Community level physiological study of algicidal bacteria in the phycospheres of Skeletonema costatum and Scrippsiella trochoidea

Bacteria in the phycosphere have a unique ecological relationship with host algae due to their utilization of algal extracellular products as nutrients. Some bacteria control the growth of algal cells and even lyse them. The diversity of bacteria and their community dynamics in the phycosphere of microalgae are still relatively little understood, especially of those associated with red tide-causing algae. In this study, scanning electron microscope (SEM) images of algal cell morphology revealed that the phycosphere bacteria of the red tide-causing algae, Skeletonema costatum and Scrippsiella trochoidea, could lyse them within 72 h. The community level physiology of the algicidal bacteria was studied using Biolog ECO microplates, a common method for the ecological study of microbial communities. The average well color development (AWCD) values of bacteria in the phycospheres of both species were low, indicating that the bacteria had low metabolic activity overall. The diversity indices were both lower than the bacterial diversity from natural environments. However, the bacteria associated with S. trochoidea demonstrated a higher AWCD value and diversity than those in the phycosphere of S. costatum. The utilization of carbon sources significantly changed at different lytic times, reflecting that the bacterial community structure changed during the algae-lysing process. These results revealed that the bacterial communities in phycospheres had a simple structure and low diversity. When the balance between algae and bacteria broke down, the total bacterial density increased while the algicidal bacteria accumulated and became the dominant species, changing the bacterial community structure in this micro-ecosystem.     

Marine bacteria antagonistic to the harmful algal bloom species Alexandrium tamarense (Dinophyceae)

As part of efforts to enhance the strategies employed to manage and mitigate algal blooms and their adverse effects, algicidal bacteria have shown promise as potential suppressors of these events. Nine strains of bacteria algicidal against the toxic dinoflagellate, Alexandrium tamarense, were isolated from the East Sea area, China. Sequence analysis of 16S rDNA showed that all the algicidal bacteria belonged to the γ-proteobacteria subclass and the genera Pseudoalteromonas (strain SP31 and SP44), Alteromonas (strain DH12 and DH46), Idiomarina (strain SP96), Vibrio (strain DH47 and DH51) and Halomonas (strain DH74 and DH77). To assess the algicidal mode of these algicidal bacteria, bacterial cells and the filtrate from bacterial cultures were inoculated into A. tamarense cultures, and fluorescein diacetate vital stain was applied to monitor the growth of the algal cells. The results showed that all the algicidal bacteria exhibited algicidal activity through an indirect attack since algicidal activity was only detected in cell free supernatants but not the bacterial cells. This is the first report of bacteria from the genus Idiomarina showing algicidal activity to the toxic dinoflagellate A. tamarense and these findings would increase our knowledge of bacterial–algal interactions and the role of bacteria during the population dynamics of HABs.     

Algicidal metabolites produced by Bacillus sp. strain B1 against Phaeocystis globosa

The bloom of Phaeocystis globosa has broken out frequently in the coastal areas of China in recent years, which has led to substantial economic losses. This study shows that Bacillus sp. strain B1, which was previously identified by our group, is effective in regulating P. globosa by excreting active metabolites. Heat stability, pH stability and molecular weight range of the algicidal compounds from strain B1 were measured and the results demonstrated that the algicidal activities of these compounds were not affected by pH or temperature variation. The algicidal compounds extracted with methanol were isolated and purified by ODS-A column chromatography and HPLC. The algicidal compounds corresponding to peaks 2–5 eluted from HPLC were further analysed by quadrupole time-of-flight mass spectrometry (Q-TOF–MS). PeakView? Software determined the compounds corresponding to peaks 2–5 to be l-histidine, o-tyrosine, N-acetylhistamine and urocanic acid on the basis of the accurate mass information, the isotopic pattern and MS–MS spectra. Furthermore, these compounds were also able to eliminate Skeletonema costatum, Prorocentrum donghaiense and Heterosigma akashiwo. This is the first report of bacteria-derived algicidal compounds being identified only by Q-TOF–MS and PeakView? Software, and these compounds may be used as the constituents of algicides in the future.     

