Marine Agar Medium Research Articles

ARDRA와 DGGE를 이용한 Halichondria panicea 해면의 공생세균 다양성

Department of Biological Science and Biotechnology, Hannam University, Daejeon 34054, Republic of Korea(Received December 23, 2015; Accepted December 24, 2015)ABSTRACT: Culture-dependent ARDRA and culture-independent DGGE were employed to investigate the bacterial community associated with the marine sponge Halichondria panicea collected from Jeju Island. A total of 120 bacterial strains associated with the sponge were cultivated using modified Zobell and Marine agar media. PCR amplicons of the 16S rRNA gene from the bacterial strains were digested with the restriction enzymes HaeIII and MspI, and then assigned into different groups according to their restriction patterns. The 16S rRNA gene sequences derived from ARDRA patterns showed more than 96% similarities compared with known bacterial species, and the isolates belonged to four classes, Alphaproteobacteria , Gammaproteobacteria , Bacteroidetes , and Firmicutes , of which Alphaproteobacteria was dominant. DGGE fingerprinting of 16S rRNA genes amplified from the sponge-derived total gDNA showed 14 DGGE bands, and their sequences showed 100% similarities compared with the sequences available in GenBank. The sequences derived from DGGE bands revealed high similarity with the uncultured bacterial clones. DGGE revealed that bacterial community consisted of seven classes, including Alphaproteobacteria , Gammaproteobacteria , Acidobacteria , Actinobacteira , Bacteroidetes , Cyanobacteria , and Chloroflexi . According to both the ARDRA and DGGE methods, three classes, Alphaproteobacteria , Gamma-proteobacteria , and Bacteroidetes , were commonly found in H. panicea. However, overall bacterial community in the sponge differed depending on the analysis methods. Sponge showed more various bacterial community structures in culture independent method than in culture-dependent method.Key words: Halichondria panicea , 16S rRNA gene, ARDRA, bacterial diversity, DGGE, marine sponge

독도 해안식물로부터 분리된 호염성 세균들의 특성 및 계통학적 분석

독도의 해안에 군락을 이룬 해안식물 근권에서 호염성 및 염내성을 가지는 세균의 분리를 위해 3종의 해안식물의 군락을 선정한 후 각 식물의 군집 하부에서 토양시료를 채취하였다. 시료는 marine broth 한천배지를 이용하여 형태학적인 구분을 통해 순수분리 되었다. 분리된 161개 세균들을 NaCl 9.0% 농도로 조정된 배지에서 생존하는 26개 균주를 선발하여 genomic DNA를 얻은 후, 16S rRNA gene sequence를 증폭하여 부분동정 하였다. 이들의 유연관계 확인을 위해 계통수를 작성한 결과, 이들은 각각 Firmicutes (30.8%), Gamma proteobacteria (53.8%), Bacteroidetes (7.7%), Alpha proteobacteria (7.7%), Actinobacteria (7.7%)에 속하였으며, 이는 기존의 독도 토양 및 해수 미생물상 연구와 특징적 차이를 보인다. 또한, 분리된 세균의 종 조성도 기존 독도 토양 및 해양연구와 유의적으로 상이함을 보였다. 이에 더하여 선발된 26개 균주들 중에서 4균주가 12.0% 이상의 염농도에서 생장하였으며, 이들 중에서 3개 균주가 15.0% 이상의 염농도에서 생장하여 극호염성의 특성을 나타내었으며, 광범위한 염분농도에서도 생장하는 특성을 보였다. 이들은 해안식물 근권에서 독도 특유의 고염분 및 염분변화라는 환경적 스트레스를 극복하며 해안식물과 어떠한 상호작용을 하는 것으로 생각된다. To study the halobacterial diversity at the rhizospheric soil of coastal plant native to Dokdo islands, several host plant were selected and its rhizospheric soil was sampled. Soil sample was diluted serially and pure isolation was done by sub-culture using marine agar media. 26 halophilic strains cultivable at the marine medium containig concentration of 9.0% sodium chloride were selected among total 161 isolates. Their partial 16S rRNA gene sequences extracted from genomic DNA were analyzed and partially identified. Furthermore, to identify their genetic relationship, phylogenetic tree was deduced. Total 26 strains were belongs to Firmicutes (30.8%), Gamma proteobacteria (53.8%), Bacteroidetes (7.7%), Alpha proteobacteria (7.7%), and Actinobacteria (7.7%). These results showed the specific difference from previous researches which has been reported the microbial flora of soil or sea water around the Dokdo islands. Furthermore, 4 among 26 halophilic strains grew at above 12.0% NaCl concentrated marine broth, and 2 strains Idiomarina abyssalis LM4H23 and Halomonas huangheensis AS4H13 grew at 15.0% concentration. These halophilic strains thought to overcoming the severe stress like high salt concentration or variation derived from Dokdo-specific climate and might have unknown, specific relationship with their host coastal plant native to Dokdo islands.

