A Saponification Method for Chlorophyll Removal from Microalgae Biomass as Oil Feedstock.

Microalgae oil is an optimal feedstock for nutraceutical, pharmaceutical and biodiesel production, but its high levels of chlorophyll limit its large-scale application. To date, few effective approaches have been developed to remove chlorophyll from microalgae oil. The main purpose of this study was to present a preprocessing method of algae oil feedstock (Scenedesmus) to remove chlorophyll by saponification. The results showed that 96% of chlorophyll in biomass was removed. High quality orange transparent oil could be extracted from the chlorophyll reduced biomass. Specifically, the proportion of neutral lipids and saturation levels of fatty acids increased, and the pigments composition became carotenoids-based. The critical parameters of chlorophyll reduced biodiesel conformed to the standards of the USA, China and EU. Sodium copper chlorophyllin could be prepared from the bleaching effluent. The results presented herein offer a useful pathway to improve the quality of microalgae oil and reduce the cost of microalgae biodiesel.

Gelatiniphilus marinus gen. nov., sp. nov., a bacterium from the culture broth of a microalga, Picochlorum sp. 122, and emended description of the genus Hwangdonia.

A Gram-stain-negative, non-motile, non-spore-forming, rod-shaped bacterium, designated strain GYP-24T, was isolated from the culture broth of a marine microalga, Picochlorum sp. 122. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain GYP-24T forms a robust cluster with H.wangdoniaseohaensis KCTC 32177T (95.8 % sequence similarity) in the family Flavobacteriaceae. Growth of strain GYP-24T was observed at 15, 22, 28, 30, 33 and 37 °C (optimal 30-33 °C), pH 6.0-10.0 (optimal pH 7.0-8.0) and in the presence of 0.5-4 % (w/v) NaCl (optimal 2-3 %). The only menaquinone of strain GYP-24T was MK-6, and the G+C content of the genomic DNA was 36.9 mol%. The major fatty acid profile comprised iso-C17 : 0 3-OH, summed feature 3 (C16 : 1 ω7c/ω6c), iso-C15 : 1 G and iso-C15 : 0. The major polar lipids of strain GYP-24T were phosphatidylethanolamine, one unidentified phospholipid, three unidentified aminolipids and three unidentified lipids. Comprehensive analyses based on polyphasic characterization of GYP-24T indicated that it represents a novel species of a new genus, for which the name Gelatiniphilus marinus gen. nov., sp. nov. is proposed. The type strain is GYP-24T (=KCTC 42903T=MCCC 1K01730T). An emended description of the genus Hwangdonia is also given.

Pyruvatibacter mobilis gen. nov., sp. nov., a marine bacterium from the culture broth of Picochlorum sp. 122.

A Gram-stain-negative, aerobic bacterium, designated strain GYP-11T, was isolated from the culture broth of a marine microalga, Picochloruma sp. 122. Cells were dimorphic rods; free living cells were motile by means of a single polar flagellum, and star-shaped-aggregate-forming cells were attached with stalks and non-motile. Sodium pyruvate or Tween 20 was required for growth on marine agar 2216.16S rRNA gene sequence analysis revealed that this isolate shared 94.07 % similarity with its closest type strain, Parvibaculum hydrocarboniclasticum EPR92T. Phylogenetic analyses indicated that strain GYP-11T represents a distinct lineage in a robust clade consisting of strain GYP-11T, alphaproteobacterium GMD21A06 and Candidatus Phaeomarinobacter ectocarpi Ec32. This clade was close to the genera Parvibaculum and Tepidicaulis in the order Rhizobiales. Chemotaxonomic and physiological characteristics, including cellular fatty acids and carbon source profiles, also readily distinguished strain GYP-11T from all established genera and species. Thus, it is concluded that strain GYP-11T represents a novel species of a new genus in the order Rhizobiales, for which the name Pyruvatibacter mobilis gen. nov., sp. nov. is proposed. The type strain of Pyruvatibacter mobilis is GYP-11T ( = CGMCC 1.15125T = KCTC 42509T).

