reductase present in the extracts. This cytochrome, with an α-peak at 548 nm, had physicochemical properties similar to the low potential c-type cytochrome, cytochrome c₅₄₉, previously described by other workers from Anacystis nidulans. The participation of such c-cytochromes in dark electron transport, possibly respiratory, of bluegreen algae is strongly indicated.     

GROWTH OF ULVA FENESTRATA (CHLOROPHYTA) IN MICROCOSMS RICH IN ZOSTERA MARINA (ANTHOPHYTA) DETRITUS1,2

In laboratory experiments discs cut from thalli of Ulva fenestrata Postels & Ruprecht grew in diameter and biomass (dry weight and ash-free dry weight) more when dead leaves of Zostera marina L. were present than when absent. A maximum increase in dry weight of 500% in 14 days occurred with Zostera present compared with only a 200% increase with no detritus. When NO^?₃ and PO^3-₄ were added the weight of an Ulva disc increased by over 800% when Zostera was present but by only 400% in controls. In general penicillin G (475 units/ml) caused a reduction in algal growth in cultures containing detritus. It is concluded that bacteria on the detritus may compete with algae for nutrients but by releasing unknown substances the bacteria promote the growth of Ulva.