Microalgae–bacteria symbiosis in microalgal growth and biofuel production: a review

Photosynthetic microalgae can capture solar energy and convert it to bioenergy and biochemical products. In nature or industrial processes, microalgae live together with bacterial communities and may maintain symbiotic relationships. In general interactions, microalgae exude dissolved organic carbon that becomes available to bacteria. In return, the bacteria remineralize sulphur, nitrogen and phosphorous to support the further growth of microalgae. In specific interactions, heterotrophic bacteria supply B vitamins as organic cofactors or produce siderophores to bind iron, which could be utilized by microalgae, while the algae supply fixed carbon to the bacteria in return. In this review, we focus on mutualistic relationship between microalgae and bacteria, summarizing recent studies on the mechanisms involved in microalgae–bacteria symbiosis. Symbiotic bacteria on promoting microalgal growth are described and the relevance of microalgae–bacteria interactions for biofuel production processes is discussed. Symbiotic microalgae–bacteria consortia could be utilized to improve microalgal biomass production and to enrich the biomass with valuable chemical and energy compounds. The suitable control of such biological interactions between microalgae and bacteria will help to improve the microalgae-based biomass and biofuel production in the future.     

Comparison of Blue Nucleic Acid Dyes for Flow Cytometric Enumeration of Bacteria in Aquatic Systems

Seven blue nucleic acid dyes from Molecular Probes Inc. (SYTO-9, SYTO-11, SYTO-13, SYTO-16, SYTO-BC, SYBR-I and SYBR-II) were compared with the DAPI (4′,6-diamidino-2-phenylindole) method for flow cytometric enumeration of live and fixed bacteria in aquatic systems. It was shown that SYBR-II and SYTO-9 are the most appropriate dyes for bacterial enumeration in nonsaline waters and can be applied to both live and dead bacteria. The fluorescence signal/noise ratio was improved when SYTO-9 was used to stain living bacteria in nonsaline waters. Inversely, SYBR-II is more appropriate than SYTO dyes for bacterial enumeration of unfixed and fixed seawater samples.     

Flow cytometric detection and quantification of heterotrophic nanoflagellates in enriched seawater and cultures

A flow cytometric protocol to detect and enumerate heterotrophic nanoflagellates (HNF) in enriched waters is reported. At present, the cytometric protocols that allow accurate quantification of bacterioplankton cannot be used to quantify protozoa for the following reasons: i) the background produced by the bacterial acquisitions does not allow the discrimination of protozoa at low abundance, ii) since the final protozoan fluorescence is much higher than the bacterioplankton fluorescence (more than 35 fold) the protozoa acquisitions lie outside the range. With an increase in the fluorescence threshold and a reduction of the fluorescence detector voltage, low fluorescence particles (bacteria) are beneath the detection limits and only higher fluorescence particles (most of them heterotrophic nanoflagellates) are detected. The main limitation for the application of the cytometric protocol developed is that a ratio of bacteria/HNF below 1000 is needed. At higher ratios, the background of larger cells of bacterioplankton makes it difficult to discriminate protozoa. The proposed protocol has been validated by epifluorescence microscopy analyzing both a mixed community and two single species of HFN: Rhynchomonas nasuta and Jakoba libera. Taking into account the required bacteria/HNF ratio cited above, the results provide evidence that the flow cytometric protocol reported here is valid for counting mixed communities of HNF in enriched seawater and in experimental micro or mesocosms. In the case of single species of HNF previous knowledge of the biological characteristics of the protist and how they can affect the effectiveness of the flow cytometric count is necessary.     

Bacterial communities of brown and red algae from Peter the Great Bay, the Sea of Japan

The structure of microbial communities of brown algae, red algae, and of the red alga Gracilaria verrucosa, healthy and affected with rotten thallus, were comparatively investigated; 61 strains of heterotrophic bacteria were isolated and characterized. Most of them were identified to the genus level, some Vibrio spp., to the species level according to their phenotypic properties and the fatty acid composition of cellular lipids. The composition of the microflora of two species of brown algae was different. In Chordaria flagelliformis, Pseudomonas spp. prevailed, and in Desmarestia viridis, Bacillus spp. The composition of the microflora of two red algae, G. verrucosa and Camphylaephora hyphaeoides, differed mainly in the ratio of prevailing groups of bacteria. The most abundant were bacteria of the CFB cluster and pseudoalteromonads. In addition, the following bacteria were found on the surface of the algae: Sulfitobacter spp., Halomonas spp., Acinetobacter sp., Planococcus sp., Arthrobacter sp., and Agromyces sp. From tissues of the affected G. verrucosa, only vibrios were isolated, both agarolytic and nonagarolytic. The existence of specific bacterial communities characteristic of different species of algae is suggested and the relation of Vibrio sp. to the pathological process in the tissues of G. verrucosa is supposed.     