Involvement of an Extracellular Protease in Algicidal Activity of the Marine Bacterium Pseudoalteromonas sp. Strain A28

The marine bacterium Pseudoalteromonas sp. strain A28 was able to kill the diatom Skeletonema costatum strain NIES-324. The culture supernatant of strain A28 showed potent algicidal activity when it was applied to a paper disk placed on a lawn of S. costatum NIES-324. The condensed supernatant, which was prepared by subjecting the A28 culture supernatant to ultrafiltration with a 10,000-M_w-cutoff membrane, showed algicidal activity, suggesting that strain A28 produced extracellular substances capable of killing S. costatum cells. The condensed supernatant was then found to have protease and DNase activities. Two Pseudoalteromonas mutants lacking algicidal activity, designated NH1 and NH2, were selected after N-methyl-N′-nitrosoguanidine mutagenesis. The culture supernatants of NH1 and NH2 showed less than 15% of the protease activity detected with the parental strain, A28. The protease was purified to homogeneity from A28 culture supernatants by using ion-exchange chromatography followed by preparative gel electrophoresis. Paper-disk assays revealed that the purified protease had potent algicidal activity. The purified protease had a molecular mass for 50 kDa, and the N-terminal amino acid sequence was determined to be Ala-Thr-Pro-Asn-Asp-Pro. The optimum pH and temperature of the protease were found to be 8.8 and 30°C, respectively, by using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate. The protease activity was strongly inhibited by phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, antipain, chymostatin, and leupeptin. No significant inhibition was detected with EDTA, EGTA, phenanthroline or tetraethylenepentamine. These results suggest that Pseudoalteromonas sp. strain A28 produced an extracellular serine protease which was responsible for the algicidal activity of this marine bacterium.     

一株溶藻细菌对海洋原甲藻的溶藻效应

从深圳大鹏湾南澳赤潮爆发海域的表层海水中分离得到1株对海洋原甲藻(Prorocentrum micans)具有溶藻活性的海洋细菌,菌株编号为N10。利用液相感染法研究了该溶藻细菌的溶藻效果和溶藻作用方式。结果表明,菌株N10能使藻细胞失去运动活性,并膨胀变形,细胞膜内物质聚集于一端,藻细胞最终破裂死亡。菌悬液接种到藻液中的量越大,初始细菌密度越高,其溶藻效果越强。菌悬液以1∶10的体积比接种到藻液中时,藻细胞在24 h的死亡率为83%,至72 h全部溶解死亡;体积比为1∶20的藻细胞在24 h的死亡率为71%,之后藻细胞密度略有波动,120 h时死亡率达77%;而体积比为1∶100的藻细胞密度在前24 h有所下降,死亡率达39%,之后藻细胞密度又开始明显上升;对照组的藻细胞密度均呈明显上升趋势。菌悬液过滤液和高温加热处理后的菌悬液过滤液对海洋原甲藻均无溶藻活性,表明菌株N10的溶藻方式为直接溶藻。通过16S rRNA序列分析并与GenBank数据进行同源性检索,并结合细菌形态及生理生化特征,菌株N10隶属于黄杆菌科(Flavobacteriaceae)中的Muricauda sp.。     

Enhanced species-specific chemical control of harmful and non-harmful algal bloom species by the thiazolidinedione derivative TD49

