Probiotic application for juvenile rainbow trout

Merrifield, D., Dimitroglou, A., Bradley, G., Baker, R. T. M., & Davies, S. J. (2010) Probiotic applications for rainbow trout (Oncorhynchus mykiss Walbaum) I. Effects on growth performance, feed utilisation, intestinal microbiota and related health criteria. Aquaculture Nutrition, 16, 504-510.

Over the years many pathogens have caused large losses in aquacultural fish stocks; with the gastrointestinal tract suggested as the main source of entry for many fish pathogens. This has created the idea for probiotic use in combating these pathogens, by strengthening the defensive barrier of the indigenous gut microbiota. As well as helping control disease, probiotics have been shown to contribute to digestive function, have been implicated in development/maturation of gut and immune systems and have been shown to promote growth. These properties have been demonstrated in fish including trout, with a majority of probiotic interest being focused on lactic acid bacteria (LAB) and Bacillus species. However, although several studies have shown health promoting properties relating to the use of probiotics, there is a lack of information concerning beneficial effects on juvenile rainbow trout. Therefore the aim of this study was to investigate the effects of three probiotic species: Enterococcus faecium, Bacillus subtilis and Bacillus licheniformis, on juvenile rainbow trout growth, performance, feed utilisation, intestinal colonisation and related health parameters.

In this study the fish were fed a selection of the three probiotic species, either E. Faecium (EF), B. subtilis and B. licheniformis (2B) or all three of the above (EF + 2B). The fish were fed equal diets and the aquarium water was changed regularly. At the end of the trial three fish per tank were euthanized and their intestines were removed, homogenised and plated onto duplicated agar plates. Furthermore, blood was taken from three fish from each tank to measure the volume percentage of red blood cells (haematocrit levels) and the number of leukocytes present. The results showed efficient utilisation of feed in all treatment groups with an increase in fish biomass by 300%. B. subtilis and B. licheniformis were shown to be more effective in enhancing growth and body composition of rainbow trout and were not found in the control fish. However, high levels of all three probiotic species were found in respective samples. Haematocrit levels were found not to have changed throughout the probiotic treatments; although a significant increase was found in leukocyte numbers from group 2B + EF, whereas no change was found in the groups fed Bacillus spp. or E. faecium separately.

The current study was successful in demonstrating the ability of all probiotic species to survive transit through the digestive tract and colonise the intestine of rainbow trout. It also shows the potential benefits of probiotic utilisation in aquaculture. I believe the future prospects of probiotic use to be highly beneficial, especially as an alternative to antibiotic use. I believe this is also supported by previous studies indicated that the presence of E. Faecium resulting in the reduction of intestinal microbiota such as E.coli, Staphylococcus aureus and Clostridium spp. However, I agree with the authors that further research should be carried out in this area to help understand the full extent of benefits associated with probiotic use.

Marine microbiologists on the crime scene!

One might not know that sometimes forensic medicines looks to marine microbiology to solve a crime, especially regarding death by drowning . In fact, to establish where the death exactly occurred, forensic scientists investigate the presence of diatoms in the cadaver.

The diatom test, which basically consists in counting and identifying diatoms found in organs and tissues considered relevant for the investigation purposes, has been often used in the past and it is currently considered the gold standard.

However, the technical reliability, sensitivity and high cost of diatom test has caused considerable controversy.

For this reason scientists are now considering a new technique to support or replace the diatoms test findings: the biolumiscent bacteria test.

Why biolumiscent bacteria?

First of all biolumiscent bacteria do not inhabit the river waters, whereas they are prevalent in the sea, where they are ubiquitous: the presence of these bacteria could give information regarding the environment where the death occured, whereas the diatom test is not always reliable.

Secondly, their isolation and culture are easy and relatively cheap.

Moreover, marine bacteria, including bioluminiscent, are smaller than diatoms so when aspirated with water such bacteria could penetrate capillary vessels in the lungs and enter the blood system, where a portion might survive and predominantly proliferate, eventually suppressing the proliferation of endogenous bacteria from the gastrointestinal tract during the agonal period and/or after death, and of other exogenous bacteria that might enter after death.

The authors of this paper contributed in solving 2 death cases.

