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Old 01/04/2016, 07:45 AM   #2510
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But that has to be "For example, Cytophaga gene sequences were associated with high-molecular-weight dissolved organic carbon", and given the title of the paper, it's not too difficult to infer what sort of DOC stimulates cytophaga growth... But I still went and looked up the abstract, natch:

Originally Posted by Gradients of coastal fish farm effluents and their effect on coral reef microbes
Coastal milkfish (Chanos chanos) farming may be a source of organic matter enrichment for coral reefs in Bolinao, Republic of the Philippines. Interactions among microbial communities associated with the water column, corals and milkfish feces can provide insight into the ecosystem's response to enrichment. Samples were collected at sites along a transect that extended from suspended milkfish pens into the coral reef. Water was characterized by steep gradients in the concentrations of dissolved organic carbon (70-160 uM), total dissolved nitrogen (7-40 uM), chlorophyll a (0.25-10 ug/l), particulate matter (106-832 ug/l), bacteria (5 x 10^5 - 1 x 10^6 cells/ml) and viruses (1-7 x 10^7 cells/ml) that correlated with distance from the fish cages. Particle-attached bacteria, which were observed by scanning laser confocal microscopy, increased across the gradient from < 0.1% to 5.6% of total bacteria at the fish pens. Analyses of 16S rRNA genes by denaturing gradient gel electrophoresis and environmental clone libraries revealed distinct microbial communities for each sample type. Coral libraries had the greatest number of phyla represented (range: 6-8) while fish feces contained the lowest number (3). Coral libraries also had the greatest number of 'novel' sequences (defined as < 93% similar to any sequence in the NCBI nt database; 29% compared with 3% and 5% in the feces and seawater libraries respectively). Despite the differences in microbial community composition, some 16S rRNA sequences co-occurred across sample types including Acinetobacter sp. and Ralstonia sp. Such patterns raise the question of whether bacteria might be transported from the fish pens to corals or if microenvironments at the fish pens and on the corals select for the same phylotypes. Understanding the underlying mechanisms of effluent-coral interactions will help predict the ability of coral reef ecosystems to resist and rebound from organic matter enrichment.
Milkfish are widely aquacultured in tropical Asia and normally feed on phyto and pods in the wild, but farmed fish are fed high-protein pellets or even given chopped-up fish and chicken guts as supplemental feed.

That's as close to a smoking gun as I can manage: ostis are associated with cyotphaga, and cytophaga are associated with recalcitrant high molecular weight dissolved organic carbon from high-protein fish poo. If flavos are the Bacteroidetes adapted to consume the remains of green algae, then marine cytophaga may be on the other end of the spectrum: a hardcore protein specialist. It looks like the ecological niche they fill is eating what's left after other bacteria have eaten all the good stuff, plus as noted they can consume bacterial cell walls, so when the good stuff runs out, cytophaga can eat the dead bacteria, too. Cytophaga are the CUC for the CUC for the CUC, if you will. That might explain this...

12/30/2015, 10:59 PM #2421
Yes. I had dry rock too.

It's a key ingredient IMO.
12/31/2015, 05:17 AM #2429
They survey I did with Monty blew my dry rock theory off the table, but it's still a likely factor in the equation.
The good stuff dies when LR dries, and the rock remains moist long enough for decomposition to begin, so it's covered in partly-degraded proteins and dead bacteria. Cytophaga heaven.

And being the CUC of last resort tracks with cytophaga being able to consume keratin, a highly recalcitrant protein found in turtle shells, hooves, feathers, fur, and your hair, tongue, skin, and fingernails -- and, interestingly, feather meal is sometimes used as a cheap source of protein in fish food pellets and can constitute up to 5-10% of milkfish feed pellets. And there are a lot of birds nesting on Saint Paul's Rocks, which presumably means a lot of feathers end up in the water... Hmmm... Are there any known inputs of keratin in our systems? That would be an interesting correlation.

But I keep thinking about all the detritus that comes out when you stir a sand bed. All that indigestible detritus... It's been through several different creatures, probably some of them more than once, and still the bacteria haven't been able to break it down. That's the very definition of recalcitrant organic carbon, and that's what gets buried in a shallow DT sand bed that's stirred by macrofauna. Indeed, burying that stuff is really pretty much the point of DT SSBs. That's why nobody wants to acknowledge that DT SSBs are a broken implementation of Shimek's DSB tech -- an automated landfill burying detritus that settles to the bottom of a DT seems rather ingenious and is undeniably convenient. Out of sight, out of mind.