Dynamics of ice algae and phytoplankton in Frobisher Bay

Summary Vertical and seasonal variations of ice algae and phytoplankton were studied in relation to their physico-chemical environments in Frobisher Bay from 1979 to 1986. The biomass, estimated by both chlorophyll a concentrations and cell counts, was greater in the ice algae than in the phytoplankton in the underlying sea-water during winter and spring. Algal distribution in the sea ice varied vertically and seasonally, while in the underlying water column the phytoplankton distribution was much less variable. The ice algal bloom occurred at the bottom of the ice, particularly in the lower 5 cm during late spring, while the phytoplankton bloom took place at depths between 1 and 10 m during early summer after the ice bloom was over. The community structure of the ice algae changed from pennate to centric diatoms as the ice melted. The centrics dominated through the fall, and then decreased as the pennates increased in dominance when the ice formed again in winter. Species diversity and number were greater in the sea ice than in the seawater, but they were similar vertically within each habitat. The evenness of the species distribution did not vary with ice thickness or water depth. Species composition, abundance and dominance of ice algae and phytoplankton continually change both vertically and seasonally. The differential abilities of the species to attain maximal growth rates under various environmental conditions may result in species succession. Evidence is given for the major role of environmental factors regulating the dynamics of ice algae and phytoplankton.     

Experimental identification and in silico prediction of bacterivory in green algae

While algal phago-mixotrophs play a major role in aquatic microbial food webs, their diversity remains poorly understood. Recent studies have indicated several species of prasinophytes, early diverging green algae, to be able to consume bacteria for nutrition. To further explore the occurrence of phago-mixotrophy in green algae, we conducted feeding experiments with live fluorescently labeled bacteria stained with CellTracker Green CMFDA, heat-killed bacteria stained with 5-(4,6-dichlorotriazin-2-yl) aminofluorescein (DTAF), and magnetic beads. Feeding was detected via microscopy and/or flow cytometry in five strains of prasinophytes when provided with live bacteria: Pterosperma cristatum NIES626, Pyramimonas parkeae CCMP726, Pyramimonas parkeae NIES254, Nephroselmis pyriformis RCC618, and Dolichomastix tenuilepis CCMP3274. No feeding was detected when heat-killed bacteria or magnetic beads were provided, suggesting a strong preference for live prey in the strains tested. In parallel to experimental assays, green algal bacterivory was investigated using a gene-based prediction model. The predictions agreed with the experimental results and suggested bacterivory potential in additional green algae. Our observations underline the likelihood of widespread occurrence of phago-mixotrophy among green algae, while additionally highlighting potential biases introduced when using prey proxy to evaluate bacterial ingestion by algal cells.Subject terms: Microbial ecology, Cellular microbiology     

Observations on interactions between naturally-collected bacteria and several species of algae

Interactions between a naturally-collected algal species and strains of bacteria with which it was closely associated were examined under controlled conditions. Three strains of bacteria, Pseudomonas, Xanthomonas and Flavobacterium, were isolated from Oscillatoria. These bacteria were grown in combination with axenic cultures of the Oscillatoria culture as well as with several additional algal species. Oscillatoria growth was stimulated by all of the bacteria, but other algal species varied in their response. Some were stimulated, but others were inhibited or unaffected by exposure to the bacterial strains. There were also observations indicating that some algae may be able to develop resistance to antagonistic bacteria. These data suggest that succession and dominance of individual algal species may be influenced by interactions with bacteria.     

In Situ Activity of Suspended and Immobilized Microbial Communities as Measured by Fluorescence Lifetime Imaging

In this study, the feasibility of fluorescence lifetime imaging (FLIM) for measurement of RNA:DNA ratios in microorganisms was assessed. The fluorescence lifetime of a nucleic acid-specific probe (SYTO 13) was used to directly measure the RNA:DNA ratio inside living bacterial cells. In vitro, SYTO 13 showed shorter fluorescence lifetimes in DNA solutions than in RNA solutions. Growth experiments with bacterial monocultures were performed in liquid media. The results demonstrated the suitability of SYTO 13 for measuring the growth-phase-dependent RNA:DNA ratio in Escherichia coli cells. The fluorescence lifetime of SYTO 13 reflected the known changes of the RNA:DNA ratio in microbial cells during different growth phases. As a result, the growth rate of E. coli cells strongly correlated with the fluorescence lifetime. Finally, the fluorescence lifetimes of SYTO 13 in slow- and fast-growing biofilms were compared. For this purpose, biofilms developed from activated sludge were grown as autotrophic and heterotrophic communities. The FLIM data clearly showed a longer fluorescence lifetime for the fast-growing heterotrophic biofilms and a shorter fluorescence lifetime for the slow-growing autotrophic biofilms. Furthermore, starved biofilms showed shorter lifetimes than biofilms supplied with glucose, indicating a lower RNA:DNA ratio in starved biofilms. It is suggested that FLIM in combination with SYTO 13 represents a useful tool for the in situ differentiation of active and inactive bacteria. The technique does not require radioactive chemicals and may be applied to a broad range of sample types, including suspended and immobilized microorganisms.     

