Flow Cytometric Analysis of Bacterial Protein Synthesis: Monitoring Vitality After Water Treatment.

Bacterial vitality after water disinfection treatment was investigated using bio-orthogonal non-canonical amino acid tagging (BONCAT) and flow cytometry (FCM). Protein synthesis activity and DNA integrity (BONCAT-SYBR Green) was monitored in Escherichia coli monocultures and in natural marine samples after UV irradiation (from 25 to 200 mJ/cm2) and heat treatment (from 15 to 45 min at 55°C). UV irradiation of E. coli caused DNA degradation followed by the decrease in protein synthesis within a period of 24 h. Heat treatment affected both DNA integrity and protein synthesis immediately, with an increased effect over time. Results from the BONCAT method were compared with results from well-known methods such as plate counts (focusing on growth) and LIVE/DEAD™ BacLight™ (focusing on membrane permeability). The methods differed somewhat with respect to vitality levels detected in bacteria after the treatments, but the results were complementary and revealed that cells maintained metabolic activity and membrane integrity despite loss of cell division. Similarly, analysis of protein synthesis in marine bacteria with BONCAT displayed residual activity despite inability to grow or reproduce. Background controls (time zero blanks) prepared using different fixatives (formaldehyde, isopropanol, and acetic acid) and several different bacterial strains revealed that the BONCAT protocol still resulted in labeled, i.e., apparently active, cells. The reason for this is unclear and needs further investigation to be understood. Our results show that BONCAT and FCM can detect, enumerate, and differentiate bacterial cells after physical water treatments such as UV irradiation and heating. The method is reliable to enumerate and explore vitality of single cells, and a great advantage with BONCAT is that all proteins synthesized within cells are analyzed, compared to assays targeting specific elements such as enzyme activity.

Assessing Stress Resilience After Smolt Transportation by Waterborne Cortisol and Feeding Behavior in a Commercial Atlantic Salmon (Salmo salar) Grow-Out Recirculating Aquaculture System.

Sampling protocols and water quality sensors have been developed to assess fish health and welfare in recirculating aquaculture systems (RASs). Still, the use of fish-based non-invasive welfare indicators, reflecting the physiological state of the fish, is limited in this type of system. Cortisol, the major stress-coping hormone in fish, diffuses through the gills. Consequently, waterborne cortisol is a potential fish-based non-invasive welfare indicator in RAS. However, its use in commercial rearing systems is sparse. In this study, we evaluated water cortisol levels and feeding behavior as welfare indicators of newly inserted smolt in a commercial RAS for harvest size Atlantic salmon. The RAS consisted of two parallel fish rearing raceways, raceways 1 and 2, sharing the same water treatment with common outlets and inlets. The smolts were inserted in raceway 1 while salmon that have been in the system for 6 months or more were kept in raceway 2. The smolt insertion period was 3 days. Samples for water cortisol levels were withdrawn the day before, 1 and 3 days after the smolt insertion period. Smolt insertion resulted in elevated water cortisol concentrations in the entire system, with the highest values in raceway 1, one day after smolt insertion. Estimated cortisol production in newly inserted smolt decreased over time, was similar to what has been reported in salmon adapting to experimental tanks. Feeding behavior indicated that the appetite was not fully resumed in the newly inserted smolts, while the appetite of fish in raceway 2 was unaffected by smolt insertion. These results, obtained in a highly intensive commercial RAS, suggest that waterborne cortisol together with feeding behavior can be used as indicators for adaptive processes associated with stress resilience in farmed fish. Thus, they are promising non-invasive indicators for assessing the impact of potential stressors on fish welfare in this type of rearing system.

Bioorthogonal Non-canonical Amino Acid Tagging Combined With Flow Cytometry for Determination of Activity in Aquatic Microorganisms.

In this study, we have combined bioorthogonal non-canonical amino acid tagging (BONCAT) and flow cytometry (FCM) analysis, and we demonstrate the applicability of the method for marine prokaryotes. Enumeration of active marine bacteria was performed by combining the DNA stain SYBR Green with detection of protein production with BONCAT. After optimization of incubation condition and substrate concentration on monoculture of Escherichia coli, we applied and modified the method to natural marine samples. We found that between 10 and 30% of prokaryotes in natural communities were active. The method is replicable, fast, and allow high sample throughput when using FCM. We conclude that the combination of BONCAT and FCM is an alternative to current methods for quantifying active populations in aquatic environments.