Analysis of Carotenoid Production by Halorubrum sp. TBZ126; an Extremely Halophilic Archeon from Urmia Lake.

Carotenoids are of great interest in many scientific disciplines because of their wide distribution, diverse functions and interesting properties. The present report describes a new natural source for carotenoid production. Halorubrum sp., TBZ126, an extremely halophilic archaeon, was isolated from Urmia Lack following culture of water sample on marine agar medium and incubation at 30 °C. Then single colonies were cultivated in broth media. After that the cells were collected and carotenoids were extracted with acetone-methanol (7:3 v/v). The identification of carotenoids was performed by UV-VIS spectroscopy and confirmed by thin layer chromatography (TLC) in the presence of antimony pentachloride (SbCl5). The production profile was analyzed using liquid-chromatography mass spectroscopy (LC-MS) techniques. Phenotypic characteristics of the isolate were carried out and the 16S rRNA gene was amplified using polymerase chain reaction (PCR). LC-MS analytical results revealed that produced carotenoids are bacterioruberin, lycopene and β-carotene. Bacterioruberin was found to be the predominant produced carotenoid. 16S rRNA analysis showed that TBZ126 has 100% similarity with Halorubrum chaoviator Halo-G*T (AM048786). Halorubrum sp. TBZ126, isolated from Urmia Lake has high capacity in the production of carotenoids. This extremely halophilic archaeon could be considered as a prokaryotic candidate for carotenoid production source for future studies.

Coralslurrinella hongkonensis gen. nov., sp. nov., a novel bacterium in the family Psychromonadaceae, isolated from the coral Platygyra carnosus

A novel bacterial strain, JLT2006T, was isolated from the scleractinian coral Platygyra carnosus, located in Hong Kong, China. Cells of this strain were Gram-negative, rod-shaped or oval-shaped and motile by the means of polar flagella. They formed faint-yellow, round colonies on marine agar medium. Phylogenetic analyses based on the 16S rRNA gene sequence indicated that the strain JLT2006T belonged to the class Gammaproteobacteria and was most closely related to Alteromonas-like bacteria of the genera Psychromonas, Pseudoalteromonas, Moritella, Shewanella and Ferrimonas, with less than 93 % sequence similarity. The predominant fatty acids were identified as C18:1x7c/C18:1x6c (23.0 %), C16:1x7c/C16:1x6c (18.2 %) and C16:0 (16.4 %). The quinone was menaquinone-7 (100 %). The polar lipids were determined to be phosphatidylglycerol, phosphatidylethanolamine, phospholipid, glycolipid and lipid. The genomic DNA G?C content was 40.3 mol%. Based on the 16S rRNA gene sequence as well as the physiological and biochemical features that separate the strain JLT2006T from other recognized bacteria, a novel species of a new genus with the name Coralslurrinella hongkonensis gen. nov., sp. nov. is proposed. The type strain is JLT2006T (=JCM 18796T = CGMCC 1.10992T).

