A STUDY OF WALL ORNAMENTATION IN CULTURES OF SCENEDESMUS

In a comparative study, six strains of Scenedesmus isolated from one small pond were examined in culture. Three of the isolates, S. denticulatus, S. dispar, and S. abundans, proved to be morphologically quite stable when studied in the laboratory. In an axenic culture of any one of the three pleomorphic strains one could find coenobia which resemble S. longispina or S. armatus. High percentages of four-celled coenobia with several ridges but with only two spines (the S. semipulcher type) were found typically in older cultures of two of the pleomorphic cultures. Long, delicate appendages (bristles) were easily detected on most coenobia in desiccated preparations of all but the S. dispar culture. The use of cultures in identifying Scenedesmus coenobia is discussed.     

SCENEDESMUS MORPHOGENESIS. TRACE ELEMENTS AND SPINE FORMATION1

When Scenedesmus culture 16, a soil organism with numerous long spines, was grown in axenic culture in a dilute laboratory medium (with approximately 30 mg/liter total salts), spines were not formed. In this medium the organism resembled S. bijugatus, whereas S. longus and S. abundans colonies were typically formed in more concentrated media. Spines were formed on daughter colonies upon transfer to the dilute medium plus additional Fe EDTA or Ca EDTA. No other individual components of the medium did this. Spineless colonies were healthy and green with a well-defined chloroplast, provided the culture was transferred often. In addition to the absence of spines, cultures in the dilute medium had a linear arrangement of cells within a colony, along with terminal cells 20% shorter than median cells. With 4-celled spiny colonies all cells were the same length and were alternately arranged.     

Nitrogen and phosphate removal by free and immobilised cells of Scenedesmus bijugatus (Kützing) from the Pinang River estuary,Penang, Malaysia

This research studied the effects of inorganic nutrient removal by free and immobilized Scenedesmus bijugatus cells, measured by algal growth (i.e., the chlorophyll a concentration) and the efficiency of the uptake of inorganic nutrients by the cells (uptake rate (b) and removal percentage) in water samples from the organically polluted Pinang River estuary (PRE). Water samples from the PRE were collected during low and high tide. S. bijugatus cells had a higher growth rate when incubated in low tide PRE water samples compared to high tide PRE water samples, with a growth rate of 0.29 µgml^?1d^?1 and 0.06 µgml^?1d^?1 for free and immobilized cells, respectively. S. bijugatus was able to more efficiently remove nitrogen, especially ammonium (81–94%), compared to phosphate (62–88%) from both low and high tide water samples. S. bijugatus cells in low tide PRE water samples recorded highest phosphate (0.36 mgL^?1d^?1 and 0.25 mgL^?1d^?1 for free and immobilized cells, respectively) and ammonium uptake rates (0.44 mgL^?1d^?1 and 0.29 mgL^?1d^?1 for free and immobilized cells respectively). Both inorganic nutrient removal and microalgal cell growth were not significantly different between free and immobilized S. bijugatus (p > 0.05). The data obtained indicated that the removal of nutrients by microalgae was affected by salinity and the immobilization technique applied may have good potential for bioremediation.     

Interaction effects of insecticide combinations on the growth ofScenedesmus bijugatus andSynechococcus elongatus

The interaction effects of insecticide combinations involving an organophosphate (monocrotophos or quinalphos) and a pyrethroid (cypermethrin or fenvalerate) were determined on the growth ofScenedesmus bijugatus (unicellular green alga) andSynechococcus elongatus (unicellular cyanobacterium). In general, an organophosphate and a pyrethroid in combinations interacted additively, antagonistically, and synergistically on the growth of the two phototrophic microorganisms. However, combinations of quinalphos and cypermethrin or fenvalerate yielded additive interaction response in onlyS. bijugatus.     

