Bacterial Degradation of Algal Toxins: Possibilities for Use in Post-Harvest Treatment? E. Turrell 1 , J. Cotterill 2 , E. Papapanagiotou 3 , J-P. Lacaze 1 , J. Graham 1 , P. Lamont 4 , Laura Morley 1 1 ab 3 1) Background Marine bacteria capable of metabolising algal toxins were isolated. Such bacteria could play a role in elimination of algal toxins from shellfish; possible applications include manipulating the shellfish bacterial flora to depurate algal toxins. 2) Toxin degradation by bacteria Bacteria were shown to have the capacity to degrade PSP toxins. Bacteria were grown with algal toxins - degradation of the toxins was monitored over time. Results from candidate bacteria for use in depurating shellfish are shown opposite: 2 3) Microencapsulation – delivery of bacteria to shellfish Microencapsulation of toxin degrading bacteria could be used to deliver a concentrated pulse of bacteria to the digestive system of toxin contaminated shellfish. The size of capsule that could be filtered & ingested by shellfish was determined. Alginate capsules of 3 different sizes (10- 50, 50-100 & 100-250μm) containing non-degradable fluorescent microbeads (as a proxy for bacteria) of 3 different colours (blue, pink, yellow) were fed to mussels & scallops (Figs. A, B, C & D). Pink microbeads were detected in faeces after 16 h with the alginate wall dissolved (Fig. F). The capsules were passed into the digestive system prior to being excreted (Figs. G & H). Good news – toxin degrading bacteria could be released from alginate capsules into the digestive system ! B C D A pink yellow blue E Visible alginate walls F 75 Klebsiella sp. SD12 67 Pseudoalteromonas sp. SD438 60 Pseudoalteromonas sp. SD424 55 Unidentified SD466 53 Stappia sp. SD335 51 Erythrobacter citreus SD314 Total toxicity (STX eq.) degradation (%) 1, Preliminary id (SSU rRNA) Isolate PSP 1 Isolates were grown in marine broth with (nM): STX (390), NEO (390), GTX-1 (636), GTX-2 (708), GTX-3 (234) & GTX-4 (210) over 5 d. Change in total PSP toxicity was determined by LC-MS-MS 4 Microbeads in faeces 4) Detoxifying shellfish – a possibility? The next stage was to develop a method of depurating shellfish by feeding a diet of microencapsulated bacteria. Bacterial isolate, SD12, shown to degrade PSP toxins in vitro was microencapsulated (50 – 100 μm) (Fig. I) & fed to scallops contaminated with PSP toxins. Empty capsules were used as a control (Fig. J). This project was supported by the: Sixth Framework Programme of the European Community (SPIES-DETOX Collective Research Project 0302790-2) I J Scallops were sampled over time (Fig. K), dissected (Fig. L) and the hepatopancreas (HP) analysed for PSP toxins using LC-MS-MS. 5) Preliminary results Variation in toxin concentrations in the HP were observed between scallops fed microencapsulated bacteria and empty capsules, although inter-animal variability was high. Fig. M shows example MRM chromatograms for NEO. Further trials will be undertaken to determine if a practical method for detoxifying cultured shellfish is possible! L K Time (minutes) 14 16 18 20 22 m/z 316.1>220.2 NEO detected at 24 h in HP of scallop fed microencapsulated bacteria NEO detected at 24 h in HP of scallop fed control capsules Bacteria inside capsules empty capsules M Both mussels & scallops ingested capsules over 2h. Smaller capsules (blue & pink beads) were readily ingested. A higher proportion of yellow capsules were detected in mussel pseudo-faeces & a higher proportion of blue capsules were detected in scallop pseudo-faeces, the alginate wall was still visible so capsules were not degraded prior to rejection (Fig E). G H Microencapsulated pink beads released in the digestive organs
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Bacterial Degradation of Algal Toxins: Possibilities for Use in Post-Harvest Treatment?
E. Turrell1, J. Cotterill2, E. Papapanagiotou3, J-P. Lacaze1, J. Graham1, P. Lamont4, Laura Morley1
1ab3
1) BackgroundMarine bacteria capable of metabolising algal toxins were isolated. Such bacteria could play a role in elimination of algal toxins from shellfish; possible applications include manipulating the shellfish bacterial flora to depurate algal toxins.
2) Toxin degradation by bacteriaBacteria were shown to have the capacity to degrade PSP toxins. Bacteria were grown with algal toxins - degradation of the toxins was monitored over time. Results from candidate bacteria for use in depurating shellfish are shown opposite:
2
3) Microencapsulation – delivery of bacteria to shellfishMicroencapsulation of toxin degrading bacteria could be used to deliver a concentrated pulse of bacteria to the digestive system of toxin contaminated shellfish. The size of capsule that could be filtered & ingested by shellfish was determined. Alginate capsules of 3 different sizes (10-50, 50-100 & 100-250µm) containing non-degradable fluorescent microbeads (as a proxy for bacteria) of 3 different colours (blue, pink, yellow)were fed to mussels & scallops (Figs. A, B, C & D).
Pink microbeads were detected in faeces after 16 h with the alginate wall dissolved (Fig. F). The capsules were passed into the digestive system prior to being excreted (Figs. G & H). Good news – toxin degrading bacteria could be released from alginate capsules into the digestive system !
B C DApink
yellow
blue
E
Visible alginate walls
F
75Klebsiella sp.SD12
67Pseudoalteromonas sp.SD438
60Pseudoalteromonas sp.SD424
55UnidentifiedSD466
53Stappia sp.SD335
51Erythrobacter citreusSD314
Total toxicity(STX eq.)
degradation (%)1,
Preliminary id(SSU rRNA)Isolate
PSP
1Isolates were grown in marine broth with (nM): STX (390), NEO (390), GTX-1 (636), GTX-2 (708), GTX-3 (234) & GTX-4 (210) over 5 d. Change in total PSP toxicity was determined by LC-MS-MS
4
Microbeads in faeces
4) Detoxifying shellfish – a possibility?The next stage was to develop a method of depurating shellfish by feeding a diet of microencapsulated bacteria. Bacterial isolate, SD12, shown to degrade PSP toxins in vitro was microencapsulated (50 – 100 µm) (Fig. I) & fed to scallops contaminated with PSP toxins. Empty capsules were used as a control (Fig. J).
This project was supported by the:Sixth Framework Programme of the European Community (SPIES-DETOX Collective Research Project 0302790-2)
I J
Scallops were sampled over time (Fig. K), dissected (Fig. L) and the hepatopancreas (HP) analysed for PSP toxins using LC-MS-MS.
5) Preliminary resultsVariation in toxin concentrations in the HP were observed between scallops fed microencapsulated bacteria and empty capsules, although inter-animal variability was high. Fig. M shows example MRM chromatograms for NEO.
Further trials will be undertaken to determine if a practical method for detoxifying cultured shellfish is possible!
LK
Time (minutes)14 16 18 20 22
m/z 316.1>220.2
NEO detected at 24 h in HP of scallop fed microencapsulated bacteria
NEO detected at 24 h in HP of scallop fed control capsules
Bacteria inside capsules
empty capsules
M
Both mussels & scallops ingested capsules over 2h. Smaller capsules (blue & pink beads) were readily ingested. A higher proportion of yellow capsules were detected in mussel pseudo-faeces & a higher proportion of blue capsules were detected in scallop pseudo-faeces, the alginate wall was still visible so capsules were not degraded prior to rejection (Fig E).
GH
Microencapsulated pink beads released in the
digestive organs
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