Antibacterial activity of pigment produced from Micrococcus luteus KF532949

The problems of synthetic pigment causing toxicity and carcinogenicity in the human body decrease its use. Therefore, interest in natural pigment production is increasing. Therefore, the marine environment has recently become an attractive research subject for many investigations, because of its rich biodiversity. Attempt has been initiated to isolate Micrococcus luteus from marina beach water. The sea water sample isolated was allowed to grow in a Zobell marine agar medium and incubated at 37 °C for one week. The isolated strain from marine source was confirmed by 16S rRNA sequencing. The GC content was found to be 58.6%. Phylogenetic tree analysis was performed to know the sequences that are closely related. The strain was assessed for pigment production in Zobell marine broth and incubated at 37 °C for two days. The swab sticks collected from hospital were streaked directly on the labelled agar plates such as eosin methylene blue, MacConkey, nutrient, blood, mannitol salt agar and incubated at 37 °C for 24 h and characterized. The antibacterial activity of crude pigment was tested against wound isolates. The individual yellow colour colonies obtained was sequenced, submitted to gene bank under the accession number KF532949. Phylogenetic analysis revealed, that the identified strain M. luteus is closely related to various Micrococcus sp. The crude pigment showed promising results against Staphylococcus sp., Klebsiella sp., Pseudomonas sp. isolated from wound. From this result, we can conclude that the isolated strain M. luteus is able to act against both gram positive and gram negative bacteria.

Algoriphagus faecimaris sp. nov., isolated from coastal sediment

A Gram-negative, non-motile, non-sporulating bacterial strain, designated LYX05(T), was isolated from coastal sediment of Qingdao, China, on the coast of the Yellow Sea. Strain LYX05(T) was aerobic and heterotrophic. The strain grew optimally at 37 °C and pH 7.5 and in the presence of 2% (w/v) NaCl. Colonies were 1-2 mm in diameter, circular, reddish orange and shiny with entire edges on marine agar medium. Cells were rods (0.3-0.5 µm wide and 0.8-1.6 µm long). The dominant fatty acids were iso-C(15:0) (40.82%) and C(16:0) (10.45%). The DNA G+C content was 42.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain LYX05(T) was phylogenetically related to the members of the genus Algoriphagus and the closest relative was Algoriphagus hitonicola 7-UAH(T) (95.8% 16S rRNA gene sequence similarity). On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain LYX05(T) was considered to represent a novel species of the genus Algoriphagus, for which the name Algoriphagus faecimaris sp. nov. is proposed. The type strain is LYX05(T) (=JCM 16561(T) =DSM 23095(T) =LMG 25474(T)).

Eionea nigra gen. nov., sp. nov., a gammaproteobacterium from the Mediterranean Sea

A novel aerobic, gram-negative bacterial strain, designated 17X/A02/237(T), was isolated from waters of the coastal north-western Mediterranean Sea. Cells were motile straight rods and formed dark-grey colonies on marine agar medium. Strain 17X/A02/237(T) contained ubiquinone Q-8 and its major fatty acids were C(16 : 1)ω7c and/or iso-C(15 : 0) 2-OH, C(18 : 1)ω7c, C(16 : 0), C(18 : 0) and C(10 : 0) 3-OH. The G+C content of the genomic DNA was 47.5 mol%. Phylogenetic analysis of the 16S rRNA gene sequence placed the strain in the class Gammaproteobacteria. Based on 16S rRNA gene sequence data, as well as physiological and biochemical characteristics, this isolate represents a novel species of a new genus, for which the name of Eionea nigra gen. nov., sp. nov. is proposed. The type strain is 17X/A02/237(T) ( = DSM 19752(T) = CIP 109759(T) = MOLA 288(T)).

