Application of Ion Torrent Sequencing to the Assessment of the Effect of Alkali Ballast Water Treatment on Microbial Community Diversity

Abstract

The impact of NaOH as a ballast water treatment (BWT) on microbial community diversity was assessed using the 16S rRNA gene based Ion Torrent sequencing with its new 400 base chemistry. Ballast water samples from a Great Lakes ship were collected from the intake and discharge of both control and NaOH (pH 12) treated tanks and were analyzed in duplicates. One set of duplicates was treated with the membrane-impermeable DNA cross-linking reagent propidium mono-azide (PMA) prior to PCR amplification to differentiate between live and dead microorganisms. Ion Torrent sequencing generated nearly 580,000 reads for 31 bar-coded samples and revealed alterations of the microbial community structure in ballast water that had been treated with NaOH. Rarefaction analysis of the Ion Torrent sequencing data showed that BWT using NaOH significantly decreased microbial community diversity relative to control discharge (p<0.001). UniFrac distance based principal coordinate analysis (PCoA) plots and UPGMA tree analysis revealed that NaOH-treated ballast water microbial communities differed from both intake communities and control discharge communities. After NaOH treatment, bacteria from the genus Alishewanella became dominant in the NaOH-treated samples, accounting for <0.5% of the total reads in intake samples but more than 50% of the reads in the treated discharge samples. The only apparent difference in microbial community structure between PMA-processed and non-PMA samples occurred in intake water samples, which exhibited a significantly higher amount of PMA-sensitive cyanobacteria/chloroplast 16S rRNA than their corresponding non-PMA total DNA samples. The community assembly obtained using Ion Torrent sequencing was comparable to that obtained from a subset of samples that were also subjected to 454 pyrosequencing. This study showed the efficacy of alkali ballast water treatment in reducing ballast water microbial diversity and demonstrated the application of new Ion Torrent sequencing techniques to microbial community studies.

Schrödinger’s microbes: Tools for distinguishing the living from the dead in microbial ecosystems

“…After PMA treatment, DNA is extracted, and amplification is typically performed by PCR. PCR-based procedures found to be compatible with PMA treatment include qPCR [ 66 , 67 ], microarrays [ 20 , 68 ], denaturing gradient gel electrophoresis (DGGE) [ 69 ], and amplicon sequencing [ 70 – 72 ]. In addition to PCR, successful PMA treatment has been reported in combination with loop-mediated isothermal amplification (LAMP) [ 73 ], multiple displacement amplification (MDA) [ 74 ], and metagenomic library construction for low-biomass clean room samples [ 39 ].…”

Attenuation and modification of the ballast water microbial community during voyages into the Canadian Arctic

“…As these treatments are already proposed and are due to become mandated in 2017 for newly built vessels travelling outside of any single nation's exclusive economic zone (IMO, ), one question that remains is how effective these treatment approaches will be. This is already an active area of research (e.g., Fujimoto et al., ), and one where next‐generation sequencing approaches, such as those used here, will play a major role. For vessels like the M/V Arctic that exclusively travel within the coastal waters of a single nation, and which will be exempt from new ballast water treatment requirements, the risk of transporting microbes via ballast water will likely remain high.…”

“…Early studies of microbial communities in ballast water were limited by difficulty in identifying species (but see Tomaru et al., ). The advent of high‐throughput sequencing and metagenomic approaches has begun to fill this knowledge gap and inform our understanding of bacterial communities world‐wide, including in ballast water (e.g., Aridgides et al., ; Brinkmeyer, ; Fujimoto et al., ; Pagenkopp Lohan, Fleischer, Carney, Holzer, & Ruiz, ). These approaches, which typically involve DNA sequencing of the bacterial 16S ribosomal RNA gene (16S rRNA) and subsequent matching of DNA sequences to known bacterial groups, provide a vastly more detailed view of the microbial community in ballast water.…”

“…However, metagenomics studies on the bacterial ballast water community are still rare. The few papers that have been published to date provide detailed information on the diversity of microbes in ballast water related either to water sources (Brinkmeyer, ; Ng et al., ) or to effects of alkali treatment (Fujimoto et al., ). None of the metagenomics papers published to date examine changes that occur over the course of a voyage, or that might be associated with BWE.…”

Efficacy of pH elevation as a bactericidal strategy for treating ballast water of freight carriers

“…Similarly, in Table 5 , there were seven similar instances. It was assumed that no cfu were present when no colonies were recovered; however, the ability to kill bacteria that are capable of entering a viable, but nonculturable state was not determined [13] . In a previous study, Starliper and Watten [7] showed that sodium hydroxide apparently destroyed bacterial cell walls because microscopically, intact cells were not detected after treatments that were the same as administered in the present study.…”