Vol. 83: 251–262, 2019 AQUATIC MICROBIAL ECOLOGY Published online October 30 https://doi.org/10.3354/ame01921 Aquat Microb Ecol OPENPEN ACCESSCCESS B12 production by marine microbial communities and Dinoroseobacter shibae continuous cultures under different growth and respiration rates Josué Villegas-Mendoza1,*, Ramón Cajal-Medrano2, Helmut Maske2 1UABC, Facultad de Ciencias Marinas, Carretera Tijuana-Ensenada km 106, Ensenada, Baja California CP 22860, Mexico 2CICESE, Carretera Tijuana-Ensenada No. 3918, Ensenada, Baja California CP 22860, Mexico ABSTRACT: In situ dissolved B12 concentration in marine ecosystems is controlled by the balance between rates of release of B12 by prokaryotes, uptake by prokaryotes and eukaryotes, and abiotic degradation. We used chemostats at a range of specific growth rates (μ, d−1; 0.1 to 1) with natural communities of prokaryotes and monospecific cultures of a B12 producer, Dinoroseobacter shibae. We measured the dissolved B12 concentration produced in the culture (B12-d), the B12 in the partic- ulate fraction (B12-p), cell concentration, respiration rate, particulate organic carbon and nitrogen (POC, PON), and the 16S amplicon composition. Total dissolved B12 concentrations (0.92 to −1 4.90 pmol l ) were comparable to those found in the surface ocean. B12-p concentration was 6 to 35 times higher than B12-d. B12-d, B12-p, and community composition showed no relation to μ for either natural populations or D. shibae. The chemostats allowed calculation of the rates of production: −1 −1 −1 −1 B12-d (0.34 ± 0.28 pmol l d ) and B12-p (5.65 ± 2.34 pmol l d ), and the B12 cell quota (900 to −1 3300 molecules cell ). In multispecies and D. shibae cultures, B12 production rates per cell in - creased with respiration rates (volumetric or per cell), and with rates of cellular organic carbon and nitrogen production. Rates increased with μ, but not the concentrations of B12-d or of B12-p. To understand the physiological and ecological dynamics of B12, concentrations alone are insufficient since they do not provide rates, which are important in understanding the dynamics between producers and consumers. KEY WORDS: Marine bacteria · Intracellular and dissolved B12 · Continuous cultures 1. INTRODUCTION prokaryotic and eukaryotic organisms (Sañudo- Wilhelmy et al. 2014). Approximately half of eukary- The presence and importance of organic com- otic phytoplankton are dependent on the B12 pro- pounds as regulators of plankton growth in the ocean duced by bacteria and archaea (Croft et al. 2005, has been known for almost 100 yr; one group of com- Sañudo-Wilhelmy et al. 2012, Helliwell 2017, Gómez- pounds that has been studied in detail is the group of Consarnau et al. 2018). Helliwell et al. (2016) showed B vitamins, specifically biotin (B7), thiamin (B1), and the rapid experimental adaptation of a B12-indepen- cobalamin (hereafter B12). Measurements of B12 in dent algae, Chlamydomonas reinhardtii, to a B12- the ocean have indicated sub-picomolar concentra- dependent clone. It has been suggested that euka - tions offshore and picomolar concentrations in coas - ryotic microorganisms that are now auxotrophic have tal regions (Menzel & Spaeth 1962, Okbamichael & lost the capacity to synthesize the B12-independent Sañudo-Wilhelmy 2004, Sañudo-Wilhelmy et al. methionine synthase (MetE) (Helliwell et al. 2011), 2006, Panzeca et al. 2009). Despite its low concentra- and it is possible that a similar mechanism exists for tion in seawater, B12 plays a vital role in auxotrophic prokaryotes. In this case, they would depend on the © The authors 2019. Open Access under Creative Commons by *Corresponding author: [email protected] Attribution Licence. Use, distribution and reproduction are un - restricted. Authors and original publication must be credited. Publisher: Inter-Research · www.int-res.com 252 Aquat Microb Ecol 83: 251–262, 2019 enzyme methionine synthase (MetH), which requires The dissolved B12 concentration in seawater re - B12 as a cofactor. Laboratory studies have indicated sults from the balance of production to consump- that the concentration of dissolved B12 required tion, in cluding B12 degradation by photochemistry to maintain axenic algal growth ranges from sub- and other processes. There is little information picomolar and picomolar (Tang et al. 2010, Bertrand available on the cellular B12 concentration (Sañudo- et al. 2012) to 15 and 37 pmol l−1 (Kazamia et al. Wilhelmy et al. 2014). Recently, Suffridge et al. 2012). These concentrations are comparable to those (2017) measured particulate and dissolved B-vita- found in the ocean (Sañudo-Wilhelmy et al. 2014). mins in cultured marine bacteria and environmen- The literature indicates that the production of B12 is tal samples and observed that the concentration of unique to prokaryotes (Warren et al. 2002, Grossman dissolved and particulate vitamins varied between 2016). In silico analysis of microbial genomes sug- microbial communities with out a definite pattern gests that the potential to