Thalassobaculum salexigens sp. nov., a new member of the family Rhodospirillaceae from the NW Mediterranean Sea, and emended description of the genus Thalassobaculum

A novel Gram-negative bacteria, named CZ41_10a(T), was isolated from coastal surface waters of the north-western Mediterranean Sea. Cells were motile, pleomorphic rods, 1.6 mum long and 0.7 mum wide and formed cream colonies on marine agar medium. The G+C content of the genomic DNA was 65 mol%. Phylogenetic analysis of 16S rRNA gene sequences placed the new isolate in the genus Thalassobaculum, a member of the family Rhodospirillaceae, class Alphaproteobacteria. Unlike Thalassobaculum litoreum CL-GR58(T), its closest relative, strain CZ41_10a(T) was unable to grow anaerobically and did not exhibit nitrate reductase activity. On the basis of DNA-DNA hybridization, fatty acid content and physiological and biochemical characteristics, this isolate represents a novel species for which the name Thalassobaculum salexigens sp. nov. is proposed. The type strain is CZ41_10a(T) (=DSM 19539(T)=CIP 109604(T)=MOLA [corrected] 84(T)). An emended description of the genus Thalassobaculum is also given.

Haliea rubra sp. nov., a member of the Gammaproteobacteria from the Mediterranean Sea

A novel aerobic, Gram-negative bacterium, designated strain CM41_15a(T), was isolated from the surface of coastal waters of the north-western Mediterranean Sea. Cells were non-motile straight rods that formed red colonies on marine agar medium. The G+C content of the genomic DNA was 65 mol %. Phylogenetic analysis of the 16S rRNA gene sequence placed the strain in the genus Haliea within the class Gammaproteobacteria. On the basis of the 16S rRNA gene sequence comparison and physiological and biochemical characteristics, strain CM41_15a(T) represents a novel species, for which the name Haliea rubra sp. nov. is proposed. The type strain is CM41_15a(T) (=DSM 19751(T)=CIP 109758(T)=MOLA 104(T)).

Nisaea denitrificans gen. nov., sp. nov. and Nisaea nitritireducens sp. nov., two novel members of the class Alphaproteobacteria from the Mediterranean Sea

Two novel Gram-negative bacteria, designated strains DR41_21(T) and DR41_18(T), were isolated from coastal, surface waters of the north-western Mediterranean Sea. The cells were motile, pleomorphic rods, 2.9 microm long and 0.9 microm wide and formed cream colonies on marine agar medium. The G+C content of the genomic DNA was 60 mol%. Phylogenetic analysis of 16S rRNA gene sequences positioned the isolates in the class Alphaproteobacteria within the family Rhodospirillaceae. The 16S rRNA gene sequence similarity of the two strains was 98.8 % but DNA-DNA hybridization indicated only 55 % relatedness. Strain DR41_21(T) was able to denitrify and possessed nirK and nosZ genes, unlike strain DR41_18(T), which possessed only nirK. These isolates represent two novel species of a new genus, Nisaea gen. nov., for which the names Nisaea denitrificans sp. nov. and Nisaea nitritireducens sp. nov. are proposed. The type strain of Nisaea denitrificans is DR41_21(T) (=DSM 18348(T)=CIP 109265(T)=OOB 129(T)) and the type strain of Nisaea nitritireducens is DR41_18(T) (=DSM 19540(T)=CIP 109601(T)=OOB 128(T)).

Balneola alkaliphila sp. nov., a marine bacterium isolated from the Mediterranean Sea

A novel aerobic, Gram-negative bacterium, designated strain CM41_14b(T), was isolated from surface waters in the coastal north-western Mediterranean Sea. Cells were non-motile, straight rods, 2.6 microm long and 0.7 microm wide and formed pale-orange colonies on marine agar medium. The G+C content of the genomic DNA was 39 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence placed the strain within the genus Balneola (phylum Bacteroidetes). On the basis of 16S rRNA gene sequence comparisons, and physiological and biochemical characteristics, the isolate represents a novel species for which the name Balneola alkaliphila sp. nov. is proposed. The type strain is CM41_14b(T) (=DSM 19538(T)=CIP 109603(T)=OOB 103(T)).