Culture experiments involving 23 algae strains were conducted to evaluate the algicidal effects of a newly developed algicidal thiazolidinedione (TD) derivative (TD49) on non-harmful and harmful algal bloom (HAB) species. We also assessed the effect of various concentrations of TD49 on various growth phases (lag, logarithmic, and stationary) of the harmful algae Heterocapsa circularisquama (Dinophyceae) and Heterosigma akashiwo (Raphidophyceae; hereafter, Heterosigma) and the non-harmful diatoms Skeletonema costatum and Chaetoceros didymus. The inhibitory ratios (%) for H. circularisquama and Heterosigma at 2.0 μM TD49 were significantly higher than those at other concentrations, and the inhibitory ratio varied depending on growth phase and species as follows: logarithmic?≥?stationary?>?lag phase for H. circularisquama and logarithmic?≥?lag?>?stationary phase for Heterosigma. Although the inhibitory ratios for C. didymus were similar to those for the two harmful algae (H. circularisquama and Heterosigma), inhibitory effects on S. costatum were not apparent at >2.0 μM in any growth phase. The algicidal activity of TD49 on the harmful and non-harmful algae was as follows: unarmored HAB species?>?armored HAB species?>?diatom species?>?cryptophyte species. TD49 was algicidal to most HABs but had a little inhibitory effect on some non-harmful algae, implying that TD49 has selective algicidal activity. Our results indicate that TD49 is potentially of use in the control of HAB species within semi-enclosed bays.     

A marine bacterium producing protein with algicidal activity against Alexandrium tamarense

Interactions between bacteria and harmful algal bloom (HAB) species have been acknowledged as an important factor of regulating the population of these algae. In the study, two strains of algicidal bacteria, DHQ25 and DHY3, were screened out because of their probably secreting algicidal proteins against axenic Alexandrium tamarense. Molecular characterization classified them to the γ-proteobacteria subclass and to the genus Vibrio and Pseudoalteromonas, respectively. After centrifugation and ultrafiltration, chromatography of the cultural supernatants of DHQ25 revealed 8 peaks by HPLC. SDS-PAGE and Native PAGE determination showed that peak 7 to be a monoband peak. Both xenic and axenic culture of A. tamarense were susceptible to the purified protein (short for P7 below) indicated by algicidal activity assay. Observation of algicidal process demonstrated that algal cells were lysed and cellular substances were released under visual fields of microscope. P7 proved to be a challenge controller of A. tamarense by the above characterizations of algicidal activity assaying and algicidal process. This is the first report of a protein algicidal to the toxic dinoflagellate A. tamarense. The findings increase our knowledge of bacterial–algal interactions and the role of bacteria during controlling HABs.     

Lysis of Antarctic algal strains by bacterial pathogen

The present paper describes the isolation, physiological and genetic characteristic of a bacterial agent which inhibits the growth of algae and causes death of laboratory cultures of Antarctic microalgal strains: prokaryotic cyanobacteria Synechocystis salina and green eukaryotic microalga Choricistis minor. The bacterial strain LB1 was isolated from algal damaged laboratory cultures of S. salina. It was established that this bacterium is obligate aerobic, Gram-positive, non-spore-forming, immotile, irregular rods with dimensions 0.3–2 μm. Our results showed that LB1 has algicidal effect to S. salina as well as to C. minor. Transmission electron microscopy observations confirmed the destruction of S. salina by the bacterium. Biochemical analysis of LB1 revealed positive reaction to d-glucose, catalase, hydrolysis of gelatin, acid production from: lactose, l-arabinose, l-ramnose, esculin and β-galactosidase. The partial sequence (1,404 bp) of the 16S rRNA gene of LB1 showed 99 % similarity with type strains of the genus Microbacterium. The results of the biochemical, antimicrobial and of 16S rRNA analysis of LB1 allowed us to identify LB1 as Microbacterium sp. Studying expression of pathogenicity of the bacteria to algal cultures will help to solve the problem of algal production for biotechnological purposes.     

