Bacterial Community Composition and Chromophoric Dissolved Organic Matter Differs with Culture Time of Skeletonema Dohrnii
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diversity Article Bacterial Community Composition and Chromophoric Dissolved Organic Matter Differs with Culture Time of Skeletonema dohrnii Yang Liu 1,2, Jinjun Kan 3, Jing Yang 4, Md Abu Noman 2 and Jun Sun 2,* 1 Institute of Marine Science and Technology, Shandong University, Qingdao 266237, China; [email protected] 2 College of Marine Science and Technology, China University of Geosciences (Wuhan), Wuhan 430074, China; [email protected] 3 Stroud Water Research Center, 970 Spencer Road, Avondale, PA 19311, USA; [email protected] 4 School of Life Science, Shanxi University, Taiyuan 030006, China; [email protected] * Correspondence: [email protected]; Tel.: +86-22-60601116 Abstract: Skeletonema dohrnii is a common red tide microalgae occurring in the coastal waters and throughout the world. The associated heterotrophic or autotrophic bacteria play vital roles in regulating algal growth, production, and physiology. In this study, we investigated the detailed bacterial community structure associated with the growth of S. dohrnii’s using high-throughput sequencing-based on 16S rDNA. Our results demonstrated that Bacteroidetes (48.04%) and Pro- teobacteria (40.66%) in all samples accounted for the majority of bacterial populations. There was a significant linear regression relationship between the abundance of bacterial phyla and culture time. Notable shifts in bacterial community composition were observed during algal growth: Flavobac- teriales accounted for the vast majority of sequences at the order level. Furthermore, the relative Citation: Liu, Y.; Kan, J.; Yang, J.; S. dohrnii Noman, M.A.; Sun, J. Bacterial abundance of Rhodobacterales was gradually reduced during the whole growth process of Community Composition and (0–12 days). However, beyond that, the relative abundance of Marinobacter was slowly increasing. It Chromophoric Dissolved Organic is noteworthy that five fluorophores (Peaks T1,T2, I, M, and A) were detected during the growth Matter Differs with Culture Time of stage of S. dohrnii. The characteristic indexes (fluorescence index, humification index, and biological Skeletonema dohrnii. Diversity 2021, 13, index) of chromophoric dissolved organic matter (CDOM) also varied with the culture time. In 150. https://doi.org/10.3390/ addition, the taxa of bacteria had certain effects on CDOM and they were inextricably linked to d13040150 each other. Academic Editor: Michael Wink Keywords: microalgae; bacteria; high-throughput sequencing; fluorophores Received: 27 February 2021 Accepted: 26 March 2021 Published: 30 March 2021 1. Introduction Publisher’s Note: MDPI stays neutral Diatoms are ubiquitous autotrophic eukaryotes in aquatic ecosystems, and they con- with regard to jurisdictional claims in tribute about 20% of the total photosynthesis of the earth [1]. Skeletonema is a genus published maps and institutional affil- of diatoms belonging to the family Skeletonemaceae (Bacillariophyta, Centricae, Disci- iations. nales, Discoideae). Skeletonema spp. are commonly found in marine and coastal waters throughout the world and known as a causative species of red tide [2–5]. Among diatoms, Skeletonema dohrnii is a cosmopolitan species widely distributed in the temperate regions of the northern and southern hemispheres [4]. In the coastal waters of China, S. dohrnii is a common red tide algae that forms long chains with a shell surface diameter up to Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. 4–12 microns [5]. It is a cold-water species that mainly occurs in winter and spring. In This article is an open access article summer and fall, S. dohrnii disappears and accumulates in bottom water and shallow distributed under the terms and sediments [3,6,7]. Their occurrence and distribution may be influenced by environmental conditions of the Creative Commons gradients such as temperature and interactions with other living organisms [2,3,5,8]. Attribution (CC BY) license (https:// Microalgae and bacteria have coexisted in aquatic habitats for more than 200 million creativecommons.org/licenses/by/ years [9], which means that the interactions between diatoms and bacteria have undergone 4.0/). a long evolutionary time scale. Bacterial communities constitute sophisticated interactions Diversity 2021, 13, 150. https://doi.org/10.3390/d13040150 https://www.mdpi.com/journal/diversity Diversity 2021, 13, 150 2 of 13 with microalgae [10], including mutualism, commensalism, antagonism, competition, and parasitism. Microalgae can provide organic matter to promote bacterial growth, or an- timicrobial substances to inhibit bacterial reproduction [11–13]. Bacteria can also produce vitamins, nutrients, and other