Light availability regulates the response of algae and heterotrophic bacteria to elevated nutrient levels and warming in a northern boreal peatland

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Green fluorescent protein-propidium iodide (GFP-PI) based assay for flow cytometric measurement of bacterial viability.

BACKGROUND: Several staining protocols have been developed for flow cytometric analysis of bacterial viability. One promising method is dual staining with the LIVE/DEAD BacLight bacterial viability kit. In this procedure, cells are treated with two different DNA-binding dyes (SYTO9 and PI), and viability is estimated according to the proportion of bound stain. SYTO9 diffuses through the intact cell membrane and binds cellular DNA, while PI binds DNA of damaged cells only. This dual-staining method allows effective separation between viable and dead cells, which is far more difficult to achieve with single staining. Although SYTO9-PI dual staining is practical for various bacterial viability analyses, the method has a number of disadvantages. Specifically, the passage of SYTO9 through the cell membrane is a slow process, which is significantly accelerated when the integrity of the cell membrane is disrupted. As a result, SYTO9 binding to DNA is considerably enhanced. PI competes for binding sites with SYTO9 and may displace the bound dye. These properties diminish the reliability of the LIVE/DEAD viability kit. In this study, we investigate an alternative method for measuring bacterial viability using a combination of green fluorescent protein (GFP) and PI, with a view to improving data reliability. METHODS: Recombinant Escherichia coli cells with a plasmid containing the gene for jellyfish GFP were stained with PI, and green and red fluorescence were measured by FCM. For comparison, cells containing the plasmid from which gfp was removed were stained with SYTO9 and PI, and analyzed by FCM. Viability was estimated according to the proportion of green and red fluorescence. In addition, bioluminescence and plate counting (other methods to assess viability) were used as reference procedures. RESULTS: SYTO9-PI dual staining of bacterial cells revealed three different cell populations: living, compromised, and dead cells. These cell populations were more distinct when the GFP-PI combination was used instead of dual staining. No differences in sensitivity were observed between the two methods. However, substitution of SYTO9 with GFP accelerated the procedure. Bioluminescence and plate counting results were in agreement with flow cytometric viability data. CONCLUSIONS: In bacterial viability analyses, the GFP-PI combination provided better distinction between current viability stages of E. coli cells than SYTO9-PI dual staining. Additionally, the overall procedure was more rapid. No marked differences in sensitivity were observed.     

Bacterial communities of some brown and red algae from Peter the Great Bay, the Sea of Japan

The structure of microbial communities of brown algae, red algae, and of the red alga Gracilaria verrucosa, healthy and affected with thallus rot, were comparatively investigated; 61 strains of heterotrophic bacteria were isolated and characterized. Most of them were identified to the genus level, some Vibrio spp., to the species level according to their phenotypic properties and the fatty acid composition of cellular lipids. The composition of the microflora of two species of brown algae was different. In Chordaria flagelliphormis, Pseudomonas spp. prevailed, and in Desmarestia viridis, Bacillus spp. The composition of the microflora of two red algae, G. verrucosa and Camphylaephora hyphaeoides, differed mainly in the ratio of prevailing groups of bacteria. The most abundant were bacteria of the CFB cluster and pseudoalteromonads. In addition, the following bacteria were found on the surface of the algae: Sulfitobacter spp., Halomonas spp., Acinetobacter sp., Planococcus sp., Arthrobacter sp., and Agromyces sp. From tissues of the affected G. verrucosa, only vibrios were isolated, both agarolytic and nonagarolytic. The existence of specific bacterial communities characteristic of different species of algae is suggested and the relation of Vibrio sp. to the pathological process in the tissues of G. verrucosa is supposed.     