Effects of phenolic compounds on growth and metabolic activities of Chlorella vulgaris and Scenedesmus bijugatus isolated from soil

Phenol and three nitrophenols (o-nitrophenol, m-nitrophenol, p-nitrophenol), commonly occurring pollutants in natural eco-systems, were tested for their toxic effects on soil isolates of Chlorella vulgaris and Scenedesmus bijugatus, growing under phototrophic, photoheterotrophic and heterotrophic conditions. The toxicity criteria included cell number, chlorophyll, total protein and carbohydrate content, ¹⁴CO₂ uptake and in vivo nitrate reductase activity. Both C. vulgaris and S. bijugatus were sensitive to the pollutants when the cultures were grown under phototrophic or heterotrophic conditions. However, the toxicity was found reversed or alleviated upon photoheterotrophic growth of the cultures. Transmission electron microscope studies revealed various cytological abnormalities in C. vulgaris in the presence of phenolics at algistatic levels.     

Salinization and wastewater effects on the growth and some cell contents of Scenedesmus bijugatus

The aim of this study is to determine the effect of salinization and wastewater stresses on the growth, some cellular contents (total soluble proteins, total soluble carbohydrates, nucleic acids, and amino acids composition) and ultrastructure using TEM of unicellular green alga Scenedesmus bijugatus. Treatment of S. bijugatus by NaCl at 10 and 50 mg L⁻¹ significantly increased the growth of this alga and its cellular macro-molecules. While, treatment above this concentration with NaCl significantly inhibited the growth and cellular macro-molecules. On the other hand, treatment by NaCl at the pre-lethal concentration (300 mg L⁻¹) had different effects on its detected amino acids. Whereas, Asp. Acid, Pro, Cys, Val, Iso-leu, leu, Phe.ala and Lys were slightly stimulated with salinization treatment. On contrast the levels of amino acids: Thr, Ser, Glu.acid, Gly, Ala, Mth, His and Arg were markedly inhibited. Ultrastructure examination of treated S. bijugatus by 300 mg L⁻¹ of NaCl for 8 days showed increase of starch granules, shrinkage of cell contents and thickening of cell wall. The recorded data indicated also that treatment by wastewater with all concentrations led to stimulatory effects on their growth and cellular macro-molecules except at 100% wastewater which had inhibitory effects on Asp., Gly., Thr., Ser., Pro., Glu., Ala., Meth., and Cyst., of S. bijugatus. Also, wastewater induced a slight change in the treated S. bijugatus as elevation in starch granules and presence of thylakoid membranes although not clear as in the control.     

Alterations of the L-forms of a sporebearing bacillus

A large pleomorphic gram-negative bacillus developed as a contaminant on blood-agar. Spores were formed in one culture. L-forms were produced with penicillin on blood-agar with 2.5% NaCl; they grew well when transplanted to agar with 0.5% NaCl. After several transplants and long incubation of the L-forms without penicillin, in three transplants small gram-negative pleomorphic bacilli grew, but no L-forms. This occurred once on blood-agar and twice on 30% gelatin. The growth obtained from these small bacilli was similar in morphology and in the physical properties of the organisms to the altered L-forms of Proteus and Salmonella. Multiplication of the pleomorphic organisms and development of branching filaments from the round forms was apparent. The original large gram-negative bacillus was regularly recovered from the L-forms, and was recovered several times from the descendants of the small bacilli. These observations are essentially similar to those made with L-forms of Proteus and with an L-form studied in 1952, indicating alterations in L-forms of bacteria which do not produce B type L-forms.     

First detailed description of Hispanomys bijugatus Mein and Freudenthal, 1971 (Rodentia, Cricetodontinae) from the Upper Aragonian of La Grive-Saint Alban (France): Biostratigraphical implications

The material of Hispanomys bijugatus (Rodentia, Cricetodontinae) from La Grive-Saint Alban (carrière Lechartier, fissure L3) is described for the first time and compared with all species of the genus known to date. As common in the Upper Aragonian populations of Hispanomys, this taxon evidences a low variability. H. bijugatus shows some progressive characters with respect to the remaining Aragonian congeneric species, such as the absence of labial and lingual cingula surrounding the upper and lower molar valleys respectively, the increase in the number of roots on the second lower molar, and the lost of mesolophs. This suggests that H. bijugatus, in spite of being one of the oldest species of the genus, is relatively derived with regard to the coeval congeneric species. Because H. bijugatus and H. decedens are believed to be closely related species within the same lineage, the fact that the former shows a more progressive dental morphology than the latter suggests that the unnamed fissure-fillings from La Grive and La Grive M (with H. decedens only) are older than La Grive L3 (with H. bijugatus only). The coexistence of both species at locality L5 suggests an intermediate age.     