Isolation and characterization of a marine algicidal bacterium against the harmful raphidophyceae <Emphasis Type="Italic">Chattonella marina</Emphasis>

A bacterial strain named AB-4 showing algicidal activity against Chattonella marina was isolated from coastal water of ULjin, Republic of Korea. The isolated strain was identified as Bacillus sp. by culture morphology, biochemical reactions, and homology research based on 16S rDNA. The bacterial culture led to the lysis of algal cells, suggesting that the isolated strain produced a latent algal-lytic compound. Amongst changes in algicidal activity by different culture filtrate volumes, the 10% (100 μl/ml) concentration showed the biggest change in algicidal activity; there, estimated algicidal activity was 95%. The swimming movements of Chattonella marina cells were inhibited because of treatment of the bacterial culture; subsequently, Chattonella marina cells became swollen and rounded. With longer exposure time, algal cells were disrupted and cellular components lost their integrity and decomposed. The released algicide(s) were heat-tolerant and stable in pH variations, except pH 3, 4, and 5. Culture filtrate of Bacillus sp. AB-4 was toxic against harmful algae bloom (HAB) species and nontoxic against livefood organisms. Bacillus sp. AB-4 showed comparatively strong activity against Akashiwo sanguinea, Fibriocapsa japonica, Heterosigma akashiwo, and Scrippsiella trochoidea. These results suggest that the algicidal activity of Bacillus sp. AB-4 is potentially useful for controlling outbreaks of Chattonella marina.     

Isolation and characterization of algicidal bacteria from <Emphasis Type="Italic">Cochlodinium polykrikoides</Emphasis> culture

In this study, we analyzed a bacterial community closely associated with Cochlodinium polykrikoides that caused harmful algal blooming in the sea. Filtration using a plankton mesh and percoll gradient centrifugation were performed to eliminate free-living bacteria. Attached bacteria were analyzed by culture-dependent and culture-independent methods. Five culturable bacterial strains were isolated and identified from the C. polykrikoides mixed bacterial community. The isolates belonged to α-Proteobacteria (Nautella sp., Sagittula sp., and Thalassobius sp.) and γ-Proteobacteria (Alteromonas sp. and Pseudoalteromonas sp.). All of the 5 isolates showed algicidal activity against C. polykrikoides and produced extracellular compounds responsible for algicidal properties after entering the stationary phase. The algicidal compounds produced by the 5 isolates were heat-stable and had molecular masses of less than 10,000 Da. Furthermore, the algicidal compounds were relatively specific for C. polykrikoides in terms of their algicidal activities. Culture-independent analysis of the bacterial community in association with C. polykrikoides was performed using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). On the basis of the PCR-DGGE profile, Sagittula sp. was identified as a dominant species in the bacterial community of C. polykrikoides.     

Resistance of the fish-killing dinoflagellate Cochlodinium polykrikoides against algicidal bacteria isolated from the coastal sea of Japan

Noxious red tides of the dinoflagellate Cochlodinium polykrikoides tend to be long lasting and cause mass mortalities of cultured and natural fish and invertebrates along the western coast of Japan and the southern coast of Korea. In order to assess the tolerance of C. polykrikoides to attack by algicidal bacteria, the effects of algicidal bacteria strains on the growth of three C. polykrikoides strains were examined in laboratory culture experiments. Algicidal bacteria used were two strains of Cytophaga (J18/M01 and AA8-2, direct attack type and wide prey range), three strains of Alteromonas (S, K, D) and one strain of Pseudoalteromonas (R, indirect attack type), which were all isolated by using Chattonella antiqua as a prey organism. Neither Cytophaga strain showed any algicidal activity. In the cases of Alteromonas and Pseudoalteromonas, some cultures of C. polykrikoides were killed, but at least 10 days or more were required for the death of this dinoflagellate. C. polykrikoides survived in the presence of algicidal bacteria in concentrations up to 10⁶–10⁷ cells ml⁻¹, which is enough for other red tide microalgae to be killed. On the contrary, the algicidal effects of bacteria on C. antiqua were detected clearly within a few days. These results imply that C. polykrikoides is resistant to the six algicidal bacteria examined, which may reflect the capacity for mixotrophy. This resistance of C. polykrikoides to algicidal bacteria could provide a selective advantage for survival compared to other microalgae susceptible to attack by algicidal bacteria and hence prolong red tides caused by this harmful dinoflagellate.     