Aliikangiella marina gen. nov., sp. nov., a marine bacterium from the culture broth of Picochlorum sp. 122, and proposal of Kangiellaceae fam. nov. in the order Oceanospirillales.

A Gram-stain-negative, non-motile, non-spore-forming, long rod-shaped bacterium, designated strain GYP-15T, was isolated from the culture broth of a marine microalga, Picochloruma sp. 122. Phylogenetic analyses revealed that strain GYP-15T shared 90.6 % 16S rRNA gene sequence similarity with its closest relative, Kangiella aquimarina KCTC 12183T, and represents a distinct phylogenetic lineage in a robust clade consisting of GYP-15T and members of the genera Kangiella and Pleionea in the order Oceanospirillales. Chemotaxonomic and physiological characteristics, including major cellular fatty acids, NaCl tolerance and pattern of carbon source utilization, could also readily distinguish strain GYP-15T from all established genera and species. Thus, it is concluded that strain GYP-15T represents a novel species of a new genus, for which the name Aliikangiella marina gen. nov., sp. nov. is proposed. The type strain of Aliikangiella marina is GYP-15T ( = MCCC 1K01163T = KCTC 42667T). Based on phylogenetic results, 16S rRNA gene signature nucleotide pattern and some physiological characteristics, the three genera Kangiella, Pleionea and Aliikangiella are proposed to make up a novel family, Kangiellaceae fam. nov., in the order Oceanospirillales.

Maribacter flavus sp. nov., isolated from a cyanobacterial culture pond.

A Gram-stain-negative, yellow, non-spore-forming, strictly aerobic bacterium, designated C3T, was isolated from a cyanobacterial culture pond. Cells were halophilic, rod-shaped and able to move by gliding. Growth of strain C3T was observed at 15-30 °C (optimum 25 °C), pH 6.0-9.0 (optimum pH 7.5), and in the presence of 1-7 % (w/v) NaCl (optimum 2-3 %). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain C3T formed a distinct lineage within the family Flavobacteriaceae and exhibited the highest similarity (95.21 %) to the type strains of Maribacter dokdonensis, Maribacter arcticus, Maribacter orientalis and Maribacter stanieri, and 'Maribacter caenipelagi' HD-44. The only isoprenoid quinone present within strain C3T was menaquinone 6 (MK-6). The G+C content of genomic DNA was 41.5 mol%. The major polar lipids were phosphatidylethanolamine and three unidentified lipids. The predominant cellular fatty acids (>5 % of the total fatty acids) were iso-C15 : 1 G, iso-C15 : 0, iso-C15 : 0 3-OH, iso-C17 : 0 3-OH, and summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c). On the basis of the phenotypic, chemotaxonomic and phylogenetic characteristics, strain C3T represents a novel species of the genus Maribacter, for which the name Maribacter flavus sp. nov. is proposed. The type strain is C3T ( = KCTC 42508T = CGMCC 1.15112T).

The influence of cultivation period on growth and biodiesel properties of microalga Nannochloropsis gaditana 1049.

This work reported for the first time the detailed impacts of cultivation period on growth dynamics and biochemical composition of a microalga strain Nannochloropsis gaditana 1049. The results shown either the biomass accumulation, lipid content, neutral lipid content, monounsaturated fatty acids composition or the favorable fatty acid profile of C16-C18 increased along with the cultivation period extension, but the lipid productivity displayed a decrease since cultured for 16 days, with the highest value reached 289.51 ± 16.34 mg L(-1) d(-1). Biodiesel properties of this microalga also changed with the cultivation period extension, with average unsaturated degree decreased from 1.24 ± 0.03 to 0.59 ± 0.02, cloud point increased from 3.39 ± 0.40 °C to 12.14 ± 0.32 °C, cetane number increased from 54.59 ± 0.20 to 58.96 ± 0.16 and iodine number reduced sharply from 105.15 ± 2.24 gI2/100g to 56.44 ± 1.76 gI2/100g, which all satisfied the specifications of biodiesel standard.