The Occurrence of Fungi in the Recently Discovered Jarkowicka Cave in the Karkonosze Mts. (Poland)

Our study is one of the very few cases of speleomycological research in recently discovered caves. The aim of this research was to assess the population size of fungal colonies and their species composition in the Jarkowicka cave, discovered in 2012. The air samples were taken from one location outside the cave and from two locations inside of it. Mycological evaluation of the rocks inside the cave was performed usingswab sampling procedure. In the Jarkowicka cave we found 22 species of fungi, including 13 isolated from air at the entrance and from the walls, and 8 species from air inside the cave. Cladosporium spp. were the fungi most frequently isolated from internal atmosphere of the Jarkowicka cave, and from the external air. On the other hand, the fungi most frequently isolated from the rocks were Mucor spp. We found several species not yet described as cave inhabitants: Hypocrea pachybasioides, Cladosporium uredinicola, and Embellisia abundans. Our study may provide a basis for comparison to other similar studies conducted in frequently visited caves by tourists.     

Denitrifying activity of Xanthobacter autotrophicus strains isolated from a submerged fixed-film reactor

Xanthobacter autotrophicus strains with the ability to reduce nitrate and nitrite to either nitrous oxide or molecular nitrogen were isolated from submerged fixed-film reactors. Isolated strains were Gram-negative rods able to grow on methanol, ethanol and sucrose. The yellow cellular pigmentation, pleomorphic appearance, and the presence of poly-β-hydroxybutyrate granules suggest that the organisms might belong to the genus Xanthobacter. Comparison of 16S rDNA gene sequences demonstrated the affiliation of the strains to X. autotrophicus species. The results show that X. autotrophicus may play a role in inorganic nitrogen removal from a denitrifying submerged filter used for the treatment of contaminated groundwater. To our knowledge, no data on denitrifying activity in X. autotrophicus strains have been reported previously.     

Ultrastructure of the endosymbionts of the whitefly,Bemisia tabaci andTrialeurodes vaporariorum

Summary The ultrastructure of the mycetocytes and mycetome micro-organisms of the sweetpotato whitefly,Bemisia tabaci Genn. andTrialeurodes vaporariorum West are described. InB. tabaci, two morphologically distinct types of micro-organisms were observed in mycetocytes. The predominant type lacked a distinct cell wall, was pleomorphic in shape with a surrounding vacuole. The second type was a coccoid organism, with inner and outer cell membranes. The coccoid organism was often found in groups of varying number within vacuoles, and in many cases appeared to be undergoing degradation. InT. vaporariorum mycetocytes, pleomorphic and coccoid organisms were found, although the coccoid micro-organism inT. vaporariorum, had a thicker cell wall than the coccoid micro-organism inB. tabaci.Abbreviations C coccoid micro-organism - P pleomorphic micro-organism     

两步法培养对丰富栅藻脂肪酸和氨基酸的影响

为了提高丰富栅藻的油脂产量和评估培养条件对其营养成分的影响,对丰富栅藻的培养基正交优化后,对其进行两步法培养并分析培养前后的脂肪酸和氨基酸的变化。结果表明：两步法培养后丰富栅藻的生物量、油脂含量和油脂产量分别达到23.57 g/L、33.49%和7.58 g/L; 丰富栅藻的单不饱和脂肪酸（MUFAs）特别是C18：1（n-9）的相对含量有所上升,但是饱和脂肪酸（SFAs）和多不饱和脂肪酸（PUFAs）的相对含量有所下降; 氨基酸特别是必需氨基酸的总量降低; 但丝氨酸和蛋氨酸的含量却略有增加。     