Algicidal Effects of a Novel Marine Pseudoalteromonas Isolate (Class Proteobacteria,Gamma Subdivision) on Harmful Algal Bloom Species of the Genera Chattonella,Gymnodinium, and Heterosigma

During a bacterial survey of the Huon Estuary in southern Tasmania, Australia, we isolated a yellow-pigmented Pseudoalteromonas strain (class Proteobacteria, gamma subdivision), designated strain Y, that had potent algicidal effects on harmful algal bloom species. This organism was identified by 16S rRNA sequencing as a strain with close affinities to Pseudoalteromonas peptidysin. This bacterium caused rapid cell lysis and death (within 3 h) of gymnodinoids (including Gymnodinium catenatum) and raphidophytes (Chattonella marina and Heterosigma akashiwo). It caused ecdysis of armored dinoflagellates (e.g., Alexandrium catenella, Alexandrium minutum, and Prorocentrum mexicanum), but the algal cultures then recovered over the subsequent 24 h. Strain Y had no effect on a cryptomonad (Chroomonas sp.), a diatom (Skeletonema sp.), a cyanobacterium (Oscillatoria sp.), and two aplastidic protozoans. The algicidal principle of strain Y was excreted into the seawater medium and lost its efficacy after heating. Another common bacterial species, Pseudoalteromonas carrageenovora, was isolated at the same time and did not have these algicidal effects. The minimum concentrations of strain Y required to kill G. catenatum were higher than the mean concentrations found in nature under nonbloom conditions. However, the new bacterium showed a chemotactic, swarming behavior that resulted in localized high concentrations around target organisms. These observations imply that certain bacteria could play an important role in regulating the onset and development of harmful algal blooms.Historically, the dynamics of marine bacterial and algal populations have been studied largely in isolation. Increasing evidence is now pointing toward a close spatial and temporal association between the two and recently attention has been focused on phagocytosis of bacteria by photosynthetic flagellates (21, 28, 30). In contrast, the importance of inhibitory or predatory bacteria in regulating populations of different algal species has received relatively little attention (9, 11). Some bacteria may selectively promote bloom formation by algal species (13), while other bacteria have algicidal effects and are involved in the termination and decomposition of algal blooms (12). The latter finding has raised the possibility of bacterial control of harmful algal blooms (19). There is little data on the occurrence of marine algicidal bacteria outside Japan, where toxic blooms are frequent events (20), and algicidal bacteria have been isolated during toxic blooms of naked dinoflagellates and raphidophytes (9).Gymnodinium catenatum (a causative organism of paralytic shellfish poisoning) is thought to have been introduced into southern Tasmania via ballast water after 1973, and in some years it has a severe negative impact on the shellfish industry (16). Previous efforts to understand and predict the seasonal and interannual variability of harmful algal blooms have largely focused on the environmental factors that affect dinoflagellate growth in the water column, notably water temperature, rainfall, and water column stability (16). Rainfall and estuarine flow patterns also largely determine the allochthonous input of dissolved organic matter (DOM), which is a source of organic carbon for bacteria (27) and is possibly involved in micronutrient dynamics that promote G. catenatum growth (3, 6). As part of a study investigating DOM, bacteria, and algal interactions in the Huon Estuary (24), we isolated two bacterial strains that we tested for possible alga-bacterium interactions by using cultures of G. catenatum. Both bacteria appeared to be Pseudoalteromonas species, which are extremely common, slightly halophilic, gram-negative bacteria found in many marine ecosystems. Preliminary observations indicated that one of the strains was extremely toxic towards G. catenatum, while the other was more benign. The aims of this study were (i) to determine the taxonomic identity of the bacteria, (ii) to document by light microscopy the sequence of algal cell lysis after exposure to an algicidal Pseudoalteromonas strain and compare this lysis to the effect of the more benign Pseudoalteromonas species, (iii) to define the minimum bacterial concentrations required for algicidal effects and compare these concentrations to concentrations in natural water samples, and (iv) to investigate the range of potential target organisms for the bacterium.     

