Non-axenic Conditions Research Articles

Organic carbon utilisation by the filamentous alga Tribonema

Filamentous algae have potential application to wastewater treatment, in particular for efficient recovery of nutrients into biomass. However, supplying inorganic carbon is a major limiting factor. The utilisation of organic carbon present in wastewater may reduce the constraints in carbon supply, however there is little knowledge of mixotrophic growth amongst filamentous algae. This study investigated the utilisation of organic carbon sources relevant to wastewater by the filamentous xanthophyte alga Tribonema. Algae growth was compared in the absence of organic carbon (autotrophic) and in presence of 0.2 g-C L-1 glucose, ethanol or acetate under mixotrophic (presence of organic carbon and light) or heterotrophic (presence of organic carbon and absence of light) conditions. To investigate direct utilisation of organic carbon and indirect utilisation via bacterial CO2-genesis, cultivation was performed under both axenic and non-axenic conditions. Tribonema was found to directly utilise glucose, which increased mixotrophic productivity and maintained growth under heterotrophic conditions. In contrast, acetate was only indirectly utilised mixotrophically in the presence of bacteria, whereas ethanol was not utilised under any conditions. The underlying mechanisms of glucose utilisation by Tribonema were also investigated by analysing its photosynthetic rate and respiration rate under glucose concentrations ranging from 0 – 100 g L-1. Based on the results, enhancements to metabolic pathways and reduced CO2 requirements provided by glucose utilisation were proposed. Despite the positive results with respect to glucose utilisation, out competition for this resource by bacteria suggest that Tribonema is more suitable for treatment of wastewater with low organic carbon concentrations, such as secondary-treated wastewater effluent.

Co-Culturing Microalgae with Roseobacter Clade Bacteria as a Strategy for Vibrionaceae Control in Microalgae-Enriched Artemia.

Bacterial communities associated with fish larvae are highly influenced by the microbiota of live prey used as feed (rotifers or Artemia), generally dominated by bacterial strains with a low degree of specialization and high growth rates, (e.g., Vibrionaceae), which can be detrimental to larvae. Co-cultivation of microalgae used in the enrichment of Artemia (e.g., Phaeodactylum tricornutum, or Chlorella minutissima) with Vibrio-antagonistic probiotics belonging to the Roseobacter clade bacteria (e.g., Phaeobacter spp. or Ruegeria spp.) was studied. The introduction of the probiotics did not affect microalgae growth or significantly modify the composition of bacterial communities associated with both microalgae, as revealed by DGGE analysis. The inoculation of P. tricornutum with Ruegeria ALR6 allowed the maintenance of the probiotic in the scale-up of the microalgae cultures, both in axenic and non-axenic conditions. Using Ruegeria-inoculated P. tricornutum cultures in the enrichment of Artemia reduced the total Vibrionaceae count in Artemia by 2 Log units, therefore preventing the introduction of opportunistic or pathogenic bacteria to fish larvae fed with them.

Absence of microbiome triggers extensive changes in the transcriptional profile of Hermetia illucens during larval ontogeny

Black soldier fly larvae (BSF, Hermetia illucens) have gained much attention for their industrial use as biowaste recyclers and as a new source of animal proteins. The functional effect that microbiota has on insect health and growth performance remains largely unknown. This study clarifies the role of microbiota in BSF ontogeny by investigating the differential genomic expression of BSF larvae in axenic conditions (i.e., germfree) relative to non-axenic (conventional) conditions. We used RNA-seq to measure differentially expressed transcripts between axenic and conventional condition using DESeq2 at day 4, 12 and 20 post-hatching. Gene expression was significantly up or down-regulated for 2476 transcripts mapped in gene ontology functions, and axenic larvae exhibited higher rate of down-regulated functions. Up-regulated microbiota-dependant transcriptional gene modules included the immune system, the lipid metabolism, and the nervous system. Expression profile showed a shift in late larvae (day 12 and 20), exposing a significant temporal effect on gene expression. These results provide the first evidence of host functional genes regulated by microbiota in the BSF larva, further demonstrating the importance of host-microbiota interactions on host ontogeny and health. These results open the door to optimization of zootechnical properties in alternative animal protein production, biowaste revalorization and recycling.

