Benthic foraminiferal community structure and its response to environmental factors revealed using high-throughput sequencing in the Zhoushan Fishing Ground, East China Sea.

Benthic foraminifera are excellent tools for monitoring marine environments and reconstructing paleoenvironments. This study investigated the structure and diversity of benthic foraminiferal communities in 20 superficial sediment samples obtained from the Zhoushan Fishing Ground (ZFG) using high-throughput sequencing based on small subunit ribosomal DNA and RNA amplification. The results revealed Rotaliida as the most dominant group, with spatial heterogeneity in foraminiferal distribution. Total benthic foraminiferal communities exhibited higher species richness and diversity compared to active communities. While heavy metal pollution in the ZFG was moderate, areas with elevated concentrations of heavy metals exhibited low diversity and richness in foraminiferal communities. Total foraminiferal community structure was primarily influenced by factors such as water depth and Hg, Pb, Cd, and Zn levels. Notably, Hg levels emerged as a critical factor impacting the structure and diversity of the active foraminiferal community. The dominant species, Operculina, exhibited tolerance toward heavy metal pollution.

Accurate Identification of Diverse N-acyl Homoserine Lactones in Marine Vibrio fluvialis by UHPLC-MS/MS.

Vibrio fluvialis is a marine opportunistic pathogen that frequently causes diseases in aquatic animals and humans. V. fluvialis can produce quorum sensing signaling molecules to coordinate cell density-dependent behavioral changes, including N-acyl homoserine lactone (AHL), which acts as a vital mediator of virulence-associated gene expression. Currently, several AHL molecules in V. fluvialis have been detected via biological and physicochemical methods, although different detection approaches have generated diverse AHL profiles. Here, we describe the AHL-producing bacterium, V. fluvialis BJ-1, which was isolated from marine sediments from the East China Sea. V. fluvialis BJ-1 could stimulate AHL-mediated ß-galactosidase synthesis of the biosensor Agrobacterium tumefaciens NTL4 (pZLR4) but could not induce violacein production in the AHL reporter strain, Chromobacterium violaceum CV026. This bacterial isolate exhibited strong AHL-producing activity at low cell density; however, the AHL activity declined when population density remained at high levels. Analysis of the AHLs by Ultra-High-Performance Liquid Chromatography tandem Mass Spectrometry demonstrated that V. fluvialis BJ-1 produced five different AHL signaling molecules, including two linear chain AHL products (C8- and C10-HSL), and three ß-carbon-oxidative AHL products (3-O-C8-, 3-O-C10- and 3-O-C12-HSL). Significantly, the present study is the first to accurately define the AHL profile of marine V. fluvialis. In future, the coupling of UHPLC to ESI-MS/MS is expected to be utilized for the accurate determination of AHL profiles in marine Vibrio.

Determination of Phthalates in Marine Sediments Using Ultrasonic Extraction Followed by Dispersive Solid-Phase Extraction and Gas Chromatography-Mass Spectrometry.

A simple, rapid and novel method has been developed and validated for determination of 16 phthalates in marine sediment samples by gas chromatography coupled to mass spectrometry. Freeze dried samples were first ultrasonic extraction by n-hexane:methylene chloride (1:1, v/v) and n-hexane:ethyl acetate (1:1, v/v) and followed by dispersive solid-phase extraction cleanup. The linearity of this method ranged from 1 to 1,000 µg/L, with regression coefficients ranging between 0.9993 and 0.9999. The limits of detection were in ng/g level, ranging between 0.1 and 0.25 ng/g (dry weight). The concentration of the total phthalates in marine sediment samples from Waters of Dongji (Zhoushan, China), Yueqing Bay (Wenzhou, China) and Coastal Waters of Yuhuan (Taizhou, China) ranged from 235.4 to 608.7 µg/kg with diisobutyl phthalate, dibutyl phthalate and di(2-ethylhexyl) phthalate being the major species, which constitutes of 94.6 and 98.1% of the total phthalates. The recoveries of spiked 16 phthalates at different concentration levels in sediment sample 3 of Waters of Dongji (Zhoushan, China) and sediment sample 3 of Yueqing bay (Wenzhou, China) were in the range of 78-117% and 83-114%, respectively, with relative standard deviations of 2.4-6.8% and 3.4-7.5% (n = 5), respectively. The performance of the proposed method was also compared with traditional Soxhlet extraction and column chromatography cleanup on the same genuine sediment samples and comparable efficiencies were obtained. It is concluded that this method can be successfully applied for the determination of phthalates in different marine sediment samples.