Haliea salexigens gen. nov., sp. nov., a member of the Gammaproteobacteria from the Mediterranean Sea

A novel aerobic, Gram-negative bacterium, designated 3X/A02/235T, was isolated from the surface of coastal waters in the north-western Mediterranean Sea. Cells were motile, straight rods, 1.6 microm long and 0.5 microm wide, and formed cream colonies on marine agar medium. The G+C content of the genomic DNA was 61 mol%. Phylogenetic analysis of the 16S rRNA gene sequence placed the strain in the class Gammaproteobacteria and within the family Alteromonadaceae. On the basis of 16S rRNA gene sequence comparisons and physiological and biochemical characteristics, this isolate represents a novel species of a novel genus, for which the name Haliea salexigens gen. nov., sp. nov. is proposed. The type strain of Haliea salexigens is 3X/A02/235T (=DSM 19537T =CIP 109602T =MOLA 286T).

Phylogenetic analysis of epiphytic marine bacteria on Hole-Rotten diseased sporophytes of Laminaria japonica

During an occurrence of Hole-Rotten Disease of Laminaria japonica in a cultivating farm in Ma Shan Shandong province, China, 42 Gram-negative epiphytic marine bacteria were isolated and purified on Zobell 2216E marine agar medium. Morphological and biochemical characteristics of each isolated bacterium were studied, and molecular identification of bacterial strains was conducted with polymerase chain reaction amplification to 16S rRNA gene sequence analysis. Based on nearly full length of 16S rRNA gene sequence analysis, the isolated strains were bacteria that belong to genus Pseudoalteromonas, Vibrio, Halomonas and Bacillus. The percentage of each group was 61.9%, 28.6%, 7.1% and 2.4% respectively. The results of pathogenicity assay showed that 12 strains could cause the disease symptoms in sporophytes of L. japonica. They belonged to the genera Pseudoalteromonas, Vibrio and Halomonas with 58.3%, 33.3%, 8.3% respectively. The results suggest that these bacteria are the dominant marine bacteria on diseased sporophytes of L. japonica and may be the potential pathogenic bacteria associated with Hole-Rotten Disease of L. japonica.

Idiomarina homiensis sp. nov., isolated from seashore sand in Korea

A halophilic, aerobic bacterium, designated PO-M2(T), was isolated from seashore sand, from Pohang, Korea and characterized on the basis of physiological and biochemical features. Phylogenetic analysis of 16S rRNA gene sequences revealed a clear affiliation of the novel strain with members of the genus Idiomarina. Sequence similarities between strain PO-M2(T) and the type strains of species belonging to the genus Idiomarina ranged from 94.3 to 95.5 %. Cells of strain PO-M2(T) were straight or slightly curved rods and formed light-yellow colonies on marine agar medium. The major isoprenoid quinone was ubiquinone (Q-8) and the predominant cellular fatty acids were C(15 : 0) iso (19.3 %), C(17 : 1)omega9c iso (11.9 %), C(17 : 0) iso (10.9 %), C(18 : 1)omega7c (10.4 %), C(16 : 0) (9.0 %) and C(16 : 1)omega7c and/or C(15 : 0) iso 2-OH (7.2 %). The G+C content of the DNA was 45.1 mol%. Based on physiological, biochemical and chemotaxonomic traits and comparative 16S rRNA gene sequence analysis, it is demonstrated that the isolate represents a novel species of the genus Idiomarina, for which the name Idiomarina homiensis sp. nov. is proposed. The type strain is PO-M2(T) (=KACC 11514(T)=DSM 17923(T)).

Balneola vulgaris gen. nov., sp. nov., a member of the phylum Bacteroidetes from the north-western Mediterranean Sea

A novel aerobic, Gram-negative bacterium, named 13IX/A01/164(T), was isolated from surface waters in the coastal north-western Mediterranean Sea. Cells were motile, straight rods, 2.5 mum long and 0.2 mum wide, and formed orange colonies on marine agar medium. The G+C content of the genomic DNA of strain 13IX/A01/164(T) was 42 mol%. Phylogenetic analysis of the 16S rRNA gene sequence placed the strain in the phylum Bacteroidetes within the family Crenotrichaceae. On the basis of 16S rRNA gene sequence comparison and physiological and biochemical characteristics, this isolate represents a novel species of a new genus, for which the name Balneola vulgaris gen. nov., sp. nov. is proposed. The type strain of Balneola vulgaris is 13IX/A01/164(T) (=DSM 17893(T)=CIP 109092(T)=OOB 256(T)).