synthesize B12 is found in between these 2 pools. In the present study, our different groups of bacteria and archaea (Rodionov et goal was to obtain information that would help to al. 2003, Doxey et al. 2015, Heal et al. 2017). Genetic interpret the dissolved and particulate B12 concen- and experimental evidence has shown that some trations found in natural waters. We used natural bacterial taxa lack the de novo B12 biosynthetic path- bacterial communities that should have included way; instead, they possess salvage pathways or as- consumers and producers as well as monospecific similate extracellular B12 precursors (Burkholder & cultures of a B12 producer, Dinoroseobacter shibae, Lewis 1968, Woodson & Escalante-Semerena 2004, to explore the coupling between dissolved and Helliwell et al. 2016). Taylor & Sullivan (2008) found particulate B12 production rates, applying continu- heterotrophic bacteria communities in the ocean that ous cultures under substrate-limited growth at dif- assimilate B12 at rates proportional to their productiv- ferent slow growing rates. D. shibae was chosen ity and biomass. To maintain proportionality, the for the mono-species culture for different reasons: ratio of B12 producers and consumers in bacterial its capacity for B12 production (Wagner-Döbler et communities should be preserved. However, it is not al. 2010), and because it has been shown to be a clear how the B12 demand of the bacterial community common member of the organotrophic marine is supplied in the ocean, considering the low growth community and has the ability to supply marine −1 rates of bacterioplankton (0.05 to 0.3 d in oligo - phytoplankton with B12 in culture (Cruz-López et trophic systems and coastal waters, respectively; al. 2018). Ducklow 2000). Currently, it is difficult to estimate the potential for B12 biosynthesis in microbial communities based on 2. MATERIALS AND METHODS their taxonomic composition (Helliwell et al. 2016, Heal et al. 2017). Bertrand et al. (2011) showed that of 2.1. Culture preparation 45 sequenced strains of Vibrio genomes, only V. splendidus LGO32, and Vibrio MED222 have the All cultures were run as chemostats (Herbert et al. potential to synthesize B12. Moreover, the potential 1956), either monospecific (Dinoroseobacter shibae) for the microbial community to produce B12 is not or inoculated with natural coastal bacterial communi- only dependent on its taxonomic composition but ties. The cultures were prepared following Cajal- also on environmental conditions such as tempera- Medrano & Maske (2005). Batches of 20 l of growth ture, availability of dissolved organic matter (DOM), media were prepared from GF/F-filtered seawater cobalt, nitrogen, and iron (Bertrand et al. 2011). aged in the dark in glass containers for at least 3 mo. The B12 biosynthesis pathway is energetically ex - The seawater was collected in the California Cur- pensive, involving 30 genes and up to 3% of cellular rent. During aging, the seawater was filtered again metabolism (Roth et al. 1996, Raux et al. 2000), there- (GF/F). At the end, the seawater was bubbled with fore only a minority of healthy bacteria are expected an ozone stream for 24 h, then activated charcoal was to exude or release B12 to the sea (Provasoli 1963, added for 24 h, which was then removed by GF/F Berland et al. 1976). One probable pathway for filtration. We could not be sure that all degraded B12 microbial producers to provide B12 to auxotrophic species were removed by the activated charcoal, prokaryotes and eukaryotes is release by lysis (cell therefore we measured the dissolved B12 concentra- death, exudation, senescence, viral processes) (Wil- tion in the growth media (see below). Inorganic nutri- helm & Suttle 1999, Karl 2002, Croft et al. 2005, 2006, ents and glucose were added as an organic carbon Droop 2007). source (NH4Cl: 30 µM; KH3PO4: 5 µM; FeCl3: 0.4 µM; Villegas-Mendoza et al.: B12 production by marine bacterial communities 253 glucose: 20 µM). Subsequently, the growth media 2.2. Enumerating bacteria and VLPs was acidified by bubbling with CO2, autoclaved for by microscopic examination 1 h at 1.5 psi (~10 kPa), cooled down to room temper- ature, and bubbled with sterile air to replenish O2 Bacterial cell abundance and VLPs were deter- back to saturation. Final growth media pH ranged mined by counting randomly selected fields, using an from 8.1 to 8.3. All containers and tubing used for the epifluorescence microscope. A 20 ml sample of cul- continuous culture chemostat systems were Teflon or ture was fixed with buffered formaldehyde (2% final silicon, as described by Cajal-Medrano & Maske concentration). Then, 0.2 to 0.5 ml of the sample was (2005). Before terminating and measuring each incubated with 4’,6-diamino-2-phenylindole (DAPI; chemostat, we as sured the sterility of the growth final concentration: 1 µg ml−1) (Porter & Feig 1980) medium by inoculating it in sterile Zobell growth and immediately filtered on 0.2 µm black poly - medium (see Fig. 1 in Cajal-Medrano & Maske 2005).