Wenzhouxiangella marina gen. nov, sp. nov, a marine bacterium from the culture broth of Picochlorum sp. 122, and proposal of Wenzhouxiangellaceae fam. nov. in the order Chromatiales.

A Gram-stain negative, non-motile, non-phototrophic, non-alkaliphilic, obligately aerobic, chemoheterotrophic, and rod-shaped bacterium, designated strain Ma-11(T), was isolated from the culture broth of a marine microalga, Picochloruma sp. 122. Phylogenetic analyses showed that strain Ma-11(T) has less than 91 % similarity to its closest relative, Thioalkalivibrio sulfidiphilus HL-EbGR7(T), represents a distinct phylogenetic lineage in the order Chromatiales, and could not be assigned to any defined families in this order. Chemotaxonomic, genetic and physiological characteristics, including major fatty acids, genomic G+C content, lack of motility, aerophilicity and chemoheterotrophicity, could readily distinguish strain Ma-11(T) from any established members of the order Chromatiales. Based on the 16S rRNA gene sequence analysis and its signature nucleotide pattern, a new family Wenzhouxiangellaceae fam. nov. comprising the genus Wenzhouxiangella gen. nov. and species Wenzhouxiangella marina sp. nov. is proposed. The type strain is Ma-11(T) (=CGMCC 1.14936(T) = KCTC 42284(T) = MCCC 1K00261(T)).

Bacterioplanes sanyensis gen. nov., sp. nov., a PHB-accumulating bacterium isolated from a pool of Spirulina platensis cultivation.

A Gram-negative, poly-3-hydroxybutyrate-accumulating rod bacterium, strain GYP-2(T), was isolated from a pool of marine Spirulina platensis cultivation, Sanya, China. Growth was observed at 10-45 °C and pH 6-10 in the presence of 1-10 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the new isolate belonged to Gammaproteobacteria and displayed 93.8-95.3 % 16S rRNA gene sequences similarities to members of the genera Thalassolituus, Oleibacter, and Oceanobacter, but house-keeping gene gyrB (encode DNA gyrase beta subunit) demonstrated that the new isolate was distantly related to Thalassolituus, Oleibacter, and Oceanobacter species (only 77-83 % gene gyrB sequences similarities).The G+C content of genomic DNA was 55 mol%. The major respiratory quinone was Q-9, while that for Oceanobacter kriegii LMG 6238(T) was Q-8. Major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. On the basis of its physiological, chemotaxonomic, and molecular properties, strain GYP-2(T) is suggested to represent a novel species of a new genus in Gammaproteobacteria, for which the name Bacterioplanes sanyensis gen. nov., sp. nov. is proposed. The type strain is GYP-2(T) (=CGMCC 1.12392(T)=KCTC 32220(T)).

Genome sequences of two pseudoalteromonas strains isolated from the South china sea.

Two Pseudoalteromonas strains, SCSIO 04301 and SCSIO 11900, were isolated from the South China Sea, and both strains form biofilms. Here we present the draft genome sequences of these two strains, which will aid the study of marine microbes that are adapted to marine sediments or are associated with eukaryotic hosts.

Pustulibacterium marinum gen. nov., sp. nov., a member of the family Flavobacteriaceae isolated from the Bashi Channel.

