Microplastics as a Novel Mode of Transportation for Potentially Dangerous ‘Hitchhiking’ Vibrio Spp.

 As the rate of global plastic production continues to increase, so too does public awareness of plastic pollution within the marine environment. Whilst the hazards surrounding larger plastic debris have received considerable attention from researchers, relatively little is known about the threats posed by much tinier ‘microplastics’ and how they might interact with marine microbial communities.

 Microplastics are generally classified as plastic fragments less than 5mm in diameter and occur when larger plastics break down due to weathering. As with larger plastic debris, microplastics in the marine environment become swiftly colonised by a mosaic of bacteria and other microscopic organisms, conglomerating to form complex biofilms. Previous studies have highlighted how marine plastic debris-associated microbial populations usually differ in composition from the surrounding bacterioplankton. As such, the term ‘Plastisphere’ has arisen, recognising plastic surfaces as novel habitats for their microbial constituents.

 Being lightweight and small in size, microplastics are suspended and transported readily by waves and currents, allowing their microbial colonisers to hitch rides over considerable distances. Accordingly, it is plausible that microplastics may serve as novel vectors for the transportation and dispersal of microbes.

 One recent study performed by Kirstein et al. investigated the occurrence of potentially human pathogenic Vibrio bacteria colonising microplastics within the North and Baltic Seas. The primary focus of the study was to detect the presence of V. cholerae, V. parahaemolyticus and V. vulnificus; known to cause disease in humans.

 Microplastic particles were collected from surface waters using a fine mesh net and the surrounding surface seawater was also sampled so that the occurrence of Vibrio spp. could be compared between microplastic-attached and bacterioplankton populations. Microplastic and filtered seawater samples were incubated individually at 37°C to allow for the selective enrichment of mesophilic Vibrio spp. Subsequently, samples displaying growth were plated onto a vibrio-selective agar media and incubated further. Resulting colonies resembling V. cholerae, V. parahaemolyticus and V. vulnificus were analysed via MALDI-TOF mass spectrometry and PCR amplification of species-specific genes in order to confirm their identification.

 Of the collected microplastics analysed within this study, 13% were found to exhibit colonisation by cultivatable Vibrio spp. Whilst V. cholerae and V. vulnificus were detected only within seawater samples, potentially pathogenic V. parahaemolyticus was isolated from a number of microplastic particles. Where V. parahaemolyticus was detected, it was generally present both attached to microplastics and within the surrounding seawater, suggesting that colonisation of microplastics by Vibrio spp. may occur from the surrounding bacterioplankton. Furthermore, sampling efforts within the Baltic Sea were unable to detect V. parahaemolyticus within surface waters yet identified it associated to a single microplastic particle. Such an observation may serve as an illustration of Vibrio spp. utilising microplastic to 'hitch' a ride, persisting into an environment where it may have otherwise been absent.

 Ultimately, Kirstein et al. have made an important contribution to the understanding of the hazards surrounding microplastic pollution by providing the first definitive proof of potentially human pathogenic Vibrio spp. within marine microplastic biofilms. As average sea surface temperatures continue to rise, the occurrence of Vibrio infection is increasing in frequency and geographical range. Moreover, as larger plastic items repeatedly fragment within the natural environment, marine microplastic concentrations are set to increase. Accordingly, global warming coupled with the emerging problem of marine microplastic could prove to be a perilous combination. In order to fully comprehend the hazards posed by microplastic-attached Vibrio spp., subsequent research could perhaps employ a metagenomic approach in order to gain a clearer understanding of Vibrio abundance, avoiding possible cultivation bias.              

Reviewed Paper:

Kirstein, I. V., Kirmizi, S., Wichels, A., Garin-Fernandez, A., Erler, R., Löder, M. & Gerdts, G. (2016). Dangerous hitchhikers? Evidence for potentially pathogenic Vibrio spp. on microplastic particles. Marine Environmental Research, 120: 1-8.

Can a marine diatom be an efficient source of omega-3 alternatively to conventional fish oil?

Omega-3 fatty acids are essential in human nutrition to ensure a well functioning of the metabolism and the cardiovascular system. Two major forms of these fatty acids are EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid). So far the main source of fish oils is the fish industry itself. But with increasing environmental challenges on one hand and economical and ethical issues on the other hand the production of fish oil is facing some difficulties. Therefore scientists are looking into marine microalgae as a potential and sustainable source for omega-3. A few research studies were already conducted showing that microalgae provide a wide range of advantages compared to fish stock but they mainly took place in southern latitudes^,2.

The aim of this study was to evaluate the suitability of microalgae as a source of EPA and DHA under northern climate conditions on a large scale. The microalga of interest was the diatom Phaeodactylum tricornutum, a robust species with some valuable characteristics, such as a high growth rate and high content in EPA.