Flow cytometric determinations of cellular substances in algae,bacteria, moulds and yeasts

The practical use of flow cytometry is shown in several microbial assays. Recent technical improvements in the optics and electronics of flow cytometric systems as well as in staining techniques permit the measurements of minute cellular components such as the cellular DNA and the protein content of bacteria, algae, moulds and yeasts. Single cell ingredients can be measured by this assay according to their specific stainability. The cell DNA was stained by propidium iodide while the cell protein was fluorochromed by fluorescein-iso-thiocyanate. The DNA synthesis of Saccharomyces cerevisiae and Saccharomyces pastorianus runs discontinuously while the protein content increases continuously during the vegetative growth. The different stages of DNA synthesis of yeast cells can be divided into two gap phases, a synthesis and a mitosis period, corresponding to Howard and Pelc's model of DNA synthesis. Living and dead cells can be counted differentially after staining with Erythrosine B. The red fluorescence of the chlorophyll in algae can readily be used to determine the chlorophyll content of these cells.     

Effect of cultural conditions on the sterols and fatty acids of green algae

Six species of green algae were grown autotrophically, photohetrotrophically, and heterotrophically and their fatty acid and sterol compositions determined. Sterol composition was higher in autotrophic than in heterotrophic plants by a factor of from 2 to over 20 in five of the six species studied. Relative amounts of various sterols did not change significantly with cultural conditions. In five of the species studied, autotrophic growth produced a significant increase in the relative proportion of linolenic acid compared to that in heterotrophic or photoheterotrophic growth. This increase was usually accompanied by a corresponding decrease in oleic or linoleic acids or both.     

饮用水微生物的安全快速检测

【目的】为了更好地分析饮用水中的微生物含量。【方法】利用流式细胞术(Flowcytometry,FCM)、ATP测定方法检测瓶装无气饮用水中的微生物数量、可同化有机碳(Assimilable organic carbon,AOC)含量以及微生物活性,并将检测结果与传统的饮用水微生物检测技术相对照。【结果】FCM方法可快速区分水样中的活性细菌和非活性细菌,AOC含量反映了水样中微生物再生能力;而ATP检测方法也能比异养细菌平板计数法(Heterotrophic plate count,HPC)更好地反映瓶装无气饮用水中的实际微生物含量。【结论】FCM、ATP测定方法要明显优于依赖于培养的传统方法。     

Rotifers as predators on components of the microbial web (bacteria,heterotrophic flagellates,ciliates) — a review

Recent investigations have shown that processes within the planktonic microbial web are of great significance for the functioning of limnetic ecosystems. However, the general importance of protozoans and bacteria as food sources for rotifers, a major component of planktonic habitats, has seldom been evaluated. Results of feeding experiments and the analysis of the food size spectrum of rotifers suggest that larger bacteria, heterotrophic flagellates and small ciliates should be a common part of the food of most rotifer species. About 10–40 per cent of rotifers' food can consist of heterotrophic organisms of the microbial web. Field experiments have indicated that rotifer grazing should generally play a minor role in bacteria consumption compared to feeding by coexisting protozoans. However, according to recent experiments regarding food selection, rotifers should be efficient predators on protozoans. Laboratory experiments have revealed that even nanophagous rotifers can feed on ciliates. Preliminary microcosm and chemostat experiments have indicated that rotifers, due to their relatively low community grazing rates compared to the growth rates of bacteria and protozoans, should generally not be able (in contrast to some cladocerans) to suppress the microbial web via grazing, though they may structure it. Filter-feeding nanophagous rotifers (e.g. brachionids) seem to be significant feeders on the smaller organisms of the microbial web (bacteria, flagellates, small ciliates), whereas grasping species (e.g. synchaetids and asplanchnids) seem to be efficient predators on larger organisms (esp. ciliates). Another important role of rotifers is their feedback effect on the microbial web. Rotifers provide degraded algae, bacteria and protozoans to the microbial web and may promote microbial activity. Additional experimental work is necessary for a better understanding of the function of rotifers in aquatic ecosystems.     

微藻与细菌作用关系的研究进展

藻类是水生环境中的初级生产者,它的生长常常伴随着细菌并受菌的影响。有研究者指出藻类和细菌有着密不可分的关系。一些研究表明与藻相关的主体细菌是特定的细菌群体,特别是α-变形菌频繁地发现,说明这类菌可能能够开启和维持共生关系。最近的研究提出了营养物质交换是菌藻共生的基础,这类相关化合物是复杂的和特定的分子,可能参与信号处理和监控作用,而不只是被动扩散。同时,这种作用很明显不是静态的,它的开启和终止可能是对环境和发育的响应。需要指出的是明确菌藻关系的作用机理还有待于进一步的深入研究,本篇综述结合新提出的理论,对细菌与微藻作用关系的研究进展进行总结,概括了微藻与菌的作用关系(进化关系,营养依赖,代谢互补和协作生物合成),这种作用关系涉及到的菌的分类(膜菌和藻际微环境菌,促生菌PGPB和溶藻菌)以及菌藻作用的应用(废水处理和生物燃料生产)的情况,并对菌藻关系的未来发展做了展望。     