The first case was a 63-year-old man found in the shallows of a river, about 230m upstream from the mouth of the river.

The second case was 52-year-old man found dead floating in a car about 4m from shore and about 800m upstream from the mouth of another river.

Blood, urine and tissue samples were screened for drugs,alcohol, diatoms and bacterial analysis. Regarding the lattest, blood samples were diluted and then cultured in different NaCl concentration; after incubation, biolumiscent colonies were visualized in the dark. Plates were then stained with cytochrome oxidase reagent, which causes marine Gram-negative bacteria rods to turn blue, but not most of the enteric bacteria that proliferate during human decomposition.

Numerous biolumiscent and blue colonies appeared in the agar plates at all NaCl concentrations for both of the 2 cadavers. Homologous analysis based on the 16S rRNA gene showed that they were composed of marine bacteria belonging the the family Vibrionaceae.

The presence of numerous marine bacteria from the blood samples suggested that the victims had aspirated marine or brackish water.

These results, together with the autopsy findings and laboratory analysis for drugs and diatoms, supported the results of police investigations, indicating that drowning was the cause of death of the two immersed cadavers.

References: Kakizaki, E., S. Kozawa, et al. (2009). "Bioluminescent bacteria have potential as a marker of drowning in seawater: Two immersed cadavers retrieved near estuaries." Legal Medicine 11(2): 91-96.

Effects of the Deepwater Horizon oil spill

The explosion of the Deepwater Horizon oil rig, in the Gulf of Mexico, in April 2010, resulted in the release of approximately 4.9 million barrels of light crude oil over a period of three months. Much of the oil was transported to the shoreline, and a significant proportion still remains within coastal ecosystems. The marine sands of the Gulf are covered with microbial biofilms, and much of the oil hydrocarbon entering marine systems is degraded by indigenous microorganisms. The aim of this study was to identify and characterise predominant oil-degrading taxa which could be used as model hydrocarbon degraders or indicators of contamination, and to investigate the in situ response of indigenous bacterial communities to oil contamination.

Sediment cores were taken from beach sands of Pensacola Beach, Florida, an area which was exposed to heavy oil contamination in June 2010 as a result of the Deep Horizon oil spill. Samples were collected in July and September 2010. Chemical analysis revealed concentrations of petroleum hydrocarbon (C₈ to C₄₀) ranging from 3.1 to 4500 mg kg^-1. The abundance of bacterial rRNA gene sequences was approximately ten times higher in oiled sand than clean sand from a control area. Sequence libraries from the contaminated sands showed high sequence identity (up to 99%) to characterized oil-degrading isolates. A total of 24 bacterial strains from 14 genera were isolated and confirmed as oil-degrading microorganisms, with Gammaproteobacteria (Alcanivorax, Marinobacter) and Alphaproteobacteria (Rhodobacteraceae) dominating. Community analysis showed an increase in abundance of rRNA gene sequences from the genus Alcanivorax, in contaminated samples, indicating a response to oil contamination.

The findings suggest that oil contamination can influence the abundance and community composition of indigenous bacteria in the Gulf beach sands. The authors also hypothesise that Alcanivorax spp. can act as indicators of early stage oil degradation, and members of Alphaproteobacteria and Gram-positive groups can act as indicators of later stages of degradation, and suggest work to further investigate this.

This paper is fairly easy to read, it sets out the aims very clearly and although quite lengthy, the methods and results are presented in a logical order, with the discussion clearly addressing the key questions. The findings are important because oil spills can have severe ecological and economic consequences, and the authors point out that whilst technologies for oil drilling have advanced rapidly, the strategies employed to respond to and assess the impacts of oil spills are trailing behind. In order to manage oil spills and their clean-up more effectively, more must be known and understood about those microbial communities which are not only affected, but also play a key role in biodegradation. This is one of the few studies to investigate bacteria in situ, rather than under laboratory conditions.

A review of: Kostka, J.E. et al. (2011) Hydrocarbon-degrading bacteria and the bacterial community response in Gulf of Mexico beach sands impacted by the Deepwater Horizon oil spill. Applied and Environmental Microbiology, 77 (22): 7962.