Structural insights into acyl-ACP selective recognition by the Aeromonas hydrophila AHL synthase AhyI.

BACKGROUND: Aeromonas hydrophila is a gram-negative bacterium and the major causative agent of the fish disease motile aeromonad septicemia (MAS). It uses N-acyl-homoserine lactone (AHL) quorum sensing signals to coordinate biofilm formation, motility, and virulence gene expression. The AHL signaling pathway is therefore considered to be a therapeutic target against pathogenic A. hydrophila infection. In A. hydrophila, AHL autoinducers biosynthesis are specifically catalyzed by an ACP-dependent AHL synthase AhyI using the precursors SAM and acyl-ACP. Our previously reported AhyI was heterologously expressed in E. coli, which showed the production characteristics of medium-long chain AHLs. This contradicted the prevailing understanding that AhyI was only a short-chain C4/C6-HSL synthase. RESULTS: In this study, six linear acyl-ACP proteins with C-terminal his-tags were synthesized in Vibrio harveyi AasS using fatty acids and E. coli produced active holo-ACP proteins, and in vitro biosynthetic assays of six AHL molecules and kinetic studies of recombinant AhyI with a panel of four linear acyl-ACPs were performed. UPLC-MS/MS analyses indicated that AhyI can synthesize short-, medium- and long-chain AHLs from SAM and corresponding linear acyl-ACP substrates. Kinetic parameters measured using a DCPIP colorimetric assay, showed that there was a notable decrease in catalytic efficiency with acyl-chain lengths above C6, and hyperbolic or sigmoidal responses in rate curves were observed for varying acyl-donor substrates. Primary sequence alignment of the six representative AHL synthases offers insights into the structural basis for their specific acyl substrate preference. To further understand the acyl chain length preference of AhyI for linear acyl-ACP, we performed a structural comparison of three ACP-dependent LuxI homologs (TofI, BmaI1 and AhyI) and identified three key hydrophobic residues (I67, F125 and L157) which confer AhyI to selectively recognize native C4/C6-ACP substrates. These predictions were further supported by a computational Ala mutation assay. CONCLUSIONS: In this study, we have redefined AhyI as a multiple short- to long-chain AHL synthase which uses C4/C6-ACP as native acyl substrates and longer acyl-ACPs (C8 ~ C14) as non-native ones. We also theorized that the key residues in AhyI would likely drive acyl-ACP selective recognition.

Effect of organic carbon to nitrogen ratio in wastewater on growth, nutrient uptake and lipid accumulation of a mixotrophic microalgae Chlorella sp.

This study investigated the biomass/lipid production, nutrient removal and fatty acid composition of an isolated mixotrophic microalga (Chlorella sp. G-9) cultured in simulated wastewater with different TOC/TN ratio. As the TOC/TN ratio of wastewater increased from 0 to 24, the growth rate of Chlorella sp. G-9 increased gradually, but did not increase further at 30. Nutrient removal was related to microalgae growth. In the wastewater with TOC/TN ratio of 24 and 30, 99.58% and 99.61% nitrogen was removed, respectively. In conditions of initial TOC/TN ratios of 24 and 30, Chlorella sp. G-9 could accumulate lipid as high as 35.3% and 36.5%, respectively. The corresponding lipid productivities were 34.2 and 32.6 mg L-1 d-1, respectively, which were 13.7 and 13.0 times higher than those in photoautotrophic condition. Increasing the initial TOC/TN ratio of the wastewater could slightly increase the saturated degree in fatty acid, thereby improving the stability of biodiesel.