Novel algicidal evidence of a bacterium Bacillus sp. LP-10 killing Phaeocystis globosa,a harmful algal bloom causing species

While searching for effective bio-agents to control harmful algal blooms (HABs), the bacterial strain LP-10, which has strong algicidal activity against Phaeocystis globosa (Prymnesiophyceae), was isolated from surface seawater samples taken from the East China Sea. 16S rDNA sequence analysis and morphological characteristics revealed the strain LP-10 belonged to the genus Bacillus. The lytic effect of Bacillus sp. LP-10 against P. globosa was both concentration- and time-dependent. Algicidal activities of different growth stages of the bacterial culture varied significantly. The lytic effect of different parts of the bacterial cultures indicated that the algal cells were lysed by algicidal active compounds in the cell-free filtrate. Analysis of the properties of the active compounds showed that they had a molecular weight of less than 1000 Da and that the active compounds were stable between −80 and 121 °C. The algicidal range assay indicated that five other algal species were also suppressed by strain LP-10, including: Alexandrium catenella, A. tamarense, A. minutum, Prorocentrum micans and Asterionella japonica. Our results suggested that the algicidal bacterium Bacillus sp. LP-10 could be a potential bio-agent to control the blooms of harmful algal species.     

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。     

A freshwater bacterial strain,Shewanella sp. Lzh-2, isolated from Lake Taihu and its two algicidal active substances,hexahydropyrrolo[1,2-a]pyrazine-1,4-dione and 2, 3-indolinedione

Cyanobacterial blooms have become a serious problem in Lake Taihu during the last 20 years, and Microcystis aeruginosa and Synechococcus sp. are the two dominant species in cyanobacterial blooms of Lake Taihu. A freshwater bacterial strain, Shewanella sp. Lzh-2, with strong algicidal properties against harmful cyanobacteria was isolated from Lake Taihu. Two substances with algicidal activity secreted extracellularly by Shewanella sp. Lzh-2, S-2A and S-2B, were purified from the bacterial culture of strain Lzh-2 using ethyl acetate extraction, column chromatography, and high performance liquid chromatography (HPLC) in turn. The substances S-2A and S-2B were identified as hexahydropyrrolo[1,2-a]pyrazine-1,4-dione and 2, 3-indolinedione (isatin), respectively, based on liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), and hydrogen-nuclear magnetic resonance (H-NMR) analyses, making this the first report of their algicidal activity toward cyanobacteria. S-2A (hexahydropyrrolo[1,2-a]pyrazine-1,4-dione) had no algicidal effects against Synechococcus sp. BN60, but had a high level of algicidal activity against M. aeruginosa 9110. The LD50 value of S-2A against M. aeruginosa 9110 was 5.7 μg/ml. S-2B (2, 3-indolinedione) showed a potent algicidal effect against both M. aeruginosa 9110 and Synechococcus sp. BN60, and the LD50 value of S-2B against M. aeruginosa 9110 and Synechococcus sp. BN60 was 12.5 and 34.2 μg/ml, respectively. Obvious morphological changes in M. aeruginosa 9110 and Synechococcus sp. BN60 were observed after they were exposed to S-2A (or S-2B) for 24 h. Approximately, the algicidal activity, the concentration of S-2A and S-2B, and the cell density of Lzh-2 were positively related to each other during the cocultivation process. Overall, these findings increase our knowledge about algicidal substances secreted by algicidal bacteria and indicate that strain Lzh-2 and its two algicidal substances have the potential for use as a bio-agent in controlling cyanobacterial blooms in Lake Taihu.     

