Living on the edge: Biofilms developing in oscillating environmental conditions

For the first time, the densities and diversity of microorganisms developed on ocean gliders were investigated using flow cytometry and Illumina MiSeq sequencing of 16S and 18S rRNA genes. Ocean gliders are autonomous buoyancy-driven underwater vehicles, equipped with sensors continuously recording physical, chemical, and biological parameters. Microbial biofilms were investigated on unprotected parts of the glider and surfaces coated with base, biocidal and chitosan paints. Biofilms on the glider were exposed to periodical oscillations of salinity, oxygen, temperature, pressure, depth and light, due to periodic ascending and descending of the vehicle. Among the unprotected surfaces, the highest microbial abundance was observed on the bottom of the glider’s body, while the lowest density was recorded on the glider’s nose. Antifouling paints had the lowest densities of microorganisms. Multidimensional analysis showed that the microbial communities formed on unprotected parts of the glider were significantly different from those on biocidal paint and in seawater

Evaluating the Reliability of Counting Bacteria Using Epifluorescence Microscopy

The common practice of counting bacteria using epifluorescence microscopy involves selecting 5–30 random fields of view on a glass slide to calculate the arithmetic mean which is then used to estimate the total bacterial abundance. However, not much is known about the accuracy of the arithmetic mean when it is calculated by selecting random fields of view and its effect on the overall abundance. The aim of this study is to evaluate the accuracy and reliability of the arithmetic mean by estimating total bacterial abundance and to calculate its variance using a bootstrapping technique. Three fixed suspensions obtained from a three-week-old marine biofilm were stained and dispersed on glass slides. Bacterial cells were counted from a total of 13,924 fields of view on each slide. Total bacterial count data obtained were used for calculating the arithmetic mean and associated variance and bias for sample field sizes of 5, 10, 15, 20, 25, 30, 35 and 40. The study revealed a non-uniform distribution of bacterial cells on the glass slide. A minimum of 20 random fields of view or a minimum of 350 bacterial cells need to be counted to obtain a reliable value of the arithmetic mean to estimate the total bacterial abundance for a marine biofilm sample dispersed on a glass slide

Microfouling on biocidal and non-biocidal antifouling coatings

Although antifouling marine paints have been used to prevent biofouling, not much is known about their effectiveness in preventing attachment of microorganisms. The current study aims at estimating the abundance of bacteria within biofilms developed on various commercial antifouling coatings in Marina Bandar Rowdha and Marina Shangri La, Oman. Coatings tested included Pettit #1863 and #1792, West Marine #11046620, #5566252 and #10175206, Hempel Hard Racing #76484, Hempel Olympic #86950, Hempasil X3 and International YBA920. All coatings were applied on clean plastic slides. Slides without any coating were used as controls. Microbial biofilms were harvested after 2, 7 and 14 days of biofouling. Bacterial density was estimated using epifluorescence microscopy. There was a significant difference between the various treatments (coatings and control) after 2, 7 and 14 days of biofouling. Although there were significant differences between both locations after 2 and 14 days of biofouling, no significant difference was observed after 7 days of biofouling at both locations. At Shangri La, the lowest bacterial density was found on International YBA920, Pettit #1792 and Hempasil X3 after 2 days, 7 days and 14 days respectively in comparison to the control treatments. However at Bandar Rowdha, International YBA920 showed the lowest bacterial density after 2 days while West Marine #10175206 showed the lowest bacterial density after both 7 days and 14 days of biofouling in comparison to the control treatment. The differential performance of tested antifouling coatings may be attributed to several factors including varying environmental conditions, difference in microfouling communities, time of exposure and physical and chemical properties of antifouling coating

Evaluating the Reliability of Counting Bacteria Using Epifluorescence Microscopy

The common practice of counting bacteria using epifluorescence microscopy involves selecting 5–30 random fields of view on a glass slide to calculate the arithmetic mean which is then used to estimate the total bacterial abundance. However, not much is known about the accuracy of the arithmetic mean when it is calculated by selecting random fields of view and its effect on the overall abundance. The aim of this study is to evaluate the accuracy and reliability of the arithmetic mean by estimating total bacterial abundance and to calculate its variance using a bootstrapping technique. Three fixed suspensions obtained from a three-week-old marine biofilm were stained and dispersed on glass slides. Bacterial cells were counted from a total of 13,924 fields of view on each slide. Total bacterial count data obtained were used for calculating the arithmetic mean and associated variance and bias for sample field sizes of 5, 10, 15, 20, 25, 30, 35 and 40. The study revealed a non-uniform distribution of bacterial cells on the glass slide. A minimum of 20 random fields of view or a minimum of 350 bacterial cells need to be counted to obtain a reliable value of the arithmetic mean to estimate the total bacterial abundance for a marine biofilm sample dispersed on a glass slide

Environmental conditions during a 6 month biofouling experiment in the Arabian Gulf

The development of a mature biofouling community on solid surfaces in the marine environment primarily involves the availability of colonizing bacterial communities and their ability to persist over time in any given environment. This study was undertaken as part of a collaborative project titled Biotechnological Applications of Marine Biofilms developing on solid surfaces in the Arabian Gulf funded by research grant (SQU-GCC/CL/17/02). The succession of marine biofouling communities (mature biofilms) on plastic panels were investigated over a period of six months in four locations in the Arabian Gulf (Fintas and Salmiya marinas in Kuwait, and Bandar Rowdha and Al Mouj marinas in Oman). Monthly assessment of the physico-chemical parameters of the seawater at each location was done using portable meters (thermometer, refractometer, pH meter, turbidity meter and conductivity/TDS meter). The concentrations of nutrients and elements were analysed using ion chromatography (IC) and inductively coupled plasma optical emission spectrometry (ICP-OES), respectively