Pathogenic Bacteria and Crabmeat Contamination in Mangrove Habitats

The aim of this paper was to establish a relation between the abundance of some pathogenic bacteria in water and sediment of mangrove habitats and the level of contamination of the meat obtained from crabs collected in the mangrove forest. Samples of water, sediment and crabs were collected monthly during both rainy and dry season at two areas of mangroves that differ in influence of tide and sewage discharge in the "Paraiba do Norte" estuary (Northeastern Brazil), a river that receive domestic, municipal and industrial sewage containing diverse pollutants such as viral and bacterial pathogens, toxic chemicals and a variety of organic and inorganic wastes.

The results showed that the two mangrove sites noticeably differ in physical, chemical and microbiological characteristics of the water and sediment. The sampling site "Camboa Grande" (CG), closer to the waste discharge and more distant from the river estuary, showed lower salinity, less dissolved oxygen and an higher incidence of total heterotrophic, coliforms and pathogenic bacteria in both water and sediment samples if compared to the control site "Ilha do Stuart" (IS), situated in the river course, at about 3.5 km distance from the waste discharge. This latter showed, during the dry season, lower salinity and higher values of BOD than CG, while temperature and pH did not differ significantly between the two sites. The organic matter content in the sediment of both the sites was similar, but total nitrogen content was much lower in IS site than CG. S. aureus was more abundant at CG, especially during the dry season and Salmonella spp. was detected only at this site. E. coli was found in all the water and sediment samples of CG, while its incidence was much lower at the site IS, where no coliforms and less total heterotrophic bacteria were observed in the dry season. Among other identified bacterial strains (92 in total), the most frequent were Enterobacter, Proteus and Pseudomonas. The average numbers of heterotrophic bacteria in the water of CG were 3.3 log CFU 100 mL-1, while their counts were lower at site IS (0.2 log CFU 100 mL-1), especially in the dry season. The counts of heterotrophic bacteria in the sediment were similar at both the locations (5.5 – 6.3 log CFU g-1) and their population size did not differ significantly between sampling sites and seasons. The authors also found a decrease of contaminant microorganisms in the river, downstream the discharge point, probably caused by the lower influence of sewage, as well as by the increase of salinity, which is known to affect the bacterial community and the microbial responses in the wastewater discharge.

The mangrove crab Ucides cordatus is the most harvested and has the highest commercial and subsistence importance to the local coastal population. These crabs are hand-picked and then transported to markets alive or already cooked. The quality of environment may affect the quantity and diversity of the microorganisms present on their surface, as well as the mode of collection and processing of products may cause increase of microbial contamination. In Brazil, pathogenic bacteria from genera Salmonella, Staphylococcus and Vibrio, are frequently found in crabmeat because crabs are usually processed in inadequate hygienic conditions, resulting in bacterial contamination. Moreover when crabs are collected, they are usually covered with mud, and then washed with mangrove water, that does not eliminate the contamination by saprophytic and pathogenic bacteria. However, in this study, no crabmeat sample showed the presence of coliforms and the counts of heterotrophic bacteria (mostly Enterobacter spp. and Pseudomonas spp.) has been always below 5 log CFU g-1. Moreover, in contrast to what has been obtained in other studies and in spite of differences between the bacterial abundance in the water and sediment, the contamination level of crabmeat did not differ significantly between the two stations and did not reflect the distribution pattern of bacteria in the environment. However, the influence of season on the number of total heterotrophic bacteria was significant and the meat of crabs captured in the dry season showed higher quantity of bacteria than the crabs collected in the rainy season. In addition, crabs must be cooked before obtaining the meat and the numbers of heterotrophic bacteria in crabmeat cooked for 60 minutes were slightly lower than in samples cooked for 30 minutes. Thus, the way of crabmeat processing and handling is more important in determining an increase in the contamination level of crabs than water or sediment pollution level. Anyhow, brazilian mangrove ecosystems are the first to suffer with urbanization and industrialization at coastal regions, and extensive mangrove ecosystems are already disappeared, putting an end to many of their important ecological functions. Therefore, the anthropogenic influence must be constantly monitored and every effort must be done to reduce human impact on these precious ecosystems.