Limited selection of sodium channel blocking toxin-producing bacteria from paralytic shellfish toxin-contaminated mussels ( Aulacomya ater)

Paralytic shellfish toxins (PSTs) are sodium channel blocking (SCB) toxins, produced by cyanobacteria, as well as by marine dinoflagellates and their associated bacteria, and cause serious health and economic concern worldwide. In a previous study, approximately 70% of the bacteria enriched from PST-contaminated shellfish tissue and isolated on marine agar medium were observed to produce SCB toxins. In the study reported here, the high percentage of cultivable toxigenic bacteria is demonstrated to be obtained through a marked selection on marine agar medium. The cultivable as well as the total bacterial diversity associated with PST-contaminated shellfish collected from the Magallanes region in the south of Chile has been analysed. Approximately 80% of bacterial isolates, analysed by restriction analysis of PCR amplified ribosomal DNA (i.e., ARDRA fingerprinting), were limited to only two genotypic OTUs (operational taxonomic unit). Sequence determination and analysis of the 16S rDNA from representative isolates of both OTUs established them to be closely related to species of the Psychrobacter genus of the γ-subclass of the Proteobacteria. The total bacterial diversity in the shellfish was further analysed, using a cultivation-independent strategy of extraction of total DNA from contaminated tissue, PCR-amplification of bacterial 16S rRNA genes, cloning of the PCR products and analysis of the cloned 16S rDNA sequence types by fingerprinting and sequencing. Only 2% of the cloned sequence types corresponded to species of the Psychrobacter genus. The 16S rDNA sequence types detected clustered with species of the γ-Proteobacteria subclass, the Cytophaga-Flexibacter-Bacteroides (CFB), the Fusobacteria and the Firmicutes phyla. The level of diversity observed within the libraries of cloned 16S rDNA was markedly greater than that observed among isolates obtained through marine agar enrichment cultures from the same shellfish tissue. Additionally the predominant cloned 16S rDNA sequence types detected from samples of the surrounding seawater demonstrated no correlation with those observed in the PST-contaminated mussels.

Survey of petroleum-degrading bacteria in coastal waters of Sunderban Biosphere Reserve

A survey of petroleum-degrading bacteria was carried out in the Indian part of deltaic Sunderbans to evaluate the distribution of the naturally occurring petroleum-degrading aerobic bacteria. Bacteriological analysis of surface water samples collected from five different locations in the Hooghly–Matla river mouth showed that, depending on the location, 0.08–2.0% of the heterotrophic bacteria culturable in marine agar medium could degrade crude petroleum hydrocarbons as the sole source of carbon. In the entire study area, the number of heterotrophic bacteria ranged from 1 × 103 to 3.8 × 105 c.f.u/ml, amongst which 2.7 × 101 to 6 × 103 c.f.u/ml were petroleum degraders. There was a maximum number of petroleum-degrading bacteria in the waters of Haldia Port and its surrounding areas, where the water is highly polluted by hydrocarbon discharges from a nearby oil refinery and from the ships docking at the port. Among the isolates, identified on the basis of their Gram reaction, morphological and biochemical tests including the use of API20E strips, Pseudomonas, Mycobacterium, Klebsiella, Acinetobacter, Micrococcus, and Nocardia were the most common petroleum degraders. Other heterotrophic bacteria included several species of Escherichia, Klebsiella, non-oil-degrading Pseudomonas, Vibrio, Streptococcus, Staphylococcus and Bacillus. Following preliminary selection, five strains, showing best growth in medium with oil fraction as sole carbon source, were chosen for estimation of the efficiency of crude oil biodegradation. The selected strains belonged to Pseudomonas (two strains), Mycobacterium (two strains), and Nocardia (one strain). These strains degraded 47–78% of Arab-Mix crude oil over a period of 20 days. The best oil-degrading isolate, a strain of Pseudomonas aeruginosa, (BBW1), was found to degrade and multiply more rapidly in crude oil than the rest. BBW1 showed profuse growth in Bushnell Haas medium containing crude oil (as sole source of carbon) at high concentrations ranging from 0.2 to 20% (v/v), with optimum at 10%. As much as 75% of the oil was degraded within 72 h of incubation with the bacteria. Physicochemical analysis showed considerable decrease in initial boiling point and carbon residue of the degraded oil. The ability to degrade crude oil was found to be associated with a single 70-kb plasmid, pBN70. Resistance to the metals Mn2+ (50 mM), Mg2+ (200 mM), Zn2+ (6 mM), Ni2+ (10 mM) and antibiotics like ampicillin (10 μg/ml), cephalexin (30 μg/ml), nitrofurantoin (300 μg/ml) and penicillin (10 U/ml) were plasmid-mediated.