Determination of microbial diversity in meju,fermented cooked soya beans,using nested PCR‐denaturing gradient gel electrophoresis

Aims: To identify the microbiota in meju, fermented cooked soya beans, that may directly affect the microbial communities of Korean fermented soya bean foods. Methods and Results: Using conventional bacterial 16S rDNA, bacilli‐specific 16S rDNA or fungi 18S rDNA‐specific primers, PCR products were amplified through a series of PCRs using the DNA extracted from ten meju samples. The amplicons were analysed using denaturing gradient gel electrophoresis (DGGE), which showed that Enterococcus durans was commonly detected in nine of ten meju samples. Bacillus subtilis was shown to be the major strain of bacilli in the samples tested. Based on the DGGE analysis of fungi in meju, we determined that Absidia corymbifera, Aspergillus sp. and Candida rugosa were the main fungi in the tested samples. Conclusions: A variety of bacterial and fungal micro‐organisms were identified in meju samples, in addition to the micro‐organisms already known to be present. Significance and Impact of the Study: This is the first report showing the differences and similarities in the populations of micro‐organisms in meju samples using nested PCR‐DGGE, a culture‐independent method. The results may be applicable to the development of improved meju, in which the indigenous micro‐organisms required for fermentation can be standardized.     

Correlations among culture times,sugar composition and biological activities of Sporothrix schenckii antigens

Glycoproteins were isolated by ethanol precipitation, Con A-sepharose 4B and DEAE-sephadex A-50 chromatography from culture filtrates of Sporothrix schenckii ATCC 10268 at incubation periods of 2, 7, and 14 days, and their chemical and immunological properties investigated. Sugar composition of the isolated glycoproteins varied with time of culture, i.e. from mostly mannose on the 2nd day of culture to increasing amounts of rhamnose and small amounts of galactose in addition to mannose on the 7th and 14th day. The changes in sugar composition also were observed to be closely related to the growth morphology of the organisms. The isolated glycoproteins showed different serological reactivity in immunodiffusion tests against rabbit anti-S. schenckii antiserum. In addition, they showed varying degree of cross-reaction with rabbit anti Klebsiella pneumoniae K47, anti Cladosporium werneckii and anti Saccharomyces cerevisiae antisera. The immunodiffusion results correlate well with sugar composition and strongly suggest the possibility that rhamnose, galactose and mannose determinants participate in the serological reaction of S. schenckii. In delayed hypersensitivity skin tests in guinea pigs immunized with S. schenckii, only Con A-binding glycoproteins were reactive. These fractions also resembled each other in amino acid content. The results from the present work indicate that the immunochemical properties of S. schenckii glycoproteins vary with incubation period, and suggest the need for standardization of sporotrichin test antigens.     

Sappinia sp. (Amoebozoa: Thecamoebida) and Rosculus sp. (SAR: Cercozoa) Isolated From King Penguin Guano Collected in the Subantarctic (South Georgia,Salisbury Plain) and their Coexistence in Culture

Two amoeboid organisms of the genera Sappinia Dangeard, 1896 and Rosculus Hawes, 1963 were identified in a sample containing king penguin guano. This sample, collected in the Subantarctic, enlarges the list of fecal habitats known for the presence of coprophilic amoebae. The two organisms were co‐isolated and subcultured for over 6 mo, with continuous efforts being invested to separate each one from the mixed culture. In the mixed culture, Rosculus cells were fast growing, tolerated changes in culturing conditions, formed cysts, and evidently were attracted by Sappinia trophozoites. The separation of the Rosculus strain was accomplished, whereas the Sappinia strain remained intermixed with inseparable Rosculus cells. Sappinia cell populations were sensitive to changes in culturing conditions; they improved with reduction of Rosculus cells in the mixed culture. Thick‐walled cysts, reportedly formed by Sappinia species, were not seen. The ultrastructure of both organisms was congruent with the currently accepted generic characteristics; however, some details were remarkable at the species level. Combined with the results of phylogenetic analyses, our findings indicate that the ultrastructure of the glycocalyx and the presence/absence of the Golgi apparatus in differential diagnoses of Sappinia species require a critical re‐evaluation.     