Reference:

Grisi, T.C.S.D. and Gorlach-Lira, K. (2010) The abundance of some pathogenic bacteria in mangrove habitats of Paraiba do Norte Estuary and crabmeat contamination of mangrove crab Ucides cordatus. Braz Arch Biol Technol 53: 227–234.

Omega 3 Supplements from Algae and Biodiesel Waste

Omega 3 is an essential fatty acid for humans. It has many important health benefits and is thought to be particularly important during early brain development. The term essential, refers to the fact that mammals are unable to produce this group of fatty acids themselves and are completely dependent on obtaining them from their diet. Currently, oily fish is the major commercial source of omega 3 but with increasing pressure on fish stocks, a lot of research has gone into finding an alternative source. Marine algae have been well studied as such a source as the omega 3 found in fish oils originate from microbes anyway. Another important aspect of these studies is the development of a commercially viable method of growing and harvesting them.

A byproduct of biodiesel manufactured from crops and microbes is crude glycerol which is heavily contaminated with soap and methanol. To purify the glycerol requires an expensive process which makes it inappropriate for use in other industries, like food and pharmaceuticals. As the biodiesel industry increases in size so does the amount of crude glycerol production so finding a way to utilise it could be beneficial.

In this study, a marine microalgae called Schizochytrium limacinum was grown because of its ability to produce one particular omega 3, docosahexaenoic acid (DHA) through fermentation. In the growth medium, the authors used crude glycerol as an energy source which they had obtained from a biodiesel refinery. As any industrial harvesting of DHA would require destruction of the cells, it is vital to maximise the growth rate by constantly adding more medium to the cell culture, which dilutes it but maintains exponential growth. As media is added, the same amount of cell suspension is pumped out for processing. It is basically a large container with an over-flow pipe and a stirring impeller. Maintaining a high composition of DHA within the cells is also important. The objective of this study was to examine the growth efficiency of S. limacinum, efficiency of crude glycerol utilisation and DHA production. This was achieved by taking daily samples from the run-off cell suspension after a number of different media dilutions and crude glycerol concentrations were used.

Results indicated that biomass productivity was maximum when approximately 30% of extra media was added daily, giving 3.88 g/L of biomass from the run off cell suspension. Maximum DHA yield (0.52 g/L/day) was also measured at the same dilution rate when the medium contained 90 g/L of crude glycerol. If crude glycerol concentration was higher than 90 g/L the DHA content decreased sharply for some unknown reason.

In conclusion, this method of DHA production for possible use as a dietary supplement seems a good alternative to eating oily fish. Especially when the recommendation is to eat 2-3 portions of oily fish a week which seems like an unrealistic demand on current fish stocks. The use of a waste product from the biodiesel industry to grow the organisms on seems a good utilisation of a product which does not have many other uses. The authors commented that the DHA yield was disapointing so I imagine that future research might focus on different species of algae to see if better yields can be achieved.

A Review of;
Ethier S, Woisard K, Vaughan and Wen Z (2011) Continuous culture of the microalgae Schizochytrium limacinum on bio-diesel crude glycerol for producing docosahexaenoic acid. Bioresource Technology. 102: 88-93.

Oil degradation by Antarctic bacteria

Human presence in Antarctica is recently increasing because of fishing and explorative activities. Consequently the risk of anthropogenic hydrocarbon contamination is increasing more and more so that petroleum hydrocarbons are potentially the most likely source of pollution in Antarctic ecosystem.

Microbial biodegradation of hydrocarbons seems to be a valid tool in the control of this kind of pollution, although the process can be affected by several factors, especially by the physico-chemical characteristics of the compound and the environmental conditions where pollution occurs.

The aim of this study was to estimate the efficiency of diesel oil utilization by marine Antarctic bacteria at high (20C) and low (4C) temperatures.

Two bacterial strains E28 and E60 were isolated from coastal seawater samples collected in Terra Nova Bay (Ross Sea, Antarctica) and characterized as psychrotrophs and oild degrading bacteria.

A bacterial suspension comprised of E28 and E60 was inoculated in a specific liquid medium containing commercial diesel oil. The liquid cultures were then incubated at high and low temperature for 60 days. At a given incubation time, bacterial abundance was estimated by colony-forming units (CFU) spread plating on Marine Agar medium.