A Gram-reaction-negative, non-spore-forming, gliding, non-translucent, colourless or yellow, aerobic and elevated-colony-forming strain, designated E403(T), was isolated from the Bashi Channel and subjected to a polyphasic taxonomic study. Strain E403(T) could grow in the presence of 0.3-8 % (w/v) NaCl, at 16-43 °C and at pH 6-9, and grew optimally at 28 °C, pH 8, in natural seawater medium. The respiratory quinones were MK-6 and MK-7. The major fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH, iso-C15 : 1 G, summed feature 3 (C16 : 1ω7c/C16 : 1ω6c), iso-C15 : 0 3-OH and C16 : 0. The DNA G+C content of strain E403(T) was 37.9 mol%. Phylogenetic analyses based on 16S rRNA gene sequences of members of the family Flavobacteriaceae showed that strain E403(T) formed a distinct evolutionary lineage within the stable cluster containing type strains Zhouia amylolytica HN-171(T) (92.2 % similarity) and Joostella marina En5(T) (92.4 % similarity). In addition to the large 16S rRNA gene sequence differences, E403(T) can also be distinguished from the reference type strains J. marina En5(T) and Sinomicrobium oceani SCSIO 03483(T) by several phenotypic characteristics and chemotaxonomic properties. On the basis of phenotypic, chemotaxonomic and phylogenetic properties, strain E403(T) is suggested to represent a novel species of a new genus in the family Flavobacteriaceae, for which the name Pustulibacterium marinum gen. nov., sp. nov. is proposed. The type strain is E403(T) (= CCTCC AB2012862(T) = CGMCC 1.12333(T) = KCTC 32192(T)).

[Comparison of bacterial diversity of polluted and unpolluted sediment by brominated flame retardant].

OBJECTIVE: By using brominated flame retardant we compared the bacterial diversity of highly polluted river sediment with that of nearby unpolluted lake. METHOD: Total DNA was extracted from unpolluted and highly polluted sediment sample by brominated flame retardant in Guiyu of China. The 16S rRNA gene was amplified by PCR using bacterial primer 27F and 1500R. The plasmid libraries of the amplicons were constructed. The positive clones with insert were screened on plates with IPTG/X-gal/Ap. Amplified ribosmal DNA restriction analysis (ARDRA) was carried out with restriction enzymes Hha I and Hinf I. Representative clones of each operational taxonomic unit based on the ARDRA patterns were selected to be sequenced. After proof reading and careful comparison to remove the chimeric sequences, the partial sequence of 16S rRNA gene were used for construction of the phylogenetic tree. RESULT: The result of blast searching showed that clones from unpolluted sediment sample belonged to alpha-Proteobacteria, beta-Proteobacteria, gamma-Proteobacteria, delta-Proteobacteria, Planctomycetes, Acidobacteria, Actinobacteria, Chloroflexi, Verrucomicrobia, Firmicutes, the predominant bacteria (30.2% of total clones) is Acidobacteria; most clones from polluted sediment belonged to alpha-Proteobacteria, beta-Proteobacteria, epsilon-Proteobacteria, delta-Proteobacteria, Planctomycetes, Acidobacteria, Actinobacteria, Chloroflexi, Bacteroidetes, Firmicutes, candidate division 0P01, candidate division OP08, the predominant bacteria (44.9% of total clones) are epsilon-Proteobacteria and Chloroflexi. CONCLUSION: Bacterial community structure of polluted sediment has distinguished feature and obviously different from the unpolluted sediment sample, which is mainly reflected in the dominant position of epsilon-Proteobacteria and Chloroflexi in the bacterial flora.

Streptomyces autolyticus JX-47 large-insert bacterial artificial chromosome library construction and identification of clones covering geldanamycin biosynthesis gene cluster.

Geldanamycin belongs to benzoquinone ansamycin antibiotic and has potent antitumor activities. In this study, a bacterial artificial chromosome (BAC) library with an average insert size of up to 150 kb was constructed from genomic DNA of Streptomyces autolyticus JX-47. A genetic-screening strategy was established using BAC end-sequencing and three pairs of primers designed to target the remote regions, gdmA1, gdmA3 and gdmRI, of the geldanamycin gene cluster. Three clones covering geldanamycin biosynthesis gene cluster were obtained, which together spanned a 250-kb genomic region, and a 150227-bp insert in the clone p4E9 was sequenced. Comparison with the reported geldanamycin gene cluster sequences from S. hygroscopicus revealed that it had the same gene arrangement and high gene homology in the polyketide synthase (PKS) region and its downstream with 84-100% DNA identity and 81-100% amino acid (AA) identity. Its DNA homology with the whole gene cluster sequence from S. hygroscopicus strain 17997 reached 99% identity. However, upstream of the PKS region exhibited great diversity, where only ORF16 was conserved, and the other genes including gdmL and gdmX were displaced.