Isolation and physiological characterization of Syntrophus buswellii strain GA from a syntrophic benzoate-degrading,strictly anaerobic coculture

A stable, syntrophic benzoate-degrading bacterial consortium was enriched from sewage sludge. It oxidized benzoate or 3-phenylpropionate to acetate, H₂ and CO₂. As hydrogen scavengers Methanospirillum hungatei and Desulfovibrio sp. were present. The benzoate-degrading bacteria of this syntrophic culture and of Syntrophus buswelli were able to grow with benzoate/crotonate or crotonate alone in the absence of a hydrogen-utilizing partner organism. If crotonate was the only substrate, acetate and butyrate were produced, while during growth on benzoate or 3-phenylpropionate crotonate served as a reducible co-substrate and was exclusively converted to butyrate. In the presence of crotonate interspecies hydrogen transfer was not necessary as a hydrogen sink. The benzoate degrader was isolated as a pure culture with crotonate as the only carbon source. The pure culture could also grow with benzoate/crotonate or 3-phenylpropionate/crotonate. The effect of high concentrations of crotonate and of acetate or butyrate on growth of the benzoate degrader was investigated. The benzoate degrader was compared with S. buswellii for its morphology, physiology and DNA base composition. Except for the fact that S. buswellii was also able to grow on cinnamate, no differences between the two organisms were detected. The isolate is named S. buswelli, strain GA.     

Metalworking fluids biodiversity characterization

Aims: Hypersensitivity pneumonitis of machinists associated with metalworking fluids (MWF) was recently linked to Mycobacterium immunogenum. In addition to Mycobacterium, impacts of continuous and massive contact to other micro‐organisms, such as Pseudomonas, were little studied. This report intended to quantify and characterize the microbial load of 44 in‐use MWF. Methods and Results: The main biodiversity of MWF was assessed using cultural methods, quantitative PCR (qPCR) and denaturing gradient gel electrophoresis (DGGE). Total bacteria concentrations ranged from undetectable to 10⁹ 16S rRNA gene copies per millilitre. Concentrations obtained by qPCR were up to five orders of magnitude higher than by culture, suggesting that MWF contamination is generally underestimated. Two samples showed high concentrations of Myco. immunogenum (1·55 × 10⁷ and 3·49 × 10⁵ 16S rRNA gene copies per millilitre). The overall biodiversity was low, as observed by culture and DGGE, and was comparable to data found in the literature. Pseudomonas pseudoalcaligenes was by far the main bacteria found in MWF samples (33 out of 44), followed by Ochrobactrum anthropi (32 out of 44). There was no significant relationship between the biodiversity profiles and the kind of MWF or equipment used, making it difficult to predict which micro‐organisms will colonize each particular MWF. Conclusions: Very high concentrations of bacteria were found in most MWF studied and limited biodiversities were observed. Many species of micro‐organisms were retrieved from MWF samples, but they were mostly colonized by Pseudomonas pseudoalcaligenes and Ochrobactrum anthropi. Significance and Impact of the Study: The major micro‐organisms observed or recovered in this study from in‐use MWF were present in very high concentrations, and thus further studies are needed to confirm their role in workers’ respiratory disorders or health‐related problems.     

