Development of Alternative Renewable Sources of Essential Fatty Acids Derived From Marine Bacteria for Use in Fish Feed

Cooke Aquaculture Inc.

Table of contents

• Executive Summary
• Introduction
• Results & Discussion
• Identification of strains
• Assay Development
• In vitro suitability assessment
• Growth Modification
• Fish Colonization trial
• Feed Formulation
• Colonization testing
• Conclusions
• Acknowledgements
• References

Executive Summary

In order to develop alternative and renewable sources of Poly Unsaturated Fatty Acids (PUFAs) for use in fish feed, we explored the use of culturable marine bacteria capable of producing DHA and EPA. This study identified several different isolates of marine bacteria that produce these essential fatty acids. None of these strains showed similarity to known opportunistic pathogens or other pathogenic bacteria. Selected strains were examined for their potential both as a source of EPA and DHA containing biomass and as a live feed supplement for use in colonization of the fish gut  to potentially produce EPA and DHA in vivo. Four strains were tested for their ability to increase the synthesis of EPA and DHA under modified growth conditions such as elevated glucose, canola oil supplementation, and cold treatment.  However none of these culture modifications yielded increases in EPA and DHA sufficient to consider these strains as a significant source of biomass for feed supplementation.  Colonization trials with two of the most promising strains, I3 (EPA producer) and I19 (EPA and DHA producer) prepared in easy to use and stable formulations, showed continued presence in the stomach and small intestine for 4-5 days following a 1 week feeding period with bacterially supplemented feed. While the titres of I3 and I19 in the gut tissues were low and unlikely to support substantial denovo synthesis of EPA and DHA for nutritive value to the fish, this initial trial presents evidence that colonization is possible and may require refinement of dosage and application strategy in order to increase the presence of these microbes in the gut to nutritionally relevant levels.

Introduction

The nutritional requirements and health benefits of PUFAs such as DHA and EPA are well established  and  include  involvement  in  brain  health,  growth  & development, immune function, and cardiovascular health.  Both in human and fish health, foods containing  PUFAs  are  essential  for  maintaining  and  promoting  good  health. A traditional source of PUFAs for fish feed supplementation has been fish oil extracted from biomass procured in the traditional capture fishery.  With declining fish stocks, an increasing consumer demand for encapsulated fish oil dietary supplements, and a rise in the interest in developing alternative energy sources (e.g. biodiesel), the supply of fish oil available for fish feed supplementation is decreasing and becoming more expensive. Hence, it is essential to explore alternative and renewable sources for PUFAs to develop a sustainable supply. It has been recognized for some time that certain types of marine bacteria are capable of producing EPA and DHA although little attention has been given to exploit them as a source of PUFAs (Jøstensen and Landfald 1997;Yano et al. 1994;Yano et al. 1997).  Indeed, a recent review of alternative sources of PUFAs give little consideration to marine bacteria as an alternative source due to an erroneous belief that all PUFA producing strains have strict requirements for high pressure and low temperatures that would not be amenable to fermentation (Ward and Singh 2005).  However, given that many isolates of these bacteria are easily grown on a simple carbon based media at room temperature, they present a viable alternate source of EPA & DHA for dietary supplementation of fish feed.  Further, due to the live nature of these organisms and the fact that many are found in the gut of marine fish in high abundance (Yano et al. 1997), it is of interest to explore the possibility that they may colonize the gut of the fish and serve to provide an ongoing source of essential PUFAs. Should colonization follow, it may be possible to top-coat feed with them in a more readily available plant-based oil containing precursors to EPA or DHA to enhance the nutritional effect.  Indeed PUFA producing bacteria have been used to enrich rotifers, Brachionus plicatilis, with EPA and DHA (Lewis et al. 1998;Nichols et al. 1996) although we have been unable to identify reports describing their use as intended here.  Hence the proposed project is intended to address this technology gap and has the following objectives:

1. To identify and characterize additional PUFA producing bacterial strains.
2. To investigate and develop their use as a source of PUFA biomass for feed supplementation.
3. To investigate and develop their use as a live feed additive.

Results & Discussion

Identification of strains

Prior to beginning this project, a small number of EPA producing marine bacteria had been identified by RPC as well as one strain that produced small amounts of DHA.  To identify additional DHA and EPA-producing strains, a selection of strains from the RPC bacterial library were chosen for fatty acid analysis. Additional Shewanella spp. were selected for testing based on the different phylogenetic groupings and all Colwellia spp. were chosen as they had been previously reported capable of synthesizing DHA (Jøstensen and Landfald 1997;Russell and Nichols 1999;Yano et al. 1994;Yano et al. 1997).  Each strain was prepared as described in the materials and methods and analysed for all fatty acids listed in.