A method to type the potential angucycline producers in actinomycetes isolated from marine sponges.

Angucyclines are aromatic polyketides with antimicrobial, antitumor, antiviral and enzyme inhibition activities. In this study, a new pair of degenerate primers targeting the cyclase genes that are involved in the aromatization of the first and/or second ring of angucycline, were designed and evaluated in a PCR protocol targeting the jadomycin cyclase gene of Streptomyces venezuelae ISP5230. The identity of the target amplicon was confirmed by sequencing. After validation, the primers were used to screen 49 actinomycete isolates from three different marine sponges to identify putative angucycline producers. Seven isolates were positively identified using this method. Sequence analysis of the positive amplicons confirmed their identity as putative angucycline cyclases with sequence highly similar to known angucycline cyclases. Phylogenetic analysis clustered these positives into the angucycline group of cyclases. Furthermore, amplifications of the seven isolates using ketosynthase-specific primers were positive, backing the results using the cyclase primers. Together these results provided strong support for the presence of angucycline biosynthetic genes in these isolates. The specific primer set targeting the cyclase can be used to identify putative angucycline producers among marine actinobacteria, and aid in the discovery of novel angucyclines.

Culture-dependent and culture-independent diversity of Actinobacteria associated with the marine sponge Hymeniacidon perleve from the South China Sea.

In this report, the diversity of Actinobacteria associated with the marine sponge Hymeniacidon perleve collected from a remote island of the South China Sea was investigated employing classical cultivation and characterization, 16S rDNA library construction, 16S rDNA-restriction fragment length polymorphism (rDNA-RFLP) and phylogenetic analysis. A total of 184 strains were isolated using seven different media and 24 isolates were selected according to their morphological characteristics for phylogenetic analysis on the basis of their 16S rRNA gene sequences. Results showed that the 24 isolates were assigned to six genera including Salinispora, Gordonia, Mycobacterium, Nocardia, Rhodococcus and Streptomyces. This is the first report that Salinispora is present in a marine sponge from the South China Sea. Subsequently, 26 rDNA clones were selected from 191 clones in an Actinobacteria-specific 16S rDNA library of the H. perleve sample, using the RFLP technique for sequencing and phylogenetic analysis. In total, 26 phylotypes were clustered in eight known genera of Actinobacteria including Mycobacterium, Amycolatopsis, Arthrobacter, Brevibacterium, Microlunatus, Nocardioides, Pseudonocardia and Streptomyces. This study contributes to our understanding of actinobacterial diversity in the marine sponge H. perleve from the South China Sea.

Isolation of high molecular weight DNA from marine sponge bacteria for BAC library construction.

Metagenomics is a powerful tool for mining the genetic repositories from environmental microorganisms. Bacteria associated with marine sponges (phylum Porifera) are rich sources of biologically active natural products. However, to date, few compounds are discovered from the sponge metagenomic libraries, and the main reason might be the difficulties in recovery of high molecular weight (HMW) DNA from sponge symbionts to construct large insert libraries. Here, we describe a method to recover HMW bacterial DNA from diverse sponges with high quality for bacterial artificial chromosome (BAC) library construction. Microorganisms concentrated from sponges by differential centrifugation were embedded in agarose plugs to lyse out the HMW DNA for recovery. DNA fragments over 436 kb size were recovered from three different types of sponges, Halichondria sp., Haliclona sp., and Xestospongia sp. To evaluate the recovered DNA quality, the diversity of bacterial DNA comprised in the HMW DNA derived from sponge Halichondria sp. was analyzed, and this HMW DNA sample was also cloned into a shuttle BAC vector between Escherichia coli and Streptomyces sp. The results showed that more than five types of bacterial DNA, i.e., Proteobacteria, Nitrospirae, Cyanobacteria, Planctomycetes, and unidentified bacteria, had been recovered by this method, and an average 100 kb size insert DNA in a constructed BAC library demonstrated that the recovered HMW DNA is suitable for metagenomic library construction.

