Bacterial Community Assembly, Succession, and Metabolic Function during Outdoor Cultivation of Microchloropsis salina

Abstract

Outdoor cultivation of microalgae has promising potential for renewable bioenergy, but there is a knowledge gap on the structure and function of the algal microbiome that coinhabits these ecosystems. Here, we describe the assembly mechanisms, taxonomic structure, and metabolic potential of bacteria associated with Microchloropsis salina cultivated outdoors. Open mesocosms were inoculated with algal cultures that were either free of bacteria or coincubated with one of two different strains of alga-associated bacteria and were sampled across five time points taken over multiple harvesting rounds of a 40-day experiment. Using quantitative analyses of metagenome-assembled genomes (MAGs), we tracked bacterial community compositional abundance and taxon-specific functional capacity involved in algal-bacterial interactions. One of the inoculated bacteria (Alteromonas sp.) persisted and dispersed across mesocosms, whereas the other inoculated strain (Phaeobacter gallaeciensis) disappeared by day 17 while a taxonomically similar but functionally distinct Phaeobacter strain became established. The inoculated strains were less abundant than 6 numerically dominant newly recruited taxa with functional capacities for mutualistic or saprophytic lifestyles, suggesting a generalist approach to persistence. This includes a highly abundant unclassified Rhodobacteraceae species that fluctuated between 25% and 77% of the total community. Overall, we did not find evidence for priority effects exerted by the distinct inoculum conditions; all mesocosms converged with similar microbial community compositions by the end of the experiment. Instead, we infer that the 15 total populations were retained due to host selection, as they showed high metabolic potential for algal-bacterial interactions such as recycling alga-produced carbon and nitrogen and production of vitamins and secondary metabolites associated with algal growth and senescence, including B vitamins, tropodithietic acid, and roseobacticides. IMPORTANCE Bacteria proliferate in nutrient-rich aquatic environments, including engineered algal biofuel systems, where they remineralize photosynthates, exchange secondary metabolites with algae, and can influence system output of biomass or oil. Despite this, knowledge on the microbial ecology of algal cultivation systems is lacking, and the subject is worthy of investigation. Here, we used metagenomics to characterize the metabolic capacities of the predominant bacteria associated with the biofuel-relevant microalga Microchloropsis salina and to predict testable metabolic interactions between algae and manipulated communities of bacteria. We identified a previously undescribed and uncultivated organism that dominated the community. Collectively, the microbial community may interact with the alga in cultivation via exchange of secondary metabolites which could affect algal success, which we demonstrate as a possible outcome from controlled experiments with metabolically analogous isolates. These findings address the scalability of lab-based algal-bacterial interactions through to cultivation systems and more broadly provide a framework for empirical testing of genome-based metabolic predictions.

Keywords: Microchloropsis; Rhodobacteraceae; algal-bacterial interactions; community assembly; metagenome-assembled genomes.

FIG 1

Experimental design of algal-bacterial coculture mesocosms and resulting algal biomass over time. (A) Duplicate 16-L algal mesocosms of M. salina inoculated with either Phaeobacter gallaeciensis BS107 or Alteromonas sp. I10 or left axenic at day 0. (B) Mesocosms were left open to bacterial dispersal and maintained for 40 days, with 5 rounds of harvest in which 90% of algal biomass was removed and 10% of biomass was retained into the subsequent round. Dashed lines indicate when DNA for metagenomes was collected. (C) M. salina biomass measured over time by chlorophyll a fluorescence (log₁₀ scale). Colors indicate initial inoculation treatment: Phaeobacter (red), Alteromonas (blue), and axenic culture (green). Points represent the mean result of each treatment at each time point sampled, with error bars indicating standard deviation. Dashed lines correspond with metagenomic sampling time points (cultivation round 3: days 17 and 19; round 5: days 34, 36, and 38).

FIG 2

Estimated relative abundance of M. salina-associated bacteria over time. Abundance of each of the 15 bacterial taxa inoculated or recruited within the M. salina algal cultivation system was estimated using the mean of the median fold coverage of metagenomic contigs from each sample (n = 30 total) against the comprehensive metagenome-assembled genome (MAG) and was normalized within each sample by transformation to a relative proportion, referred to as the “relative coverage.” (A to F) Facets represent MAG coverage averaged across duplicate mesocosms ± standard deviation for each inoculation treatment, i.e., Phaeobacter inoculated (A and B), Alteromonas inoculated (C and D), and uninoculated axenic control (E and F) with 5 time points sampled for each treatment (round 3: days 17 and 19; round 5: days 34, 36, and 38). Higher-abundance taxa are shown in the upper row (A, C, E, and G), and lower-abundance taxa are represented in the bottom row (B, D, F, and H). (G and H) Box plots show distribution of MAG relative coverage system-wide for all samples, sorted in decreasing order of abundance. Colors represent MAG taxonomic groups as follows: class Alphaproteobacteria in warm colors, including Rhodobacteraceae (purples) (MSM6, MSM9, MSM11, MSM12, MSM13, MSM14, MSM15), Sphingomonadaceae (red) (MSM4), and unclassified Rickettsiales (orange) (MSM10); class Bacteroidia in greens, including Cyclobacteriaceae (MSM7, MSM8) and Flavobacteraceae (MSM5); and class Gammaproteobacteria in blues, including Alteromonadaceae (MSM1), Methylophagaceae (MSM3), and Oleiphilaceae (MSM2). An asterisk indicates the MAG recovered from one of two inoculated strains.

FIG 3

Biogeochemical capabilities of M. salina-associated bacteria. The bacterial biogeochemical capacity was determined by pathway marker gene presence (>75%). (A) Bacterial capabilities for specific metabolic pathways within the carbon cycle, including carbon fixation, respiration, methanotrophy, and carbon monoxide oxidation. (B) Bacterial metabolic capacities for steps of the nitrogen cycle, including assimilation, mineralization, and denitrification. MAGs are grouped by their respective taxonomic classifications by class and by family within each class grouping (α = Alphaproteobacteria: R = Rhodobacteraceae [purples], S = Sphingomonadaceae [red], U = unclassified Rickettsiales [orange]; B = Bacteroidia: C = Cyclobacteriaceae [yellow], F = Flavobacteraceae [greens]; γ = Gammaproteobacteria [blues]: A = Alteromonadaceae, M = Methylophagaceae, O = Oleiphilaceae). Relative coverage averaged system-wide (n = 30 samples) is indicated for each MAG. Arrows show flow of C and N via bacterial metabolism, weighted for thickness based on the number of MAGs capable of that specific pathway. Dashed gray arrows indicate input or conversion from nonbacterial processes.

FIG 4

Biphasic outcomes of algal-bacterial interactions depending on community consortia. Here, we show a hypothetical community-based conceptual model building upon a previously described algal-bacterial biphasic interaction, supported by our genome-based predictions and experimental evidence of algal outcome under various bacterial conditions. Microalgae produce p-coumaric acid (pCA), which induces a metabolic response in bacteria, with some bacteria capable of tolerance via pCA degradation or conversion of pCA into algal growth-promoting (tropodithietic acid [TDA]) or growth-inhibiting (roseobacticide) compounds. (A) In the presence of exogenous pCA, the bacterium Phaeobacter gallaeciensis, capable of roseobacticide production, inhibited algal growth in comparison to axenic cultures free of bacterial cells. (B) A synthetic bacterial community comprised of 8 isolates, capable of pCA degradation among other metabolic characteristics, also inhibited algal growth in the presence of pCA. (C) In combination, P. gallaeciensis and the pCA degrader community promoted algal growth in comparison to axenic algal cultures.