Evaluation of Aurantiochytrium mangrovei Biomass Grown on Digestate as a Sustainable Feed Ingredient of Sea Bass, Dicentrarchus labrax, Juveniles and Larvae

The use of microalgae as a sustainable source of n-3 long-chain polyunsaturated fatty acids (LC-PUFA) as an alternative to fish oils from small pelagic fish (e.g., anchovy, sardine) has received growing interest in the past few years. The present study aimed to: (i) produce Aurantiochytrium mangrovei biomass by heterotrophic fermentation using a medium containing anaerobic digestion liquid effluent, and (ii) evaluate a biomass rich in n-3 LC-PUFA and good quality proteins as a feed ingredient for sea bass juveniles and larvae. Two 800 L bioreactors were used to produce Aurantiochytrium biomass in non-axenic conditions. Biomass was then filtered through a crossflow filtration system (300 Kda ceramic membrane) and freeze-dried. Sea bass juveniles (32.7 ± 4.2 g) were fed both a control diet and a diet containing 15% of freeze-dried A. mangrovei biomass for 38 days. Juvenile survival percentage was 90% on average in both dietary conditions. Similar growth was observed between fish fed with both diets, demonstrating the feasibility to replace 15% of a standard fish feed by Aurantiochytrium biomass. The liver of sea bass juveniles fed with the A. mangrovei diet contained significantly higher proportions of 22:6n-3, 22:5n-6, and 20:4n-6 than those fed with the control diet, while the proportions of 16:0, 16:1n-7, and 18:1n-9 were significantly lower. The secondary oxidation, as measured by malonylaldehyde (MDA) content, in the liver and muscle of juveniles fed with the microalgae diet tended to be higher than in fish fed the control diet, but the differences were not statistically significant. Although the larvae survival percentage was low for all the tanks after 41 days of rearing, the inclusion of 15% of hydrolyzed A. mangrovei biomass in the larvae micro-diet did not impair the development of sea bass larvae and only marginally affected their lipid composition. In the future, we have to further optimize a sustainable workflow between Aurantiochytrium cultivation and fish feed production and confirm the zootechnical and biochemical results.

Feeding Selectivity of Aphelenchoides besseyi and A. pseudogoodeyi on Fungi Associated with Florida Strawberry.

Aphelenchoides besseyi and A. pseudogoodeyi are foliar nematodes associated with commercial strawberry production in Florida, United States. The reproductive and feeding habits of these two nematode species were assessed on Florida isolates of the fungi Botrytis cinerea, Colletotrichum gloeosporioides, Macrophomina phaseolina, and Neopestalotiopsis rosae, which are pathogenic to strawberry, and nonpathogenic isolates of Fusarium oxysporum and Monilinia fructicola grown on potato dextrose agar in Petri dishes. Each culture was inoculated with six specimens of mixed life stages of A. besseyi or A. pseudogoodeyi and incubated at 24°C under axenic and nonaxenic conditions 23 and 31 days after inoculation, respectively. A. besseyi reproduction rates were greater on strawberry-pathogenic isolates of B. cinerea, C. gloeosporioides, and N. rosae than on the nonpathogenic isolates of F. oxysporum and M. fructicola. In contrast, reproductive rates of A. pseudogoodeyi did not vary among fungi cultures. For both nematode species, M. phaseolina was a poor host because it did not produce mycelium on the media used. Our findings indicate that A. besseyi is more selective in its fungal-feeding preference than A. pseudogoodeyi. Additionally, A. pseudogoodeyi eggs and juveniles were significantly more numerous than adults. Yet, for A. besseyi, adult stages were more abundant. Fungi aid in the maintenance of soil-dwelling populations of these two nematode species. Removing fungus-infected strawberry plant residues is both a desirable and effective management practice to limit A. besseyi in central Florida commercial strawberry fields.

