Elsevier

Algal Research

Volume 40, June 2019, 101500
Algal Research

Bacterial communities protect the alga Microchloropsis salina from grazing by the rotifer Brachionus plicatilis

https://doi.org/10.1016/j.algal.2019.101500Get rights and content

Highlights

  • Unpredictable pond crashes limit the economic feasibility of microalgal biofuel.

  • Seven bacterial communities are shown to protect microalgae from rotifer grazing.

  • We conducted community profiling to compare protective and nonprotective cultures.

  • Bacterial taxa putatively responsible for protective function were identified.

  • Algal cultivation may benefit from protective microbes that mitigate pond crashes.

Abstract

Open algal ponds are likely to succumb to unpredictable, devastating crashes by one or several deleterious species. Developing methodology to mitigate or prevent pond crashes will increase algal biomass production, drive down costs for algae farmers, and reduce the risk involved with algae cultivation, making it more favorable for investment by entrepreneurs and biotechnology companies. Here, we show that specific algal-bacterial co-cultures grown with the green alga Microchloropsis salina prevented grazing by the marine rotifer, Brachionus plicatilis. We obtained seven algal-bacterial co-cultures from crashed rotifer cultures, maintained them in co-culture with Microchloropsis salina, and used a microalgal survival assay to determine that algae present in each co-culture were protected from rotifer grazing and culture crash. After months of routinely diluting and maintaining these seven algal-bacterial co-cultures, we repeated the assay and found the opposite result: none of the seven bacterial communities protected the microalgae from rotifer grazing. We performed 16S rRNA gene amplicon sequencing on the protective and nonprotective co-culture samples and identified substantial differences in the makeup of the bacterial communities. Protective bacterial communities consisted primarily of Alphaproteobacteria (Rhodobacteraceae) and Gammaproteobacteria (Marinobacter, Pseudomonas, Methylophaga) while nonprotective bacterial communities were less diverse and missing many putatively crucial members. We compared the seven protective communities with the seven nonprotective communities and we correlated specific bacterial amplicon sequence variants with algal protection. With these data, our future work will aim to define and develop an engineered-microbiome that can stabilize industrial Microchloropsis salina cultures by protecting against grazer-induced pond crashes.

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.

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