Microorganisms in ballast water: Disinfection, community dynamics, and implications for management.

Increasing concerns have accelerated the development of international regulations and methods for ballast water management to limit the introduction of non-indigenous species. The transport of microorganisms with ballast water has received scientific attention in recent years. However, few studies have focused on the importance of organisms smaller than 10 µm in diameter. In this work, we review the effects of ballast water transport, disinfection, and the release of microorganisms on ecosystem processes with a special focus on heterotrophic bacteria. It is important to evaluate both direct and indirect effects of ballast water treatment systems, such as the generation of easily degradable substrates and the subsequent regrowth of heterotrophic microorganisms in ballast tanks. Disinfection of water can alter the composition of bacterial communities through selective recolonization in the ballast water or the recipient water, and thereby affects bacterial driven functions that are important for the marine food web. Dissolved organic matter quality and quantity and the ecosystem status of the treated water can also be affected by the disinfection method used. These side effects of disinfection should be further investigated in a broader context and in different scales (laboratory studies, large-scale facilities, and on the ships).

Ultraviolet radiation as a ballast water treatment strategy: Inactivation of phytoplankton measured with flow cytometry.

This study investigates different UV doses (mJ/cm(2)) and the effect of dark incubation on the survival of the algae Tetraselmis suecica, to simulate ballast water treatment and subsequent transport. Samples were UV irradiated and analyzed by flow cytometry and standard culturing methods. Doses of ≥400 mJ/cm(2) rendered inactivation after 1 day as measured by all analytical methods, and are recommended for ballast water treatment if immediate impairment is required. Irradiation with lower UV doses (100-200 mJ/cm(2)) gave considerable differences of inactivation between experiments and analytical methods. Nevertheless, inactivation increased with increasing doses and incubation time. We argue that UV doses ≥100 mJ/cm(2) and ≤200 mJ/cm(2) can be sufficient if the water is treated at intake and left in dark ballast tanks. The variable results demonstrate the challenge of giving unambiguous recommendations on duration of dark incubation needed for inactivation when algae are treated with low UV doses.

Flow cytometric applicability to evaluate UV inactivation of phytoplankton in marine water samples.

Disinfection of microbes is of importance to prevent the spread of pathogens and non-indigenous species in the environment. Here we test the applicability of using flow cytometry (FCM) to evaluate inactivation of the phytoplankter Tetraselmis suecica after UV irradiation and labeling with the esterase substrate 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM). Non-irradiated and UV irradiated samples were analyzed with the plate count technique and FCM for 24 days. The numbers of colony forming units were used as a standard to develop a FCM protocol. Our protocol readily distinguishes live and dead cells, but challenges were encountered when determining whether UV damaged cells are dying or repairable. As damaged cells can represent a risk to aquatic organisms and/or humans, this was taken into account when developing the FCM protocol. In spite of the above mentioned challenges we argue that FCM represents an accurate and rapid method to analyze T. suecica samples.

Recolonization by heterotrophic bacteria after UV irradiation or ozonation of seawater; a simulation of ballast water treatment.

Transport of ballast water with ships represents a risk for introduction of foreign species. If ballast water is treated during uptake, there will be a recolonization of the ballast water by heterotrophic bacteria during transport. We investigated survival and succession of heterotrophic bacteria after disinfection of seawater in the laboratory, representing a model system of ballast water treatment and transport. The seawater was exposed to ultraviolet (UV) irradiation, ozone (2 doses) or no treatment, incubated for 16 days and examined with culture-dependent and -independent methods. The number of colony-forming units (CFU) was reduced below the detection level after disinfection with UV and high ozone dose (700 mV), and 1% of the initial level for the low ozone dose (400 mV). After less than 3 days, the CFU was back or above the starting point for the control, UV and low ozone treatment, whereas it took slightly more than 6 days for the high ozone treatment. Disinfection increased substrate availability and reduced cell densities. Lack of competition and predation induced the recolonization by opportunistic bacteria (r-strategists), with significant increase in bacterial numbers and a low diversity (based on DGGE band pattern). All cultures stabilized after the initial recolonization phase (except Oz700) where competition due to crowding and nutrient limitation favoured bacteria with high substrate affinity (K-strategists), resulting in higher species richness and diversity (based on DGGE band pattern). The bacterial community was significantly altered qualitatively and quantitatively and may have a higher potential as invaders in the recipient depending on disinfection method and the time of release. These results have implications for the treatment strategy used for ballast water.