The results of these analyses indicated that an additional DHA producing strain of marine bacteria was identified. Strain FB14 was found to produce 1.6% DHA as a percentage of the total fatty acid content.  Other EPA-producing strains were also identified with the highest producing up to nearly 18% EPA of total fatty acids. The majority of strains were found to contain between 5-12% EPA in these initial screens.  Based on these results, 13 strains were selected for further work in order to identify strains which could be cultured selectively if used with in vivo feeding trials and/or developed into a productive source of biomass.

Assay Development

To begin development of a selective assay, each strain was examined for sensitivity to 10 different antibiotics utilizing disc diffusion assays. The results indicated that nearly all strains were resistant to one or more antibiotics thereby allowing for the use of a semi-solid agar media supplemented with an antibiotic specific to each strain to act as a selective media in assaying for gut colonization in feeding trials.

In vitro suitability assessment

To evaluate selected strains for in vivo use, each was plated on several media types and examined for growth and production of potential virulence factors.  Strains were evaluated for growth on BA and BA supplemented with 2% salt.  Salt supplementation of standard culture media is often required for growth of marine bacteria.  BA is used as an assessment of potential virulence since virulent strains of bacteria will often produce hemolytic activity, visualized as a zone of clearing around bacterial growth on plates. All selected strains grew well on BA or BA+salt and did not produce any detectable hemolytic activity. Selected strains were also evaluated for growth on TCBS and TCBS+seawater to determine if they were capable of surviving in the gut as this media is often used as a diagnostic for the cultivation of enteric Vibrio spp.  Results indicated  that  the  majority  of  strains  were  capable  of  growing  on  this  media  and therefore may survive in the gut.  However, it was evident from this analysis that FB14 did not grow well, if at all, on either BA or TCBS media.

Further characterization of strains was also conducted using API 20E  biochemical testing  strips to determine if any might be identical to known pathogenic strains. Results of this analysis revealed that while the majority showed similarity to the S. putrefaciens group, none were shown to be identical. Further similarity analysis was conducted through comparison of 16S rDNA genes that included the S. putrefaciens and S. alga opportunistic pathogens. Results indicated that none of the selected strains were identical to either of these pathogens and that all selected strains were dissimilar enough to be classified as members of phylogenetically distinct clades.

Growth Modification

Based on yield information from the preliminary screens and information from the selectivity assays and in vitro suitability assays, two DHA-producing strains and two EPA-producing strains were chosen for growth modification experiments in an attempt to develop these strains as a viable source of PUFA biomass.

As a first step at increasing the yield of PUFA’s, growth media was supplemented with the basic carbon source, glucose.  Following culture and preparation of cells, total fatty acids were extracted and compared.  Results indicated that glucose supplementation of culture media did not have any positive effect on the production of PUFA’s. In fact, it appeared to have had a negative effect on production of EPA and DHA.  The precise reason for this is unclear although it is tempting to speculate that the bacterial strains utilize and store lipids as an energy source and a surplus of glucose may negate the need to synthesize and store energy as lipid.

Growth media was also supplemented with canola oil which has one of the highest contents of α-linolenic acid (~7%; ALA), a precursor to DHA and EPA.  It was of interest to determine if the bacterial strains would be capable of utilizing or converting this readily available precursor into the longer chain EPA and DHA. Results indicated that increasing the amount of canola oil only served to decrease the amount of EPA but increased the proportion of ALA in the lipids. Only I19 showed a marginal increase in the amount of DHA with 1% supplementation but none with 5%.

Curiously, strain FB14 failed to produce EPA or DHA in both the glucose and oil supplementation experiments. The reason for this remains unclear.

The precise reason for the declines in DHA and EPA is unclear although it is likely that the bacterial cells may be able to substitute ALA for the other longer chain lipids in their membranes.  It is also tempting to speculate that the bacterial strain may store ALA for use as an energy source or as a store for the future production of the longer chain fatty acids should the cells be exposed to the proper environmental conditions. It is interesting to note that no additional solubilization agents were added to the culture media to facilitate uptake or access to the lipids by the bacterial cells.  Hence it is likely that they possess the necessary enzymatic machinery to solubilize and incorporate ALA into their membranes.  This was particularly evident for strain I3 in that following culture for 48 hrs, a large proportion of the added canola oil appeared to be partially homogenized with the surrounding culture media.  Analysis if this fraction showed  it  to  be  largely  ALA  so  it  is  possible  that  I3  is  capable  of solubilizing the canola oil more readily than the other strains.