The study took place in Norway for a period of six months categorized in three different seasons covering spring, summer and autumn. As test subjects three strains of the diatom P. tricornutum were used, two local strains (M28 and B58) and one commercial Spanish strain called Fito. The marine microalgae were cultivated in big outdoor flat panel photobioreactors to enable the observation of strain specific responses to the environmental characteristics. Each panel was observed intensively regarding the pH, the temperature as well as the nitrate and phosphate concentrations. The difficulty of the study consisted in maintaining the conditions in the panel at a steady state. Due to some technical problems related to the choice of equipment and methods the pH level fluctuated heavily at some points of the experiment.

However the study lead to some interesting findings. The highest biomass production with a correlating high content of EPA was observed in the spring season because of the highest irradiance. The three strains differed in their growth rate but weren’t consistent during all three seasons. Compared to M28 and B58, Fito significantly showed the highest EPA content.

The article showed that a cultivation of marine microalgae on a large scale is possible in northern latitudes. However, this is efficient only during half of the year, when the irradiance is high. This raises the question if algal omega-3 is beneficial enough from an economical point of view?

Even if the productivity of diatoms and the content of EPA is stronger in southern located studies a big disadvantage is the cost intensive cooling necessary because of the strong irradiance. In general the cultivation of marine diatoms is still quite expensive compared to the conventional fish industry. I think that a lot of improvements regarding the methods and equipment will be needed. Another study showed that the acyl lipid composition could be influenced by the age of the cells or by the use of biolistic transformation². Maybe this could be used in further attempts to increase the EPA content in the diatoms. In my opinion improvements regarding the experimental set-up could also have a great impact on the efficiency of the study. The use of helical instead of flat panel photobioreactor¹, or even chemostats could be a way to enhance the stability of the cultures.

In sum, more research on this subject will be needed even though the potential of marine microalga as a source of omega-3 is undeniable.

Article Reviewed

Steinrücken, P., Prestegard, S. K., de Vree, J. H., Storesund, J. E., Pree, B., Mjøs, S. A., & Erga, S. R. (2018). Comparing EPA production and fatty acid profiles of three Phaeodactylum tricornutum strains under western Norwegian climate conditions. Algal research, 30, 11-22.

https://www.sciencedirect.com/science/article/pii/S2211926417308846

References

¹ Fernández, F. A., Hall, D. O., Guerrero, E. C., Rao, K. K., & Grima, E. M. (2003). Outdoor production of Phaeodactylum tricornutum biomass in a helical reactor. Journal of Biotechnology, 103(2), 137-152.

https://www.sciencedirect.com/science/article/pii/S0168165603001019

² Alonso, D. L., Belarbi, E. H., Fernández-Sevilla, J. M., Rodríguez-Ruiz, J., & Grima, E. M. (2000). Acyl lipid composition variation related to culture age and nitrogen concentration in continuous culture of the microalga Phaeodactylum tricornutum. Phytochemistry, 54(5), 461-471.

https://www.sciencedirect.com/science/article/pii/S0031942200000844

³ Hamilton, M. L., Haslam, R. P., Napier, J. A., & Sayanova, O. (2014). Metabolic engineering of Phaeodactylum tricornutum for the enhanced accumulation of omega-3 long chain polyunsaturated fatty acids. Metabolic engineering, 22, 3-9.

https://www.sciencedirect.com/science/article/pii/S1096717613001225

More information about the molecular method biolistic transformation:

https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/gene-gun

Quorum sensing: Gram-negative bacteria are positive for Gracilaria dura carpospore liberation. 

Macroalgae release extracellular substances, which provides nutrition for a variety of microorganisms. Bacteria are the most abundant of microbial groups on macroalgae and are found on the external surfaces and internal tissues of macroalgae. Bacterial communities have shown to impact the growth, morphogenesis, and reproduction on the macroalgae: Ulva and Gracilaria spp. – which are the most common macroalgae found across global coastlines. 

Microbial communities do not live in isolation, but rather rely on intercellular interactions to thrive (Johnson et al., 2016). For bacteria, quorum sensing is a method of intercellular communication. Communication is coordinated by the detection and production of signal molecules called autoinducers; autoinducers regulate gene expression in response to bacterial population density. These signal molecules are chemically diverse, but the most widespread are N-acyl homoserine lactones (AHLs), which are found in Gram-negative bacteria. Gram-positive bacteria do not use AHL systems but rely on peptide signals instead, which are highly species-specific.  

Bacterial quorum sensing can impact the development of macroalgae species, which may include: facilitation in the settlement of zoospores, root stimulation, root and shoot growth, root nodule formation, pathogenic resistance, and even plant defence. Additionally, Authors note that prior studies found associations between AHLs on red algae Acrochaetium spp. for carpospore liberation, but knowledge was limited. The study by Singh et al. (2015) aimed to investigate the impact of bacterial quorum sensing on carpospore liberation for green and red macroalgae.  