Activities of superoxide dismutase (SOD) isoforms during growth of <Emphasis Type="Italic">Scenedesmus</Emphasis> (chlorophyta) species and strains grown in batch-cultures

Growth of Scenedesmus species and strains, grown for 28 days in mineral BBM medium in batch-cultures, displayed sigmoidal kinetics that comprised a lag, exponential and declining growth phases. Total SOD activity in these autotrophically cultured organisms, which oscillated within 0.6 – 1.4 Umg protein⁻¹, was rather species-specific and only to some extent depended on the growth phase. Contrary, three S. obliquus strains: wild type 276-6, mutant with blocked PS I (strain 56.80) and mutant with blocked PS II (strain 57.80), cultured for 7 days on BBM medium supplemented with bacto-tryptone and yeast extract (BBM+) turned out to be time-dependent and to have several times higher total SOD activity than one obtained for Scenedesmus grown autotrophically. Regardless of the media composition, the phase of growth and studied organism, dominant isoforms of total SOD were together determined Fe- and Mn-SOD. Profiles of SOD isoforms, obtained after PAGE analysis of all autotrophically and exponentially growing organisms, revealed that one Mn-SOD and one Cu/Zn-SOD bands located on gels at the same position whereas location of three bands of Fe-SOD depended on the strain. This suggests the presence of two different groups of Fe-SODs in analyzed organisms. Identical SOD profiles found in two S. armatus strains (276-4a and 276-4d) and S. subspicatus correspond well with their taxonomic position. The SOD profile of S. armatus B1-76 distinctly differed from two other S. armatus strains but was identical to S. microspinal B1-76 and S. quadricauda G-15 despite the fact that there were significant growth rate differences between these three species. SODs profiles of S. acutus 437 and S. obliguus 453 were species-specific. In S. obliquus strains cultured on BBM+ medium, there are four SOD bands: one slightly visible band of Mn-SOD, two intensive bands of Fe-SOD and one band of Cu/Zn-SOD. The above finding suggests that antioxidant response of algae kept in batch-cultures differs according to medium composition and the SOD activity mainly restricted to chloroplasts.     

Inhibition of byssal formation in Semimytilus algosus (Gould, 1850) by a film-forming bacterium isolated from biofouled substrata in northern Chile

Abstract

Semimytilus algosus is a small mussel species that fouls artificial culture systems of the scallop Argopecten purpuratus (Lamarck, 1819) in the north of Chile. Since biofouling organisms are a serious problem in culture, competing with the scallops for food and oxygen, environmentally- friendly methods are required to mitigate the effects of this fouling in the culture systems. The present study reports the evaluation of the inhibitory effect of biofilms and extracellular products (EP) of the bacterium Alteromonas strain Ni1-LEM on the byssal formation of S. algosus juveniles. Laboratory bioassays were carried out to determine the reattachment, exploratory behaviour and/or byssal thread production of the mussel in plastic Petri dishes containing bacterial biofilms, different dilutions of EP, and EP incorporated in a test substratum. It was concluded from the results that culture supernatants of the Alteromonas tested had an inhibitory effect on reattachment by S. algosus.     

Application of 13C-[2] - and 13C-[1,2] acetate in metabolic labelling studies of yeast and insect cells

The advantage of using ¹³C-labelled glucose in metabolic studies is that it is an important carbon and energy source for almost all biotechnologically and medically important organisms. On the other hand, the disadvantage is its relatively high cost in the labelling experiments. Looking for cheaper alternatives we found that ¹³C-[2] acetate or ¹³C-[1,2] acetate is a prospective compound for such experiments. Acetate is well incorporated by many organisms, including mammalian and insect cell cultures as preferred source of acetyl-CoA. Our experimental results using ¹³C NMR demonstrated that acetate was efficiently incorporated into glutamate and alanine secreted by the insect cell culture. Using D-stat culture of Saccharomyces uvarum on glucose/¹³C-acetate mineral media we demonstrated that the labelling patterns of proteinogenic amino acids can be well predicted on the basis of specific substrate consumption rates using the modified scheme of yeast metabolism and stoichiometric modelling. According to this scheme aspartate and alanine in S. uvarum under the experimental conditions used is synthesised in the mitochondria. Synthesis of alanine in the mitochondria was also demonstrated for Spodoptera frugiperda. For both organisms malic enzyme was also operative. For S. uvarum it was shown that the activity of malic enzyme is sufficient for supporting the mitochondrial biosynthetic reactions with NADPH.