A third set of experiments focused on increasing the yields of DHA in strains FB14 and I19 by using cold temperatures since previous work had shown increased synthesis with cold temperatures.  It is believed this effect is a result of the fact that the longer chained poly unsaturated fatty acids promote better membrane flexibility under cold temperatures.  To test this, both strains were cultured for 55 hrs at 10°C then harvested and subjected to fatty acid analysis.   Results indicated that cold did not have any positive effect on the production of DHA but may have had a slight increasing effect on EPA production in I19. Cold temperatures appeared to stop synthesis of EPA and DHA in FB14.  It should be noted that growth at this temperature significantly reduced the yield of bacterial cells in that timeframe.

Following all trials to increase the yields of EPA and DHA, the yields were calculated using the best case data. Given the very low yields and growth of biomass, it was concluded that these levels were simply not high enough to represent a viable source of PUFA for use as a feed supplement, even when the wet weight was factored out.  Hence, the portion of the project to feed fish bacterial biomass was concluded and focus was placed on determining if strains could be used as a live feed supplement to colonize the gut of Atlantic salmon smolts.

Fish Colonization trial

Examination of all data from the selection assay development together with the in vitro suitability assays, it was evident that the best DHA producing strain, FB14, appeared ill suited for in vivo use and would not lend itself to selective plating through the use antibiotic containing media. However, I19 did possess all of the desired characteristics from the assays and assessments so was chosen as one strain for in vivo colonization trials. A second strain, I3, was chosen based on the fact that it produced the highest amounts of EPA, possessed desirable characteristics from the in vitro suitability assays and showed resistance to one antibiotic so could be selectively plated during in vivo trials. Hence for the analysis of fish stomach and small intestine for colonization by bacterial strains I3 and I19, selective media were prepared consisting of TCBS agar supplemented with either 50 µg/ml amoxicillin (for I19) or 30 µg/ml tetracycline (for I3). Prior to the commencement of the trial, Atlantic salmon smolts obtained through other projects at RPC were sampled and their stomach and intestine swabbed onto the culture plates.  All plates showed no growth of bacteria indicating a high likelihood that the plates would be highly selective for I3 and I19. The combination of TCBS and antibiotics  provided  a  doubly  selective  media  compared  to  one  containing  only antibiotic.  In terms of strain dosage, it was decided that a high dose would be desirable so 1E+8 cells/g feed was chosen.

Feed Formulation

Several preliminary tests with the dry feed pellets established that they were capable of absorbing up to 6-9% liquid while still maintaining their integrity.  Further, these volumes also facilitated easy and thorough coating of feed pellets by simple shaking so would help to ensure even distribution of the bacterial cells over the feed.   Hence it was planned to formulate the bacteria into a solution which could then be added to the feed pellets and allowed to absorb.

To identify a possible storage solution for the bacteria that had a reasonable shelf life and could be readily mixed with feed on demand, bacteria were harvested and resuspended in different solutions.  In preliminary tests following 2 days of storage in various solutions, PBS was found to have retained the highest cell viability. To determine the shelf life of bacterial strains, both I3 and I19 were grown in 400 ml scale batches, harvested and resuspended in PBS then stored in a refrigerator at 4°C for up to 12 days, a duration fully covering the planned feeding trial.  At various points during storage, samples were collected and titred to determine loss of viability. Results indicated that I19 retained 100% viability after 12 days whereas I3 lost approximately 20% viability after 4 days.  Feed dosages for I3 were adjusted accordingly to compensate for losses and fresh stock was prepared for use on day 4 of the trial.

Experiments to determine the shelf life of bacteria coated onto feed were conducted to determine whether it was advantageous to coat large batches of feed at a time as opposed to coating a new batch of feed immediately prior to feeding. The results of this analysis were inconclusive since it was not possible to determine accurate titres of bacteria coated onto feed since both strains appeared to associate very tightly to the feed particles when homogenized in PBS. When bacteria spiked feed samples were homogenized, much of the feed remained insoluble and the majority of the bacterial cells  were  recovered  from  this  insoluble  material  as  opposed  to  the  soluble  PBS fraction. As  a  result  accurate  titres  were  not  obtainable  making  an  accurate assessment of shelf life difficult. Hence it was decided for the purposes of this trial, to make fresh bacteria spiked feed before each feeding.

To prevent possible loss of bacterial cells upon addition of feed to the tank water, it was decided to coat the pellets in fish oil. An oil topcoat would also help to increase the palatability of the feed since it was unknown how the fish would react to the flavor of the bacterial supplement. Several w:w ratios (1-5%) of oil to feed were tested and it was found that 1% was sufficient in providing good coverage without excess. To test whether the oil might have a detrimental effect on the strains, bacteria were grown in liquid culture media containing up to 5% fish oil for 24 hrs at 20°C. Following growth, the optical density of each culture was compared to cells grown in media without oil and no difference could be detected indicating that the oil had no detrimental effect on the cells.