Nitrifying bacterial growth inhibition in the presence of algae and cyanobacteria

Nitrifying bacteria, cyanobacteria, and algae are important microorganisms in open pond wastewater treatment systems. Nitrification involving the sequential oxidation of ammonia to nitrite and nitrate, mainly due to autotrophic nitrifying bacteria, is essential to biological nitrogen removal in wastewater and global nitrogen cycling. A continuous flow autotrophic bioreactor was initially designed for nitrifying bacterial growth only. In the presence of cyanobacteria and algae, we monitored both the microbial activity by measuring specific oxygen production rate (SOPR) for microalgae and cyanobacteria and specific oxygen uptake rate (SOUR) for nitrifying bacteria. The growth of cyanobacteria and algae inhibited the maximum nitrification rate by a factor of 4 although the ammonium nitrogen fed to the reactor was almost completely removed. Terminal restriction fragment length polymorphism (T‐RFLP) analysis indicated that the community structures of nitrifying bacteria remained unchanged, containing the dominant Nitrosospira, Nitrospira, and Nitrobacter species. PCR amplification coupled with cloning and sequencing analysis resulted in identifying Chlorella emersonii and an uncultured cyanobacterium as the dominant species in the autotrophic bioreactor. Notwithstanding their fast growth rate and their toxicity to nitrifiers, microalgae and cyanobacteria were more easily lost in effluent than nitrifying bacteria because of their poor settling characteristics. The microorganisms were able to grow together in the bioreactor with constant individual biomass fractions because of the uncoupled solids retention times for algae/cyanobacteria and nitrifiers. The results indicate that compared to conventional wastewater treatment systems, longer solids retention times (e.g., by a factor of 4) should be considered in phototrophic bioreactors for complete nitrification and nitrogen removal. Biotechnol. Bioeng. 2010;107: 1004–1011. © 2010 Wiley Periodicals, Inc.     

Does Microorganism Stoichiometry Predict Microbial Food Web Interactions After a Phosphorus Pulse?

Knowledge of variations in microbial food web interactions resulting from atmospheric nutrient loads is crucial to improve our understanding of aquatic food web structure in pristine ecosystems. Three experiments mimicking atmospheric inputs at different nitrogen/phosphorus (N/P) ratios were performed in situ covering the seasonal biological succession of the pelagic zone in a high-mountain Spanish lake. In all experiments, abundance, biomass, algal cell biovolume, P-incorporation rates, P-cell quota, and N/P ratio of algae strongly responded to P-enrichment, whereas heterotrophic bacteria remained relatively unchanged. Ciliates were severely restricted when a strong algal exploitation of the available P (bloom growth or storage strategies) led to transient (mid-ice-free experiment) or chronic (late ice-free experiment) P-deficiencies in bacteria. In contrast, maximum development of ciliates was reached when bacteria remained P-rich (N/P < 20) and algae approached Redfield proportions (N/P approximately 16). Evidence of a higher P-incorporation rate supports the proposition that algae and bacteria shifted from a mainly commensalistic-mutualistic to a competitive relationship for the available P when bacterial P-deficiency increased, as reflected by their unbalanced N/P ratio (N/P > 20-24). Hence, the bacterial N/P ratio proved be a key factor to understand the algae-bacteria relationship and microbial food web development. This study not only demonstrates the interdependence of life history strategies, stoichiometric nutrient content, and growth but also supports the use of bacterial N/P thresholds for diagnosing ciliate development, a little-studied aspect worthy of further attention.     

Flow cytometric enumeration and immunophenotyping of hematopoietic stem and progenitor cells

Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells (HPC) is widely used for evaluation of graft adequacy of peripheral blood stem cell grafts, and is also useful in planning the apheresis sessions necessary to obtain these grafts. The state-of-the-art method to enumerate CD34+ cells makes use of a multiparameter definition of HPC based on their light scatter characteristics and dim expression of CD45, and the use of counting beads to derive the concentration of CD34+ cells directly from the flow cytometric assessment. This method can be extended with a viability stain and additional markers for further immunological characterization of CD34+ cells, and has been successfully implemented in multicenter trials. Thus, the lower threshold of a safe HPC graft in terms of short- and long-term hematopoiesis may be more accurately defined.