Bacteria enhance the production of extracellular polymeric substances by the green dinoflagellate Lepidodinium chlorophorum

High biomasses of the marine dinoflagellate Lepidodinium chlorophorum cause green seawater discolorations along Southern Brittany (NE Atlantic, France). The viscosity associated to these phenomena has been related to problems in oyster cultivation. The harmful effect of L. chlorophorum might originate from the secretion of Extracellular Polymeric Substances (EPS). To understand whether the EPS are produced by L. chlorophorum or its associated bacteria, or if they are a product of their interaction, batch cultures were performed under non-axenic and pseudo-axenic conditions for three strains. Maximum dinoflagellate cell abundances were observed in pseudo-axenic cultures. The non-sinking fraction of polymers (Soluble Extracellular Polymers, SEP), mainly composed of proteins and the exopolysaccharide sulphated galactan, slightly increased in pseudo-axenic cultures. The amount of Transparent Exopolymer Particles (TEP) per cell increased under non-axenic conditions. Despite the high concentrations of Particulate Organic Carbon (POC) measured, viscosity did not vary. These results suggest that the L. chlorophorum-bacteria interaction could have a detrimental consequence on the dinoflagellate, translating in a negative effect on L. chlorophorum growth, as well as EPS overproduction by the dinoflagellate, at concentrations that should not affect seawater viscosity.

Metabolic engineering of phosphite metabolism in Synechococcus elongatus PCC 7942 as an effective measure to control biological contaminants in outdoor raceway ponds

BackgroundThe use of cyanobacteria and microalgae as cell factories to produce biofuels and added-value bioproducts has received great attention during the last two decades. Important investments have been made by public and private sectors to develop this field. However, it has been a challenge to develop a viable and cost-effective platform for cultivation of cyanobacteria and microalgae under outdoor conditions. Dealing with contamination caused by bacteria, weedy algae/cyanobacteria and other organisms is a major constraint to establish effective cultivation processes.ResultsHere, we describe the implementation in the cyanobacterium Synechococcus elongatus PCC 7942 of a phosphorus selective nutrition system to control biological contamination during cultivation. The system is based on metabolic engineering of S. elongatus to metabolize phosphite, a phosphorus source not normally metabolized by most organisms, by expressing a bacterial phosphite oxidoreductase (PtxD). Engineered S. elongatus strains expressing PtxD grow at a similar rate on media supplemented with phosphite as the non-transformed control supplemented with phosphate. We show that when grown in media containing phosphite as the sole phosphorus source in glass flasks, the engineered strain was able to grow and outcompete biological contaminants even when the system was intentionally inoculated with natural competitors isolated from an irrigation canal. The PtxD/phosphite system was successfully used for outdoor cultivation of engineered S. elongatus in 100-L cylindrical reactors and 1000-L raceway ponds, under non-axenic conditions and without the need of sterilizing containers and media. Finally, we also show that the PtxD/phosphite system can be used as selectable marker for S. elongatus PCC 7942 transgenic strains selection, eliminating the need of antibiotic resistance genes.ConclusionsOur results suggest that the PtxD/phosphite system is a stable and sufficiently robust strategy to control biological contaminants without the need of sterilization or other complex aseptic procedures. Our data show that the PtxD/phosphite system can be used as selectable marker and allows production of the cyanobacterium S. elongatus PCC 7942 in non-axenic outdoor reactors at lower cost, which in principle should be applicable to other cyanobacteria and microalgae engineered to metabolize phosphite.

Effect of Glycerol Concentration and Light Intensity on Growth and Biochemical Composition of Arthrospira (Spirulina) Platensis: A Study in Semi-Continuous Mode with Non-Aseptic Conditions

In this study, Arthrospira platensis was grown in the presence of different glycerol concentrations (0.5–9 g/L) under three light intensities (5, 10 and 15 Klux) in semi-continuous mode and under non-axenic conditions. The aim of this study was to investigate the growth performance, the biomass biochemical composition and any interactions between A. platensis and bacteria that would potentially grow as well on glycerol. The results here show that glycerol did not have any positive effect on biomass production of A. platensis. In contrast, it was observed that by increasing glycerol concentration the growth performance of A. platensis was restricted, while a gradual increase of bacteria population was observed, which apparently outcompeted and repressed A. platensis growth. Chlorophyll fluorescence measurements (Quantum Yields) revealed that glycerol was not an inhibiting factor per se of photosynthesis. On the other hand, cyanobacterial biomass grown on glycerol displayed a higher content in proteins and lipids. Especially, protein productivity was enhanced around 15–35% with the addition of glycerol compared to the control. In distinction, carbohydrate and photosynthetic pigments (phycocyanin and chlorophyll-α) content decreased with the increase of glycerol concentration. The results here suggest that A. platensis did not utilize glycerol for biomass production but most probably as metabolic energy carrier towards synthesis of proteins and lipids, which are more energy consuming metabolites compared to carbohydrates. The study revealed that the addition of glycerol at amounts of 0.5–1.5 g/L could be a strategy to improve protein productivity by A. platensis.