The study analysed bacterial species abundances and presence of autoinducers from epiphytic and endophytic bacteria on Ulva and Gracilaria spp. Samples were collected from two different locations (Indian coastal areas) and three seasons. Additionally, AHL-containing culture filtrates were measured from cystocarp plantlets of Gracilaria dura, to analyse carpospore liberation. Bacterial isolates were identified by 16S rRNA gene amplification and sequencing. AHL production and separation was measured by a process of inoculation and centrifugation. AHL identification was measured by liquid chromatography electrospray ionization mass/mass spectrometry and electrospray ionization mass spectrometry.  

Gammaproteobacteria occurred across all samples, seasons, and locations. This group is thought to be highly responsible for the formation of biofilms, which can determine the production of quorum sensing-mediated genes. Of the bacteria analysed, seven isolates were found to produce AHLs in G. dura; this includes Shewanella algae, which produced five different types of AHLs alone. The study found that carpospore liberation increases with the relative increase in concentration of C4- and C6-HSL, up to 10 µm. However, C8-, C10-, 3-oxo-C12-HSL, and culture filtrates of Gram-positive bacterium Bacillus flexus showed no influence on carpospore liberation.  

It appears that in this study, the short acyl chain molecules enhanced carpospore liberation when compared to longer acyl chain molecules. Short acyl chain molecules appear to have higher diffusion and solubility rates in water, which allows them to be actively taken up into plant roots and transported through shoots. 

Overall, quorum sensing efficiency does not only benefit bacterial phenotypes, but has an impact on the reproductive output of macroalgal species. The findings of this study infer that the diffusion ability, stability, and availability of AHLs are important factors that help to function carpospore liberation from G. dura. 

Referenced material:  

Johnson, W.M., Soule, M.C.K., and Kujawinski, E.B. (2016) “Evidence for quorum sensing and differential metabolite production by a marine bacterium in response to DMSP.” The ISME journal, 10(9), pp.2304. 

Article reviewed: 

Singh, R.P., Baghel, R.S., Reddy, C.R.K., and Jha, B. (2015) “Effect of quorum sensing signals produced by seaweed-associated bacteria on carpospore liberation from Gracilaria dura.” Frontiers in plant science, 6, pp.117. 

Bacterial isolate from the Hawaiian bobtail squid eggs inhibits Vibrios

The Hawaiian Bobtail Squid (Euprymna scolopes) is characteristic in its association with the bioluminescent bacterium Vibrio fischeri and is a model organism for host-bacterial associations. There are however more associations of bacteria within the squid then vibrio fischeri alone.

Recent studies of this organism have revealed a second association of bacteria in the reproductive organ, the accessory nidamental gland (ANG) of female Squid. This consortium is dominated by the clade Rhodobacteracea (roseobacter) (Collins et al, 2012, 2015), which is known to contain antimicrobial properties in free-living bacteria cultures. Although cultures in associations to test the antimicrobial property of the bacteria had not been undertaken.

The Bacterium are held within epithelium tubules in the accessory nidamental gland which connects to the nidamental gland that secretes a jelly coat surrounding the fertilized eggs of the squid.

E. scolopes has a cool trick to protect its eggs from damage when laid in the environment away from the protection of the parent where predation, pathogens and fouling are all risks. Bacteria are secreted onto the Jelly Coat of the egg from the ANG before being laid in the environment. These bacteria contain antimicrobial properties to prevent fouling of the eggs.

Gromek et al, (2016) characterize the genome and secondary metabolites of a new strain of roseobacter bacteria, isolated from the Jelly coat of E. scolopes eggs (JC1).

In the study, they obtain Hawaiian bobtail squids from the sand flats in Oahu Hawaii. Once in captivity eggs were flash frozen on the 11th day of development from one female individual. 10 eggs are thawed and the embryos are removed. The jelly coats where isolated and sterilized. The bacteria were isolated and cultured, the genome was sequenced and analysed using MasterPure DNA Purification kit.

Using taxonomic analysis of the 16s rRNA sequence they indicated the strain JC1 belonged to the  Leisingera genus. They then go on to confirm the presence of indigoidine  (which has antimicrobial properties) by mass spectroscopy in this strain. In a co-culture with 5 species within the  Vibrionaceae family, the zone of inhibition around JC1  was shown to be significant around V.  fischeri, P. leiognathi, V. parahaemolyticus, V. anguillarum, and V. harveyi. 

It indicates that this new strain may be the active antimicrobial action in the defence against the eggs in Hawaiian bobtail squid.

Inhibition of representatives of the Vibrio genus may lead to interesting results in the inhibition of  Vibrio cholorea, which may be an interesting direction to take the research.