substances that promote microalgal growth [14,15], and produce substances that inhibit the growth of microalgae [16,17]. Around algal cells, there usually exists a special micro-environment called ‘phycosphere’, which is inhabited by the specific microbial communities. The phycosphere is the main place for the interactions between algae and bacteria [18]. Some phycosphere bacteria are common to certain mi- croalgae, while unique to other microalgae, and have the interspecies specificity between microalgae and bacteria. For example, bacteria in the phycosphere of Skeletonema costatum mainly include Alphaproteobacteria, Gammaproteobacteria, and Bacteroidetes [19,20]. Studies have shown that bacterial community composition in phycosphere differs from the attached bacteria in the water. Furthermore, bacterial density and community structure will also change with different periods of microalgal growth stage and changes in the phy- toplankton community [20]. Basically, regardless of the interactions between microalgae and bacteria, changes in bacterial community are related to the physiological state of algae in different periods [20,21] and the relationship between microalgae and bacteria plays an important role in the biogeochemical cycles and algal bloom processes [22]. Therefore, bacterial communities are important for an in-depth understanding of biogeochemical cycles [22]. Currently, most of the research has mainly focused on the population distribution, morphology, and biodiversity of Skeletonema [23–27]. Meanwhile, the phycosphere bacteria of some Skeletonema have been studied as well [28,29]. However, the bacterial community composition and chromophoric dissolved organic matter of S. dohrnii has not been reported. Therefore, we established a batch culture system to explore the relationship between eukaryotic diatoms and bacteria using the 16S rDNA amplicon sequencing-based on next- generation sequencing platforms. Primarily, we chose a symbiotic system to obtain a linear relationship between the microalgae and bacteria in this study. 2. Materials and Methods 2.1. Microalgae Collection, Pre-Culture Conditions and Experimental Set-Up Skeletonema dohrnii was isolated from the central Yellow Sea in China, where it is the predominant species occurring in spring blooms and generally found all year round [5]. During the experiments, S. dohrnii was cultured in the artificial seawater (ASW) medium [30] in transparent conical flasks at 18 ◦C. The light intensity and photoperiod were 55.50 µmol photons m−2 s−1 and 14 h:10 h (light: dark) cycles under cool white fluorescent light, respectively. Microalgal cultures were shaken manually at 12 am every day to prevent sedimentation. S. dohrnii was harvested during the logarithmic growth phase. The microalgae cells were collected by centrifugation at 2000× g for 5 min. The harvested microalgal cells were washed with sterilized deionized water and centrifuged at 2000× g for 5 min again. Then S. dohrnii’s cells were inoculated to 2000 mL transparent conical flasks containing 1800 mL of artificial seawater, and the initial microalgal biomass concentration was about 4.25 × 108 cells L−1. In this study, S. dohrnii in artificial seawater was represented by Z1-Z30. The samples were taken daily until the 6th day. Also, the other three samples were collected on days 8, 10, and 12 (G8, G9, and G10). All the materials and media were sterilized before experiments. All experiments were performed in triplicates. 2.2. Determination of Microalgal Growth and Collection of Bacteria When shaking the culture solution every day, 100 µL of microalgae solution was collected into the blood cell counting chamber and microalgal cells were immediately counted using an inverted microscope (Olympus BX51, Olympus, Tokyo, Japan). Bacterial counts were determined via plate count [31]. Diversity 2021, 13, 150 3 of 13 At the beginning of the experiment, 150 mL of microalgal solution was taken daily from the samples. The microalgal culture solutions were filtered through a 3.0 µm sterile mixed cellulose ester membrane (47 mm, Millipore, MA, USA) to collect the attached bacteria. The filtered samples (the membranes with S. dohrnii and attached bacteria) were then stored in the cryogenic vial (2 mL, Corning Incorporated, New York, USA) and kept at −80 ◦C in the freezer until DNA extraction. 2.3. DNA Extraction, PCR Amplification, and Sequencing Total genomic DNA was extracted using the DNeasy PowerWater (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. DNA quantity and quality were analyzed by a Nanodrop spectrophotometer ND-2000. The extracted genomic DNA was stored at −80 ◦C for further analyses. Bacterial community structure was char- acterized by amplifying V3 and V4 regions of 16S rRNA genes using the prokaryotic- specific forward primer 343F 50-TACGGRAGGCAGCAG-30 and