Bacterial endophytes of mangrove propagules elicit early establishment of the natural host and promote growth of cereal crops under salt stress

Mangroves, dominating tropical intertidal zones and estuaries, are among the most salt tolerant plants, and propagate through reproductive units called propagules. Similarly to plant seeds, propagules may harbor beneficial bacteria. Our hypothesis was that mangroves, being able to grow into seawater, should harbor bacteria able to interact with the host and to exert positive effects under salt stress, which could be exploited to improve crop production. Therefore, we isolated bacterial endophytes from mangrove propagules with the aim to test whether these bacteria have a beneficial potential on their natural host and on different crops such as barley and rice, cultivated under salt stress. The 172 bacterial isolates obtained were screened for plant growth promotion (PGP) activities in vitro, and the 12 most promising isolates were tested on barley under non-axenic conditions and salt stress. Gordonia terrae KMP456-M40 was the best performing isolate, increasing ear weight by 65%. Based on the in vivo PGP activity and the root colonization ability, investigated by fluorescence in situ hybridization and confocal microscopy, three strains were additionally tested on mangrove propagule germination and on rice growth. The most effective strain was again G. terrae KMP456-M40, which enhanced the root length of mangrove seedlings and the biomass of salt-stressed rice under axenic conditions up to 65% and 62%, respectively. We demonstrated that propagules, the reproductive units of mangroves, host beneficial bacteria that enhance the potential of mangrove seedlings establishment and confer salt tolerance to cereal crops.

Heterotrophic cultivation of T. obliquus under non-axenic conditions by uncoupled supply of nitrogen and glucose

A fed-batch strategy is proposed to produce microalgae biomass under non-axenic heterotrophic conditions. The strategy induces the alternation of N-deplete (Glucose-replete) and N-replete (Glucose-deplete) cultivation phases by the periodic and uncoupled supply of glucose and NO3− to the culture. Cultivation of the microalga T. obliquus with this strategy reduced the ratio of the bacteria to microalgae cell concentration from 1.6, attained by conventional photoautotrophic cultivation, to 0.03. During the N-deplete phase, microalgae duplication stopped and biomass concentration increased 1.9 times, while during the N-replete phase, microalgae duplicated halving their average size and losing about 25% of their weight. The process proved to be effective under several consecutive cycles. Biomass productivity until 6.1 g/Ld and biomass concentration until 26 g/L were achieved. The results demonstrate that the proposed strategy can effectively prevent bacterial contamination, paving the way to the large scale production of microalgae biomass under non-axenic heterotrophic conditions.

Microalgae cultivation in sugarcane vinasse: Selection, growth and biochemical characterization

Sugarcane ethanol is produced at large scale generating wastes that could be used for microalgae biomass production in a biorefinery strategy. In this study, forty microalgae strains were screened for growth in sugarcane vinasse at different concentrations. Two microalgae strains, Micractinium sp. Embrapa|LBA32 and C. biconvexa Embrapa|LBA40, presented vigorous growth in a light-dependent manner even in undiluted vinasse under non-axenic conditions. Microalgae strains presented higher biomass productivity in vinasse-based media compared to standard Bold’s Basal Medium in cultures performed using 15L airlift flat plate photobioreactors. Chemical composition analyses showed that proteins and carbohydrates comprise the major fractions of algal biomass. Glucose was the main monosaccharide detected, ranging from 46% to 76% of the total carbohydrates content according to the strain and culture media used. This research highlights the potential of using residues derived from ethanol plants to cultivate microalgae for the production of energy and bioproducts.