References

Collins, A. J., Fullmer, M. S., Gogarten, J. P., & Nyholm, S. V. (2015). Comparative genomics of Roseobacter clade bacteria isolated from the accessory nidamental gland of Euprymna scolopes. Frontiers in microbiology, 6, 123.

Collins, A. J., LaBarre, B. A., Wong Won, B. S., Shah, M. V., Heng, S., Choudhury, M. H., … Nyholm, S. V. (2012). Diversity and Partitioning of Bacterial Populations within the Accessory Nidamental Gland of the Squid Euprymna scolopes. Applied and Environmental Microbiology, 78(12), 4200–4208. http://doi.org/10.1128/AEM.07437-11

Article Reviewed

------------------------------

Gromek, S. M., Suria, A. M., Fullmer, M. S., Garcia, J. L., Gogarten, J. P., Nyholm, S. V., & Balunas, M. J. (2016). Leisingera sp. JC1, a Bacterial Isolate from Hawaiian Bobtail Squid Eggs, Produces Indigoidine and Differentially Inhibits Vibrios. Frontiers in Microbiology, 7, 1342. http://doi.org/10.3389/fmicb.2016.01342

Coastal Microbial Mats… The Truth Behind the Ocean’s Nitrogen Fixation.

Coastal Microbial Mats… the truth behind the Ocean’s nitrogen fixation.

Microbial mats (MM’s) are important to the foundation of the oceans, they carry out a variety of important processes, an example of this is nutrient cycling. A large part of this is down to the nitrogen fixation which is fuelled by microbial groups of cyanobacteria that are within layers of MM’s. These cyanobacterium groups are known as active diazotrophic communities. Despite how important the cyanobacterium are, there is not a lot of known information on these microbes, this study was undertaken to shed light on this.

 The research took place at Elkhorn Slough estuary, Monterey Bay, CA, USA in October of 2009. The approach was to establish what was fuelling the nitrogen fixation within microbial mats and to gain a better understanding of what caused nitrogen fixation and how.   

It had long been assumed that all cyanobacterium played a heavy role in the full N² fixation from microbial mats. This hypothesis was proven via cultivation base studies (Paerl et al., 1991; Bebout et al., 1993). This however didn’t allow a clear understanding of how these diazotrophic communities worked within complex ecosystems, this has now been explored.

To explore this topic in its entirety a wide range of analysis and methodology was used, enabling a vast amount of results. This included biogeochemical, molecular and high-resolution secondary ion ma In-situ water the nutrient and keep as close to the outside conditions as possible.

Biogeochemical (ARA’s):

Two separate 10mm width by 10mm length mat cores and horizontally layered into 3 triplicate layers. The 3 triplicate mat cores were tested every 3 hours, 3 control mat cores where also taken used as a negative control.  The 3 test cores where separated and incubated within ethylene, this was later measured with gas chromatography to assess the depth distribution of nitrogenase activity.

N-15 incubation – the triplicate mat core samples were placed into 14ml serum jars topped with a stopper to test Gas exchanged for N-15. These were incubated in dark for 10h before samples being split in half for analysis. Half of sectioned cores were analysed by IMRS, the other half of sectioned cores were kept for later Nano-Sims analysis.

N-15 incubation of culture of cyanobacteria was also undertaken using the same techniques mentioned above however these where incubated at a temperature of 22˚C and where tested on a 8/16h day to night light cycle.

Molecular:

DNA and RNA were co-extracted from the upper 2mm of mat cores this was achieved by combining phenol–chloroform extraction with parts of the RNeasyMini and QIAamp DNA Mini Kit used for this analysis. The upper parts of the mat cores showed to have the most N-fixation, which is why this part of the core was sampled.  RNA reverse transcribed with reverse transcriptase enzyme into a single stranded complementary DNA (cDNA), this was analysed via ss spectrometry (NanoSIMS) techniques.  

Findings showed that Cyanobacteria filaments are able to split into heterocystous (cells that are specialized nitrogen-fixing and usually formed during nitrogen starvation periods) and vegetative cells, this means that they are splitting the nitrogen and photosynthesis into separate cells. This was established when heterocystous cells became apparent. Photosystem II was found to be not present but photosystem I was in high numbers as well as the presence of ATP and luciferin.

Because of this, Cyanobacteria are important during the night time due to the highest rates of nitrogen fixation happening during the night in comparison to the day time where photosynthesis has the higher advantage of sunlight. Results also demonstrate that within the presence of oxygen nitrogenase enzyme cyanobacteria are inhibited. 

This study has allowed for new discoveries to be made within the microbial field and has allowed microbiologists and like-minded ocean scientists to gain better understanding of ocean processes. Thorough analysis techniques, such as using a combination of single and standard cell analysis it has enabled researchers to link cell functions and characteristics to recognize previously unidentified microbial groups that work among complex ecosystems and how these microbes link into key ocean processes.