Bacterial communities protect the alga Microchloropsis salina from grazing by the rotifer Brachionus plicatilis
Graphical abstract
Introduction
One major problem facing algal production systems is unpredictable and complete loss of an algal crop. A ‘pond crash’ can be caused by a wide range of factors, including weather, water chemistry, contamination by other algae, infection, and grazing [1,2]. Closed algal photobioreactor systems are less prone to contamination (by viruses, fungi, protozoans, detrimental microbes, etc.) but are difficult to decontaminate when they do become infected and they require a high capital cost [1,3]. Open algal ponds require substantially lower capital costs but are more prone to contamination and are more likely to succumb to crashes due to one or several deleterious species [4]. Annually, pond crashes account for up to 30% loss of algal yield [5], which substantially drives up the cost per unit of biofuel production.
Developing methodologies to address pond crashes is critical for continued efforts in algal cultivation. Recently, high-throughput amplicon sequencing (Illumina) has been used to identify eukaryotic taxa associated with algal pond crashes that may serve as early bio-indicators of an impending crash [2]. Similarly, others have investigated algal pond pests to further characterize detrimental biological invaders [5]. Additionally, the inefficacy of most current practices invites the development of innovative intervention strategies towards algal pond pest control. Standard industrial strategies are largely focused on physical methods (e.g., filtration and sonication) [4] and the addition of various chemicals agents (e.g., quinine, formaldehyde, ammonia, and hydrogen peroxide) [6] to monocultures of algae to prevent or treat pond crashes. These chemical additives are often expensive, require routine re-applications due to photo- or biodegradation (e.g., Rotenone) [7], and have unacceptable off-target effects including harm to algal crops, change of pond chemical composition, and ecotoxicity to wildlife. New methods and updated biotechnologies are necessary for continuous, dependable, and persistent crop protection to reduce algal pond loss.
Recent efforts in agriculture, aquaculture, and human medicine have explored the potential effectiveness of microbiome restoration or manipulation towards combating disease or loss of function [[8], [9], [10]]. There is a wealth of understanding from the aquatic microbial ecology field relating to the beneficial interactions between microalgae and bacteria, including for example, increased growth via remineralization of nutrients [11], exchange of essential vitamins [12], and increased lipid and bioproduct synthesis [13]. Yet in industrial algal cultivation, the algal microbiome – the bacterial community associated with algal cultures – has been largely undervalued and accordingly understudied. However, numerous studies support the idea that certain ‘probiotic’ bacterial strains could provide benefits in algal cultivation, specifically to protect against grazing and stabilize algal systems. The bacterially-produced alkaloid violacein inhibits growth and survival of several freshwater ciliates, flagellates, and rotifers [14]. Similarly, prodigiosin, a secondary metabolite produced by Pseudoalteromonas rubra, acts as a chemical defense system for the microbe. Indeed, P. rubra along with P. piscicida, P. luteoviolacea, and members of the Photobacteriaceae and Vibrionaceae families have been found to have marked toxicity to model eukaryotes [15].
Here, we present data that supports the use of bacterial communities in algal growth production ponds, as a novel approach to mitigate pond crashes. We selected the marine rotifer Brachionus plicatilis, which is capable of consuming 200 microalgal cells per minute and doubling in population within 1–2 days [16], as our model. Our intent with this work was to determine if bacterial communities can reduce grazing of Microchloropsis salina by the marine rotifer, Brachionus plicatilis. Such pest mitigation approaches utilizing protective algal-bacterial co-cultures could potentially be implemented without the additional expense of standard interdiction techniques. We aim to understand the implications of the algal microbiome to support industrial algal production systems.
Section snippets
Axenic algae and rotifer cultures
Axenic stock culture of Microchloropsis salina CCMP 1776 was purchased from the National Center for Marine Algae and Microbiota (NCMA at Bigelow Laboratory, ME, USA); axenicity was defined by NCMA standards and M. salina cultures were not tested. Axenic M. salina and co-cultures of M. salina and bacteria were grown in modified ESAW medium [17] containing 1.65 × 10−3 M nitrate and 6.72 × 10−5 M phosphate and no silica at 20 °C, a light intensity of 100 μmol m−2 s−1 and with a 16:8 h light:dark
Algal-bacterial co-cultures protective against rotifer grazing
The algal-bacterial co-cultures (henceforth referred to as 1–7) were initially screened for protective qualities by assessing algal clearance by rotifers. When high algal densities were evident, samples were examined under low-magnification light microscopy and 1–7 contained several dead or impaired rotifers (see Supplementary video 1, Supplementary video 2). Encouraged by this observation, 1–7 were screened using a 24-well plate microalgal growth assay in order to quantitatively assess
Conclusion
Our data support that inoculating production ponds with a diverse, protective microbiome may be a novel, low-cost method to reduce the frequency of pond crashes. We describe the establishment of algal-bacterial co-cultures and demonstrate their effectiveness in reducing microalgal loss to rotifer grazing. The establishment of protective algal-bacterial communities may be applicable to additional algal-grazer pairs and, therefore, may be a preferable, less expensive method than standard
Author contributions
C.L.F. collected and analyzed the data and wrote the manuscript, C.S.W. analyzed the data and wrote the manuscript, J.A.K. analyzed the data and edited the manuscript, P.D.L. and T.W.L. collected and cultured the original algal-bacterial co-cultures and performed the original experiments, K.L.S. aided in statistical analysis and manuscript revisions, R.K.S., X.M., and T.W.L. contributed to analysis and interpretation of data and edited the manuscript.