Efficiency of Some Bacterial Strains in Potassium Release from Mica and Phosphate Solubilization under In Vitro Conditions

Phosphorus and potassium (K) are major essential macronutrients for biological growth and development. Application of beneficial microorganisms to soil is one approach to enhance crop growth. In this study, the ability of five bacterial strains, including four strains of Pseudomonas sp. (S10-3, S14-3, S19-1, and S21-1) and one strain of Azotobacter sp. SP16, to release K from muscovite and biotite was investigated. Furthermore, phosphate solubilization by these strains was measured when an insoluble source of P [Ca₃(PO₄)₂] was added to the medium. Among the bacterial strains, the highest average K release (73% higher than control) was observed with Pseudomonas sp. S14-3. The average amount of K released from biotite was 37% higher than that from muscovite in inoculated treatments. The enhanced release of mineral K might be attributed to the release of organic acids from the bacteria, a mechanism which plays a pivotal role in solubilizing phosphate from inorganic sources. The results confirmed the enhanced phosphate solubilization by the bacterial strains in the presence of muscovite. The highest P solubilizing activity (67% higher than control) was found in S21-1 and S14-3 strains. Concentrations of both K and P in the liquid phase were increased by increasing the time of experiment. X-ray diffraction analysis of muscovite specimens inoculated with S14-3 strain revealed a partial transformation of these minerals through the presence of 19.5 Å peak on the diffractogram of the magnesium-saturated sample. This may be due to the release of K from the interlayer space and subsequent filling with a number of bacterial metabolites. The findings of this research suggest K depletion from mica in the presence of bacteria, but further investigations are needed to clarify the mechanisms involved.     

Characterization of an algicidal bacterium Brevundimonas J4 and chemical defense of Synechococcus sp. BN60 against bacterium J4

As part of efforts to enhance the strategies explored to eliminate the adverse impacts of cyanobacterial blooms, we isolated an algicidal bacterium, J4, from Lake Taihu. Analysis of 16S rDNA sequence revealed that strain J4 belonged to the genus Brevundimonas. Bacterium J4 exhibited algicidal activity mainly through excretion of extracellular algicidal compounds that were further extracted with methanol and purified by silica gel chromatography and high performance liquid chromatography (HPLC). The compounds showed thermal stability, strong polarity and water solubility in J4 cultures. Study on the algicidal activity of J4 against two dominant cyanobacterial bloom-forming species in Lake Taihu showed that J4 exhibited lower algicidal rate against Synechococcus sp. BN60 (48.6%, t = 6 days) than against Microcystis aeruginosa 9110 (91.8%, t = 6 days). Additionally, rapid reduction in cell density of J4 was observed in co-cultures of Synechococcus sp. BN60 and bacterium J4 but not observed in co-cultures of M. aeruginosa 9110 and bacterium J4 during algicidal process, which was the main reason why the algicidal rate of J4 against BN60 was lower than against 9110. The reduction in cell density of J4 resulted from inducible production of antimicrobial-like compound secreted by Synechococcus sp. BN60 in co-cultures of Synechococcus sp. BN60 and bacterium J4, which reflected a kind of chemical defense from cyanobacteria (BN60) against algicidal bacteria (J4). However, M. aeruginosa 9110 had no chemical defense against J4, suggesting that whether cyanobacterial chemical defense occurs or not between cyanobacteria and algicidal bacteria depends on specific cyanobacteria–algicidal bacteria pairs. These results show that not only one-sided algicidal effect but also two-sided reciprocal inhibition interactions exist between algicidal bacteria and cyanobacteria, indicating the complexity of cyanobacteria–algicidal bacteria interactions in Lake Taihu and the need to take the cyanobacterial defensive responses into consideration when assessing potential use of algicidal bacteria.     

Isolation, identification and characterization of algicidal bacteria against Stephanodiscus hantzschii and Peridinium bipes for the control of freshwater winter algal blooms