Monthly succession of marine biofouling communities in the Arabian Gulf

This study was undertaken as part of a collaborative project titled Biotechnological Applications of Marine Biofilms developing on solid surfaces in the Arabian Gulf funded by research grant (SQU-GCC/CL/17/02). The succession of marine biofouling communities (mature biofilms) on plastic panels were investigated over a period of six months in four locations in the Arabian Gulf (Fintas and Salmiya marinas in Kuwait, and Bandar Rowdha and Al Mouj marinas in Oman). Monthly assessment of the physico-chemical parameters of the seawater at each location was done using portable meters (thermometer, refractometer, pH meter, turbidity meter and conductivity/TDS meter). The concentrations of nutrients and elements were analysed using ion chromatography (IC) and inductively coupled plasma optical emission spectrometry (ICP-OES), respectively. After each month, the developed biofilm on each panel was quantitatively and qualitatively analyzed as follows; total wet weight, abundance of bacteria using epifluorescence microscopy, chlorophyll a concentrations using spectrophotometry, percent coverage of macrofoulers, and presence or absence of signs of grazing based on visual observations and/or photodocumentation (using Image J software), which was also used to assess the presence/absence and dominance of macrofouling species. Additionally, the composition of the microbial community was investigated using 16S amplicon sequencing, resulting in supplementary data sets for bacterial community composition, predicted bacterial metabolic pathways (Bowman and Ducklow 2015, doi:10.1371/journal.pone.0135868), and presence/absence of microalgae based on their chloroplast 16S

Long-term microfouling on commercial biocidal fouling control coatings

<div><p>The current study investigated the microbial community composition of the biofilms that developed on 11 commercial biocidal coatings, including examples of the three main historic types, namely self-polishing copolymer (SPC), self-polishing hybrid (SPH) and controlled depletion polymer (CDP), after immersion in the sea for one year. The total wet weight of the biofilm and the total bacterial density were significantly influenced by all coatings. Pyrosequencing of 16S rRNA genes revealed distinct bacterial community structures on the different types of coatings. Flavobacteria accounted for the dissimilarity between communities developed on the control and SPC (16%) and the control and SPH coatings (17%), while Alphaproteobacteria contributed to 14% of the dissimilarity between the control and CDP coatings. The lowest number of operational taxonomic units was found on Intersmooth 100, while the lowest biomass and density of bacteria was detected on other SPC coatings. The experiments demonstrated that the nature and quantity of biofilm present differed from coating to coating with clear differences between copper-free and copper-based biocidal coatings.</p></div

Limitation of Microbial Processes at Saturation-Level Salinities in a Microbial Mat Covering a Coastal Salt Flat

Hypersaline microbial mats are dense microbial ecosystems capable of performing complete element cycling and are considered analogs of early Earth and hypothetical extraterrestrial ecosystems. We studied the functionality and limits of key biogeochemical processes, such as photosynthesis, aerobic respiration, and sulfur cycling, in salt crust-covered microbial mats from a tidal flat at the coast of Oman. We measured light, oxygen, and sulfide microprofiles as well as sulfate reduction rates at salt saturation and in flood conditions and determined fine-scale stratification of pigments, biomass, and microbial taxa in the resident microbial community. The salt crust did not protect the mats against irradiation or evaporation. Although some oxygen production was measurable at salinities of ≤30% (wt/vol) in situ, at saturation-level salinity (40%), oxygenic photosynthesis was completely inhibited and only resumed 2 days after reducing the porewater salinity to 12%. Aerobic respiration and active sulfur cycling occurred at low rates under salt saturation and increased strongly upon salinity reduction. Apart from high relative abundances of Chloroflexi, photoheterotrophic Alphaproteobacteria, Bacteroidetes, and Archaea, the mat contained a distinct layer harboring filamentous Cyanobacteria, which is unusual for such high salinities. Our results show that the diverse microbial community inhabiting this salt flat mat ultimately depends on periodic salt dilution to be self-sustaining and is rather adapted to merely survive salt saturation than to thrive under the salt crust. IMPORTANCE Due to their abilities to survive intense radiation and low water availability, hypersaline microbial mats are often suggested to be analogs of potential extraterrestrial life. However, even the limitations imposed on microbial processes by saturation-level salinity found on Earth have rarely been studied in situ. While abundance and diversity of microbial life in salt-saturated environments are well documented, most of our knowledge on process limitations stems from culture-based studies, few in situ studies, and theoretical calculations. In particular, oxygenic photosynthesis has barely been explored beyond 5 M NaCl (28% wt/vol). By applying a variety of biogeochemical and molecular methods, we show that despite abundance of photoautotrophic microorganisms, oxygenic photosynthesis is inhibited in salt-crust-covered microbial mats at saturation salinities, while rates of other energy generation processes are decreased several-fold. Hence, the complete element cycling required for self-sustaining microbial communities only occurs at lower salt concentrationsISSN:0099-2240ISSN:1098-533