Conflict of interest
The authors have no financial conflicts of interest.
Acknowledgements
Thank you to Annette LaBauve and Anupama Sinha for assisting with protocol development and Illumina sequencing. Sandia National Laboratories is a multi-mission laboratory managed and operated by National Technology and Engineering Solutions of Sandia, LLC., a wholly owned subsidiary of Honeywell International, Inc., for the U.S. Department of Energy's National Nuclear Security Administration under contract DE-NA0003525. Algal-bacterial co-culture development, characterization, and
Statement of informed consent, human/animal rights
No conflicts, informed consent, human or animal rights applicable.
References (55)
- et al.
Pond crash forensics: presumptive identification of pond crash agents by next generation sequencing in replicate raceway mass cultures of Nannochloropsis salina
Algal Res.
(2016) - et al.
A financial assessment of two alternative cultivation systems and their contributions to algae biofuel economic viability
Algal Res.
(2014) - et al.
The contamination and control of biological pollutants in mass cultivation of microalgae
Bioresour. Technol.
(2013) - et al.
Taking advantage of rotifer sensitivity to rotenone to prevent pond crashes for alga-biofuel production
Algal Res.
(2015) - et al.
A PCR-based toolbox for the culture-independent quantification of total bacterial abundances in plant environments
J. Microbiol. Methods
(2010) - et al.
Bacterial community changes in an industrial algae production system
Algal Res.
(2018) - et al.
Algae-bacteria interactions: evolution, ecology, and emerging applications
Biotechnol. Adv.
(2016) - et al.
Pseudomonas aeruginosa disrupts Caenorhabditis elegans iron homeostasis, causing a hypoxic response and death
Cell Host Microbe
(2013) - et al.
Vibrios associated with routine productions of Brachionus plicatilis
Aquaculture
(1997) - et al.
Effect of bacteria on growth and biochemical composition of two benthic diatoms Halamphora coffeaeformis and Entomoneis paludosa
J Exp Marine Biol Ecol
(2017)
Rotifers enriched with mixed algal diet promote survival, growth, and development of barramundi larvae, Lates calcarifer (Bloch)
Aquaculture Reports
Effect of nutritional status and concentration of Nannochloropsis gaditana as enrichment diet for the marine rotifer Brachionus sp
Aquaculture
Nutrient profiles of rotifers (Brachionus sp.) and rotifer diets from four different marine fish hatcheries
Aquaculture
Biofuels from algae: challenges and potential
Biofuels
Parasites in algae mass culture
Front Microbio
Contamination in the low cost open algae ponds for biofuels production
Indust Biotech
Building the crops of tomorrow: advantages of symbiont-based approaches to improving abiotic stress tolerance
Front. Microbiol.
Exploring fish microbial communities to mitigate emerging diseases in aquaculture
FEMS Microbio Ecol
The human microbiome: at the interface of health and disease
Nat. Rev. Genet.
Interactions between bacteria and algae in aquatic ecosystems
Ann Rev Ecol Sys
Impact of microalgae-bacteria interactions on the production of algal biomass and associated compounds
Mar Drugs
Toxicity of violacein-producing bacteria fed to bacterivorous freshwater plankton
Limnol. Oceanogr.
Toxicity of bioactive and probiotic marine bacteria and their secondary metabolites in Artemia sp. and Caenorhabditis elegans as eukaryotic model organisms
App Env Microb
Fundamental studies on physiology of rotifer for its mass culture – I: filter feeding of rotifer
Bul Japan Soc Sci Fish
Evolution of an artificial seawater medium: improvements in enriched seawater, artificial water over the last two decades
J. Phycol.
Rotifers
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