Microbial aspects of linear alkylbenzene sulfonate degradation in coastal water

AbstractViable and total bacteria were determined during linear alkylbenzene sulfonate (LAS) degradation in coastal seawater. Viable bacteria were determined by plate counts on marine agar media, while total bacteria were determined by flow cytometry after SYTO‐13 staining. LAS degradation was monitored by high‐performance liquid chromatography analysis. Seawater samples with LAS at 5 mg/L, incubated in the laboratory (20°C, 100 rpm, 30 d), showed in most cases a similar evolution in bacterioplankton abundance over time, characterized by three phases: (i) a progressive increase in bacterial density; (ii) a later decrease; and (iii) a fluctuating stationary phase. Bacterioplankton degraded the LAS by growing to populations with a high percentage of viable bacteria. The bacteria were rapidly grazed by protozoa, preventing anomalous high bacterial growth and ensuring the later channeling of LAS carbon to upper trophic levels.

Use of the 5-cyano-2,3-ditolyl tetrazolium chloride reduction test to assess respiring marine bacteria and grazing effects by flow cytometry during linear alkylbenzene sulfonate degradation

Viable, total and metabolically active bacteria were determined during linear alkylbenzene sulfonate degradation in coastal seawater. Viable bacteria were estimated by plate counts on marine agar media while the total and metabolically active bacteria were determined with the nucleic acid stain SYTO-13 and the tetrazolium salt 5-cyano-2,3-ditolyl tetrazolium chloride, respectively, in double stain procedures analyzed by flow cytometry. The double stain SYTO-13/5-cyano-2,3-ditolyl tetrazolium chloride is a rapid and simple method that discriminates bacterioplankton populations according to nucleic acid content and formazan formation. Linear alkylbenzene sulfonate degradation was monitored by high-performance liquid chromatography analysis. Bacterioplankton degraded linear alkylbenzene sulfonate by growing to communities with a high percentage of viable and metabolically active bacteria. The bacteria produced were rapidly grazed by protozoa; however, the grazing took place mostly on metabolically active cells, which were larger than the rest of the population.

Use of the 5-cyano-2,3-ditolyl tetrazolium chloride reduction test to assess respiring marine bacteria and grazing effects by flow cytometry during linear alkylbenzene sulfonate degradation

Viable, total and metabolically active bacteria were determined during linear alkylbenzene sulfonate degradation in coastal seawater. Viable bacteria were estimated by plate counts on marine agar media while the total and metabolically active bacteria were determined with the nucleic acid stain SYTO-13 and the tetrazolium salt 5-cyano-2,3-ditolyl tetrazolium chloride, respectively, in double stain procedures analyzed by flow cytometry. The double stain SYTO-13/5-cyano-2,3-ditolyl tetrazolium chloride is a rapid and simple method that discriminates bacterioplankton populations according to nucleic acid content and formazan formation. Linear alkylbenzene sulfonate degradation was monitored by high-performance liquid chromatography analysis. Bacterioplankton degraded linear alkylbenzene sulfonate by growing to communities with a high percentage of viable and metabolically active bacteria. The bacteria produced were rapidly grazed by protozoa; however, the grazing took place mostly on metabolically active cells, which were larger than the rest of the population.