Genomic Barcode-Based Analysis of Exoelectrogens in Wastewater Biofilms Grown on Anode Surfaces

The most energy-demanding step of wastewater treatment is the aeration-dependent elimination of organic carbon. Microbial fuel cells (MFCs) offer an alternative strategy in which carbon elimination is conducted by anaerobic microorganisms that transport respiratory electrons originating from carbon oxidation to an anode. Hence, chemical energy is directly transformed into electrical energy. In this study, the use and stability of barcodecontaining exoelectrogenic model biofilms under non-axenic wastewater treatment conditions are described. Genomic barcodes were integrated in Shewanella oneidensis, Geobacter sulfurreducens, and G. metallireducens. These barcodes are unique for each strain and allow distinction between those cells and naturally occurring wild types as well as quantification of the amount of cells in a biofilm via multiplex qPCR. MFCs were pre-incubated with these three strains, and after 6 days the anodes were transferred into MFCs containing synthetic wastewater with 1% wastewater sludge. Over time, the system stabilized and the coulomb efficiency was constant. Overall, the initial synthetic biofilm community represented half of the anodic population at the end of the experimental timeline. The part of the community that contained a barcode was dominated by G. sulfurreducens cells (61.5%), while S. oneidensis and G. metallireducens cells comprised 10.5% and 17.9%, respectively. To the best of our knowledge, this is the first study to describe the stability of a synthetic exoelectrogenic consortium under non-axenic conditions. The observed stability offers new possibilities for the application of synthetic biofilms and synthetically engineered organisms fed with non-sterile waste streams.

Production of co-polymers of polyhydroxyalkanoates by regulating the hydrolysis of biowastes

Production of polyhydroxyalkanoate (PHA) co-polymers by Bacillus spp. was studied by feeding defined volatile fatty acids (VFAs) obtained through controlled hydrolysis of various wastes. Eleven mixed hydrolytic cultures (MHCs) each containing 6 strains could generate VFA from slurries of (2% total solids): pea-shells (PS), potato peels (PP), apple pomace (AP) and onion peels (OP). PS hydrolysates (obtained with MHC2 and MHC5) inoculated with Bacillus cereus EGU43 and Bacillus thuringiensis EGU45 produced co-polymers of PHA at the rate of 15–60mg/L with a 3HV content of 1%w/w. An enhancement in PHA yield of 3.66-fold, i.e. 205–550mg/L with 3HV content up to 7.5%(w/w) was observed upon addition of OP hydrolysate and 1% glucose (w/v) to PS hydrolysates. This is the first demonstration, where PHA co-polymer composition, under non-axenic conditions, could be controlled by customizing VFA profile of the hydrolysate by the addition of different biowastes.

Production of algal biomass, chlorophyll, starch and lipids using aquaculture wastewater under axenic and non-axenic conditions

Although co-cultivation of algae with aquaculture products has the potential to reduce water use and pollutant discharges while producing energy feedstocks and other end-products, little research has been carried out in this area. Maintaining axenic conditions (algal monocultures without other microorganisms) in algal culturing systems using aquaculture wastewater as a nutrient source would not be practical. This study examined the effects of the use of aquaculture wastewater as a nutrient source on biomass development of three algae cultures (indigenous mixed species consortium, Chlorella sp. and Scenedesmus) under axenic and non-axenic conditions. Biomass development was assessed by cell growth, chlorophyll, starch and lipid production. The presence of aquaculture microorganisms decreased biomass productivity in Chlorella but not in the other algae cultures. Non-axenic conditions had no effect on overall starch and chlorophyll production; however, significantly higher lipid contents were achieved under non-axenic conditions for Chlorella and the indigenous culture. The higher algal lipid content for these cultures under non-axenic conditions may have been due to competition with bacteria for nutrients. The presence of bacteria was required for effective removal of organics, while effective nitrogen removal was observed in all systems containing algae. Results from this study also show that algae harvesting should be timed to coincide with the peak production of the desired target end-product (biomass, chlorophyll, starch or lipid).