Biodegradation of diesel was quantified using gas-cromatography analysis and was expressed as the percentage of diesel oil degraded compared to the amount of the remaining fraction in the appropriate abiotic control samples (liquid medium uninoculated).

After 60 days of incubation up to 90% of the commercial oil disappeared from the culture medium inoculated with strains E28 and E60 overall, although the response slightly varied according to the strains and the temperatures.

The two isolates differed in the biodegradion dynamics: E28 was generally more efficient at 4C than at 20C during the first period of incubation, after that the biodegradation rapidly increased at 20C, while E60 degradation was always higher at the higher temperature.

These results prove that the temperature has a relevant effect on the biodegradation process in marine environment. Both viable counts and degradation efficiency were higher at 20C than at 4C, likely due to the decrease in enzymatic activity at low temperature and the change in the physico-chemical parameters of the oil (viscosity).

Neverthless psychrotrophic micro-organisms seem to possess an intrinsic advantage in the oil degradation at low temperature, thus more in situ investigations need to be done in order to estimate their real efficiency in degrading oil.

Isolation and characterisation of crude-oil degrading bacteria off Iran coast

Biodegradation is the utilisation of microbes to eliminate thousands of xenobiotic pollutants, including crude-oil, from the environment. Stimulating the growth of these microorganisms is a promising means of accelerating the detoxification of oil-polluted areas with a minimum impact on the ecological systems. Due to hydrocarbons being immiscible in water, growing the microbes on hydrocarbons has its difficulties. Many bacteria produce biosurfactants which overcome this problem; biosurfactants reduce surface tension, by accumulating at the interface, increasing surface area, which leads to increased bioavailability and subsequent biodegradation of the hydrocarbons.
Alkane hydroxylase is a key enzyme; it introduces an oxygen atom into the substrate. Based on phylogenetic analysis there are three main types of alkane hydroxylases; I, II and III, which degrade short, medium or long alkane chains respectively.

Oil pollution is particularly problematic in acute oil producing areas of the world, such as the Persian Gulf, Iran. Another marine environment in Iran which suffers from oil pollution, although not quite as bad, is the Caspian Sea. This paper aims to i) present bacterial strains present at these sites and evaluate them for production of biosurfactants and ii) analyse distribution of alkane hydroxylase genes in the isolated strains.

The authors isolated 25 bacterial strains, 11 of which (4 from the Caspian Sea and 7 from the Persian Gulf) showed higher growth rates on crude-oil. These were selected for further study.
The isolates were molecularly identified; only two were found to belong to the group obligate hydrocarbonoclasticus (OHCB), the remaining isolates were not related. The level of crude-oil biodegradation from these 11 species ranged from high (82%, 71% and 68%) to low (33% and 36%).
Each isolate degraded alkanes with a medium length (C12-18) to a greater extent than short chains (C9-11) and long chains (C19-25), this is because short-chained alkanes are toxic to most bacteria and long-chained alkanes are solid.
Results from ‘drop collapsing’ and ‘oil displacement’ tests indicate a direct relationship between cell-surface hydrophobicity and biosurfactant production and the emulsification activity and crude-oil biodegradation.
Results also showed that type II alkane hydroxylase enzymes were more prevalent in bacterial isolates from the Persian Gulf.

This paper concludes by saying that crude-oil-degrading bacteria have a high level of diversity and variability in biodegradation abilities. The use of these bacteria for bioremediation purposes will facilitate the better management of oil pollution in the Persian Gulf and Caspian Sea.

Overall an enjoyable read. I think this paper makes much sense; the isolates native to the problematic area were isolated and further researched to gain as much information from them as possible. It would be interesting to see what conditions these bacteria favour most, and then work on recreating these and possibility introducing them to polluted waters if a very bad pollution episode happens. This research not only needs to happen at all major oil producing areas of the world, but also in areas where oil spill migrate too.


A review of:
Hassanshahian, M., Emitiazi, G. and Cappello, S. (2012) Isolation and characterisation of crude-oil-degrading bacteria from the Persian Gulf and Caspian Sea. Marine Pollution Bulletin 64 7-12.