Exploring and exploiting microbial diversity through metagenomics for natural product drug discovery.

Microorganisms of millions species exist in every corner of the Earth, and form a dynamic genetic reservoir that are not clearly revealed and categorized due to barrier in current cultivation technology. Their applications in biomedical and environmental aspects are more than satisfactory. However, the situation has drastically changed during the turn of the century because of the rapid development of phylogenetic studies based on rRNA sequencing independent of standard laboratory cultivation. More recently, high throughput sequencing technology which enables direct sequencing of community DNA for metagenomic analyses are making a direct impact on our understanding of microbial diversity, ecology, and secondary metabolism. In this review, we highlight some recent progress and innovation on metagenomic research with an emphasis on natural product drug discovery. The rapid path of accumulating decoded metagenomics would be an efficient guide on direct access to the genomes of numerous non-culturable microorganisms for their genomic diversity and associated chemical prosperity for potential medicinal applications.

[Polyketide antibiotics produced by polyketide synthase in streptomyces--a review].

Polyketides have played an important role in antibiotic drug discovery with most antibacterial drugs being derived from a natural product or natural product lead. Furthermore, the biosynthetic gene clusters for numerous bioactive polyketides have been intensively studied over the past 15 years. This paper focuses on the polyketide drugs approved by US-FDA and takes a general view in the antibiotics produced by polyketide synthase in streptomyces.

[Marine microbial metagenomics: progress and prospect].

Preliminary statistics showed that there are more than one million species of microbes in marine environments that formed a dynamic genetic reservoir, among which the majority are not revealed and categorized due to barrier in cultivation techniques. However, the situation has changed in recent years because of the rapid development of phylogenetic studies based on small ribosomal RNA and rDNA sequencing independent to standard laboratory cultivation. These changes have significantly altered our understanding about microbial diversity and microbial ecology. In this review, we highlight some of recent progress and innovation in research on microbial diversity, and propose a metagenomic scheme as an alternative to overcome some of the barriers that still remain for exploitation of marine microbial diversity for its enormous potential in pharmaceutical applications. We believe that rapid progress in marine metagenomics allows direct access to the genomes of numerous non-cultivable microorganisms for their associated chemical prosperity.

Diversity of culturable actinobacteria isolated from marine sponge Haliclona sp.

This study describes actinobacteria isolated from the marine sponge Haliclona sp. collected in shallow water of the South China Sea. A total of 54 actinobacteria were isolated using media selective for actinobacteria. Species diversity and natural product diversity of isolates from marine sponge Haliclona sp. were analysed. Twenty-four isolates were selected on the basis of their morphology on different media and assigned to the phylum Actinobacteria by a combination of 16S rRNA gene based restriction enzymes digestion and 16S rRNA gene sequence analysis. The 16S rRNA genes of 24 isolates were digested by restriction enzymes TaqI and MspI and assigned to different groups according to their restriction enzyme pattern. The phylogenetic analysis based on 16S rRNA gene sequencing showed that the isolates belonged to the genera Streptomyces, Nocardiopsis, Micromonospora and Verrucosispora; one other isolate was recovered that does not belong to known genera based on its unique 16S rRNA gene sequence. To our knowledge, this is the first report of a bacterium classified as Verrucosispora sp. that has been isolated from a marine sponge. The majority of the strains tested belong to the genus Streptomyces and three isolates may be new species. All of the 24 isolates were screened for genes encoding polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS). PKS and NRPS sequences were detected in more than half of the isolates and the different "PKS-I-PKS-II-NRPS" combinations in different isolates belonging to the same species are indicators of their potential natural product diversity and divergent genetic evolution.