Colonization testing

To test whether feed spiked with I3 and I19 bacteria could introduce these strains to the gut of Atlantic salmon and promote colonization, supplemented feed was fed to fish three times per day for 1 week. During this time, fish fed normally and showed no adverse reaction to the supplemented feed either at the beginning or end of the feeding period.   Further, no mortalities were recorded during the feeding period or after. Following the 1st week feeding with supplemented feed, 5 fish per treatment were sampled per day for 5 days. Fish samples were sent to the lab for necropsy and titration of I3 and I19 strains in both the stomach and small intestine. The results indicated that target growth (i.e. colonies matching the colony morphology of I3 and I19) were identified in some fish over the course of the 5 day sampling period. Target growth for I19 was obtained in some fish for all five days of the sampling period with day 3 showing growth in all five fish sampled. I3 fed fish showed target growth in fish up to day 4 of sampling. Some target growth was also observed in control fish. However, this occurred in only 2 fish at low colony levels at two different points in the sampling period.  All fish sampled prior to the beginning of the feeding trial tested negative for target growth.

Detailed examination of the culture results indicated that the titre of the target growth was much lower than expected and the relative frequency of positive fish and tissue distribution was also lower. Given that each fish on average received 2.15E+8 cells per day for I3 and 2.26E+8 cells per day for I19 (assuming 100% consumption distributed across all fish evenly), it was expected that cell titres would have been higher, at least initially and then taper off.  However, cell titres for targeted growth remained fairly consistent throughout the sampling period.  Tissue distribution for target growth occurred in both the stomach and lower intestine with the majority of cases showing distribution in one tissue or the other but rarely both together in the same fish (only 2 occurrences).  The frequency of target growth detection for I3 was roughly even between the stomach and small intestine while I19 showed the majority of target growth in the stomach samples.  Given these results, it was decided that denaturing gradient gel electrophoresis (DGGE) profiling of gut microflora would not be conducted as it is unlikely  that  significant  differences  would  be  observed  given  the  low  level  of colonization.

Possible explanations for the observed levels of target growth could be that the initial dosage was not high enough given that these strains had not originated from the gut of Atlantic salmon smolts.  Perhaps a higher and more prolonged dose was required to obtain higher colonization rates since it may be difficult for non-native strains to become established in the gut among the innate gut microflora.  Examination of the feed records indicated that the fish missed 4 of the scheduled 21 feedings which may have had some impact.  Also, given the affinity of I3 and I19 for the feed (see above), it is possible that I3 and I19 have a high affinity for the gut tissues and are difficult to dislodge during tissue homogenization which resulted in low titres.

Review of the necropsy report done on fish at the time of sampling indicated that fish from the first two days of sampling were healthy and showed no signs of disease. However, at day 3 of sampling, it was evident that fish had been off feed for approximately 24 hours and fish at subsequent sampling time points were also off feed. When discussed with Northeast Nutrition aquaculture staff, they had indicated that the fish had gone off feed due to sampling stress. The impact of this on the presence of target growth is unknown although it is apparent that strains did continue to persist in the stomach and small intestine despite the lack of feed.  Whether strains would have been flushed with continued feeding is unknown.

Conclusions

In summary, a number of EPA- and DHA-producing strains of Shewanella and Colwellia spp. were identified that do not share any similarity to known opportunistic pathogens or possess any apparent virulence properties. While the growth conditions tested in this report failed to increase the levels of DHA and EPA and precluded their use as a source of biomass for feed supplementation, it remains possible that other growth supplements or conditions may function to stimulate increased production. The majority of strains identified here were unsuitable for in vivo colonization trial yet two promising strains possessed the necessary selection criteria to proceed with testing. The formulations developed for I3 and I19 strains were such that they could be stored for up to 4 days for I3 and 12 days for I19 in a refrigerator and be mixed with feed as a top coat supplement to a regular diet as needed. This formulation did appear to promote colonization of the gut although the dosage and frequency of application appeared to result in low titres and infrequent colonization. With the goal of colonization being to have the bacterial strains producing DHA and EPA in the fish gut, it is uncertain whether these levels and the frequency of colonization would be sufficient to have an impact on the levels of DHA and EPA in the fish tissues. The results of this study will be published in the Cooke Aquaculture news letter. Future work involving extended feeding trials and increased dosages will help to determine if a nutritional impact on PUFA levels are achievable.

Acknowledgements

We would like to thank the Department of Fisheries and Oceans Canada AIMAP program for funding this  project.    Thanks  also  to Jason  Holmes,  Elizabeth  Jones, Jessica Jones, Samantha Atkinson and Rebecca Liston for their excellent technical assistance during various parts of this project.

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