Five strains (HYY0510-SK04, HYY0511-SK09, HYK0512-SK12, HYK0512-PK04 and HYY0512-PK05) of algicidal bacteria against the harmful bloom forming diatom Stephanodiscus hantzschii and dinoflagellate Peridinium bipes, were isolated. Among these strains, HYY0510-SK04, HYY0511-SK09 and HYK0512-SK12 have an effective algicidal activity for S. hantzschii, while HYK0512-PK04 and HYY0512-PK05 have an algicidal effect against P. bipes. Sequence analysis of 16S rDNA showed that HYY0510-SK04 and HYY0511-SK09 were closely related to Acidovorax delafieldii ATCC 17505^T. HYK0512-SK12, HYK0512-PK04 and HYY0512-PK05 showed high homology with Variovorax paradoxus IAM 12373^T (98.9%), Hydrogenophaga palleronii ATCC 49743^T (98.8%) and Pseudomonas plecoglossicida ATCC 700383^T (98.3%), respectively. HYY0510-SK04, HYY0511-SK09 and HYK0512-SK12 degraded S. hantzschii cells within two weeks when those bacteria were inoculated at densities of ≥10⁷cells mL⁻¹ to the lag or logarithmic growth phase of the algal culture. HYK0512-PK04 and HYY0512-PK05 degraded more than 90% of P. bipes cells within 14 and 8 days, respectively, when these bacteria were inoculated at densities of ≥10⁷cells mL⁻¹. Among the five bacterial strains, HYK0512-SK12 and HYY0512-PK05 showed the most effective growth inhibition of all the algae and cyanobacteria tested. Biochemical assays revealed that the main algicidal substance from all isolates were likely to be extracellular substances. These results indicate that the bacterial strains isolated for this study are potential agents for the control of harmful algal blooms in eutrophic reservoirs.     

Efficiency of natural and engineered bacterial strains in the degradation of 4-chlorobenzoic acid in soil slurry

Two bacterial strains, the natural isolate Arthrobacter sp. FG1 and the engineered strain Pseudomonas putida PaW340/pDH5, were compared for their efficiency in the degradation of 4-chlorobenzoic acid in a slurry phase system. The recombinant strain was obtained by cloning the Arthrobacter sp. FG1 dehalogenase encoding genes in P. putida PaW340. In the slurry inoculated with pre-adapted cultures of Arthrobacter sp. FG1, the 4-chlorobenzoic acid degradation was found to be slower than that observed in the slurry inoculated with the recombinant strain P. putida PaW340/pDH5, regardless of the presence or absence of soil indigenous bacteria. Slurry inoculated with mixed cultures of Arthrobacter sp. FG1 and the 4-hyroxybenzoic acid degrader P. putida PaW340 did not show any improvement in 4-chlorobenzoic acid degradation.     

Toxic Effect of a Marine Bacterium on Aquatic Organisms and Its Algicidal Substances against Phaeocystis globosa

Harmful algal blooms have caused enormous damage to the marine ecosystem and the coastal economy in China. In this paper, a bacterial strain B1, which had strong algicidal activity against Phaeocystis globosa, was isolated from the coastal waters of Zhuhai in China. The strain B1 was identified as Bacillus sp. on the basis of 16S rDNA gene sequence and morphological characteristics. To evaluate the ecological safety of the algicidal substances produced by strain B1, their toxic effects on marine organisms were tested. Results showed that there were no adverse effects observed in the growth of Chlorella vulgaris, Chaetoceros muelleri, and Isochrystis galbana after exposure to the algicidal substances at a concentration of 1.0% (v/v) for 96 h. The 48h LC₅₀ values for Brachionus plicatilis, Moina mongolica Daday and Paralichthys olivaceus were 5.7, 9.0 and 12.1% (v/v), respectively. Subsequently, the algicidal substances from strain B1 culture were isolated and purified by silica gel column, Sephadex G-15 column and high-performance liquid chromatography. Based on quadrupole time-of-flight mass spectrometry and PeakView Software, the purified substances were identified as prolyl-methionine and hypoxanthine. Algicidal mechanism indicated that prolyl-methionine and hypoxanthine inhibited the growth of P. globosa by disrupting the antioxidant systems. In the acute toxicity assessment using M. mongolica, 24h LC₅₀ values of prolyl-methionine and hypoxanthine were 7.0 and 13.8 g/L, respectively. The active substances produced by strain B1 can be considered as ecologically and environmentally biological agents for controlling harmful algal blooms.