Mechanism of phosphate solubilization and antifungal activity of Streptomyces spp. isolated from wheat roots and rhizosphere and their application in improving plant growth

The application of plant-growth-promoting rhizobacteria (PGPR) at field scale has been hindered by an inadequate understanding of the mechanisms that enhance plant growth, rhizosphere incompetence and the inability of bacterial strains to thrive in different soil types and environmental conditions. Actinobacteria with their sporulation, nutrient cycling, root colonization, bio-control and other plant-growth-promoting activities could be potential field bio-inoculants. We report the isolation of five rhizospheric and two root endophytic actinobacteria from Triticum aestivum (wheat) plants. The cultures exhibited plant-growth-promoting activities, namely phosphate solubilization (1916 mg l(-1)), phytase (0.68 U ml(-1)), chitinase (6.2 U ml(-1)), indole-3-acetic acid (136.5 mg l(-1)) and siderophore (47.4 mg l(-1)) production, as well as utilizing all the rhizospheric sugars under test. Malate (50-55 mmol l(-1)) was estimated in the culture supernatant of the highest phosphate solublizer, Streptomyces mhcr0816. The mechanism of malate overproduction was studied by gene expression and assays of key glyoxalate cycle enzymes - isocitrate dehydrogenase (IDH), isocitrate lyase (ICL) and malate synthase (MS). The significant increase in gene expression (ICL fourfold, MS sixfold) and enzyme activity (ICL fourfold, MS tenfold) of ICL and MS during stationary phase resulted in malate production as indicated by lowered pH (2.9) and HPLC analysis (retention time 13.1 min). Similarly, the secondary metabolites for chitinase-independent biocontrol activity of Streptomyces mhcr0817, as identified by GC-MS and (1)H-NMR spectra, were isoforms of pyrrole derivatives. The inoculation of actinobacterial isolate mhce0811 in T. aestivum (wheat) significantly improved plant growth, biomass (33%) and mineral (Fe, Mn, P) content in non-axenic conditions. Thus the actinobacterial isolates reported here were efficient PGPR possessing significant antifungal activity and may have potential field applications.

Ecobiotechnological Approach for Exploiting the Abilities of Bacillus to Produce Co-polymer of Polyhydroxyalkanoate.

Ecobiotechnological approach is an attractive and economical strategy to enrich beneficial microbes on waste biomass for production of Polyhydroxyalkanoate (PHA). Here, six strains of Bacillus spp. were used to produce co-polymers of PHA from pea-shells. Of the 57 mixed bacterial cultures (BCs) screened, two of the BCs, designated as 5BC1 and 5BC2, each containing 5 strains could produce PHA co-polymer at the rate of 505-560mg/l from feed consisting of pea-shell slurry (PSS, 2% total solids) and 1% glucose (w/v). Co-polymer production was enhanced from 65-560mg/l on untreated PSS to 1,610-1,645mg/l from PSS treated with defined hydrolytic bacteria and 1% glucose. Supplementation of the PSS hydrolysate with sodium propionate enabled 5BC1 to produce co-polymer P(3HB-co-3HV) with a 3HV content up to 13% and a concomitant 1.46-fold enhancement in PHA yield. Using the principles of ecobiotechnology, this is the first demonstration of PHA co-polymer production by defined co-cultures of Bacillus from biowaste as feed under non-axenic conditions.

MiniMax, a new diminutive Glycine max genotype with a rapid life cycle, embryogenic potential and transformation capabilities

We developed Glycine max cv MiniMax (PI643148) that has a rapid life cycle, short stature and characteristic simple sequence repeat (SSR) markers that could make it useful for mutant screening, functional genomics, genetic mapping and other studies involving soybeans. We demonstrate that MiniMax is able to make somatic embryos (SEs) that rapidly develop into plantlets. Thus, the rapid cycling habit carries over into aspects of plant regeneration. Chimaeras (having transformed roots with untransformed aerial stocks) have been produced rapidly under non-axenic conditions using Agrobacterium rhizogenes-mediated transformation. Part of these experiments involved the engineering an enhanced green fluorescent protein (eGFP) reporter cassette outside the multi-cloning site of a plant expression vector, permitting non-invasive visual screening of the transformed roots. The rapid cycling growth habit of MiniMax, its ability to efficiently generate SEs and ability to be transformed should prove useful for basic aspects of G. max molecular and genetic research.

Interactions of planktonic algae and bacteria: effects on algal growth and organic matter dynamics

Interactions between phytoplankton and heterotrophic bacteria have been intensively studied, but many aspects of these interactions are still unknown because most of the studies were performed under non-axenic conditions. Therefore, we investigated the growth and release of dis- solved organic matter of the marine diatom Thalassiosira rotula in axenic culture in comparison to T. rotula cultures inoculated with either a natural marine bacterial community (German Wadden Sea) or 3 different bacterial isolates. The isolates affiliated to the Roseobacter group (HP50), Hyphomonas (HP48) and Flexibacteriaceae (HP49) and were previously obtained from a T. rotula culture inoc- ulated with a natural marine bacterial community (German Wadden Sea). To test whether the availability of inorganic nutrients, vitamins and trace metals affects algal growth, organic matter release and interactions with heterotrophic bacteria, we performed experiments with either Guil- lard's f/2 or f/10 medium, which differ in their inorganic nutrient, vitamin and trace metal concentra- tion by a factor of 5. The bacterial community promoted growth of T. rotula in both media, as shown by the higher algal numbers relative to the axenic cultures, but also led to a rapid decline after the growth phase. Isolate HP50 promoted algal growth in the f/2 medium, but inhibited growth in the f/10 medium, whereas isolate HP48 showed the opposite patterns. Isolate HP49 prevented algal growth in both media. T. rotula exhibited distinct release patterns of dissolved organic carbon (DOC), dissolved amino acids, dissolved neutral carbohydrates, transparent exopolymer particles (TEP) and protein-containing particles (Coumassie Blue-stainable particles, CSP), which were modulated by the bacteria added. TEP and CSP were produced by the growing alga only in the presence of bac- teria, indicating that the bacterial modification of algal exudates and/or the bacterial decomposition of the alga are prerequisites for formation of these microparticles. Our results show that the presence of the bacterial community and of specific populations have distinct effects on the growth and organic matter release of T. rotula and presumably also on other algae.

Algae–bacteria interactions and their effects on aggregation and organic matter flux in the sea

Aggregation of algae, mainly of diatoms, is an important process in marine pelagic systems, often terminating phytoplankton blooms and leading to the sinking of particulate organic matter in the form of marine snow. This process has been studied extensively, but the specific role of heterotrophic bacteria has largely been neglected, mainly because field studies and most experimental work were performed under non-axenic conditions. We tested the hypothesis that algae-bacteria interactions are instrumental in aggregate dynamics and organic matter flux. A series of aggregation experiments has been carried out in rolling tanks with two marine diatoms typical of temperate regions (Skeletonema costatum and Thalassiosira rotula) in an axenic treatment and one inoculated with marine bacteria. Exponentially growing S. costatum and T. rotula exhibited distinctly different aggregation behavior. This was reflected by their strikingly different release of dissolved organic matter (DOM), transparent exopolymer particles (TEP) and protein-containing particles (CSP), as well as their bacterial biodegradability and recalcitrance. Cells of S. costatum aggregated only little and their bacterial colonization remained low. Dissolved organic matter, TEP and CSP released by this alga were largely consumed by free-living bacteria. In contrast, T. rotula aggregated rapidly and DOM, TEP and CSP released resisted bacterial consumption. Experiments conducted with T. rotula cultures in the stationary growth phase, however, showed rapid bacterial colonization and decomposition of algal cells. Our study highlights the importance of heterotrophic bacteria to control the development and aggregation of phytoplankton in marine systems.

Interactions between marine snow and heterotrophic bacteria: aggregate formation and microbial dynamics

Macroscopic aggregates (marine snow) contribute to new production and nutrient dynamics in the upper ocean and vertical fluxes of organic matter to the deep ocean. To test whether microorganisms play a significant role in phytoplankton aggregate formation we studied particle abundance and size as well as abundance, colonization behaviour, and community composition of bacteria during the growth of 2 marine diatoms (Thalassiosira weissflogii and Navicula sp.) under axenic and non-axenic conditions. Community composition of free-living and attached bacteria during phytoplankton growth and aggregation was studied by amplification of 16S rRNA gene frag- ments and denaturing gradient gel electrophoresis (DGGE). Our results show that the presence of bacteria was a prerequisite for aggregation of T. weissflogii but not of Navicula sp. Occurrences of distinct populations of free-living and attached bacteria depended on phytoplankton growth and aggregation dynamics. The community composition of especially attached bacteria significantly differed between the 2 algal cultures. Our study suggests that phytoplankton aggregation and verti- cal fluxes are closely linked to interactions between the marine phytoplankton and the ambient microbial community.