I gather many hobbyists are under the impression that burying detritus in a DT SSB feeds beneficial denitrifying bacteria. As should be apparent by now, nothing could be further from the truth. Bacteria that specialize in denitrification are just one small part of a diverse population, and they tend to limit themselves. The less NO3 there is available, the less effectively denitrifiers can compete for organic carbon. That's why LR and Old School UGF setups won't zero out NO3 by themselves, for example, and that's why DyMiCo filters, which are built to maintain the proper redox conditions to favor denitrifying bacteria, have to inject labile DOC directly into the sand bed to help them do their job. Same thing with sulfur denitrators: as NO3 levels get low, flow through the media has to be increased to counteract the dropoff in efficiency that results from the denitrifying bacteria being at a competitive disadvantage. And because denitrifying bacteria live in the transition zone where hypoxia becomes anoxia, aerobic and facultatively aerobic saprophytes, including vibrio, get first crack at any labile organic carbon that reaches the substrate. (...I suspect this is why a lot of hobbyists have trouble keeping shrimp, incidentally: benthic livestock in general, and especially animals that like to root around in the sand looking for tasty morsels to eat, are directly exposed not only to high levels of potentially pathogenic bacteria but also to chemicals released by the bacterial decomposition of detritus, such as ammonia and weak organic acids, that stress shrimp and make them vulnerable to opportunistic infections. The same thing happens in wild populations of lobsters where the benthic sediments are enriched with organic matter from river discharges and algae blooms, for example, and it's a common problem with aquacultured shrimp, as well. Chitinolytic -- chitin-disassembling -- bacteria associated with black spot/brown spot disease include species from the genera aeromonas, pseudomonas, flavobacterium, and vibrio... Ring any bells? Many vibrio species show chemotaxis towards chitin, including V. cholerae, and as noted some can grow using chitin as their only source of organic carbon.)

Originally Posted by natas
My Dino disappear at night much like everyone else. My understanding here is that they go into the water column at night. How do we explain how they always end up in the same place the next morning? I literally have spots on the sand that show no Dino. The spots that are showing disappear at night and look the same (sometimes bigger) the next morning.
Dinos return to the same spots every morning because that's where the food is. The sand bed bacteria are not a single, uniform community but rather a patchwork distribution of competing populations that shift in accordance with the available food in any particular spot, and I hypothesize that the spots where cytophaga become dominant are the spots where ostreopsis dinos set up shop. Indeed, it may be enough that cytophaga becomes dominant among the bacteroidetes/CFB group bacteria without displacing gamma-pros as the numerically dominant group overall... In any event, once the dinos have seized a piece of ground and established a bacteria farm there, they do their best to hold on to it, and if the dino population grows, their bacteria farms have to grow, too.

Originally Posted by DNA
Did one more of those wonderful and elaborate tests of mine.
I placed a square plastic on the sandbed and left it there for couple of days.

Yesterday I removed it and today I have a perfect square free of dinos, but edged by dense mat of dinos.

Covering the sand would cause it to remain anoxic, which would give obligate and aerotolerant anaerobes an advantage over facultative anaerobes. The shift in the bacteria population destroyed that part of the ostis' bacteria farm -- no food, no dinos. Either the dinos' food bacteria are dead, or they're reduced in numbers too much to support a bloom on the sand above, or they've thrown in their lot with their fellow heterotrophs and will no longer refrain from eating dinos. And if you used transparent plastic, it may be that the phototrophic niche in the surface layer of the sand has been taken over by photoheterotrophs. If you left a transparent piece of plastic in place long enough, I suspect you'd eventually see cyano growing on the sand, as many species are facultative anaerobes and can use sulfur as a terminal electron receptor at night.

While they may only be able to get their farms started by opportunistically exploiting a cytophaga-dominated benthic bacterial community, if the benthic dino holobiont includes vibrio, that could explain how they kill corals...

Originally Posted by Regulation of microbial populations by coral surface mucus and mucus-associated bacteria
Caribbean populations of the elkhorn coral Acropora palmata have declined due to environmental stress, bleaching, and disease. Potential sources of coral mortality include invasive microbes that become trapped in the surface mucus and thrive under conditions of increased coral stress. In this study, mucus from healthy A. palmata inhibited growth of potentially invasive microbes by up to 10-fold. ... This result suggests that coral mucus plays a role in the structuring of beneficial coral-associated microbial communities and implies a microbial contribution to the antibacterial activity described for coral mucus. ...

This study shows that mucus collected from Acropora palmata has antibiotic activity against (1) Gram-positive and Gram-negative bacteria, (2) a number of potentially invasive microbes (including microbes from Florida Keys canal water, African dust, and surrounding sea water), and (3) a pathogen implicated in white pox disease of A. palmata. This result suggests that healthy A. palmata employ a biochemical mechanism for disease resistance that may act as a primary defense against pathogens. In contrast, mucus collected from A. palmata during a period of increased water temperature did not show significant antibiotic activity against the same suite of sources and tester strains, suggesting that the protective mechanism employed by A. palmata is lost when temperatures increase. ...

Collectively, these results suggest that coral mucus provides a hostile environment for some bacteria and a nurturing environment for others, illustrating that the mucus plays an important role in structuring microbial communities on the coral surface. ...

The antibiotic properties of coral mucus, and the potential for mucus to select a discrete set of commensal bacteria, were lost at increased temperatures during a bleaching event. Mucus was taken from corals sustained at a mean daytime sea surface temperature of 28 to 30C for 2 mo prior to collection. Vibrios were the predominant species cultured from the mucus of apparently healthy Acropora palmata tissue during this event. Vibrios were also predominant in the water column during this period, representing 85% of the cultured isolates. Less than 2% of bacteria isolated from the surface of A. palmata during this period produced antibiotic activity. These findings illustrate a temporal shift in the protective qualities of coral mucus, and a composition shift from beneficial bacteria to vibrio dominance under conditions of increased temperature. Vibrios present during this event included those involved in temperature dependent bleaching of corals, such as Vibrio shiloi and V. coralyticus as well as numerous vibrios known to be opportunistic to other marine organisms. ... However, as mucus was collected from apparently healthy coral tissue, and not bleached tissue, this provides evidence that a community shift to vibrio dominance may occur prior to zooxanthellae loss.
Vibrio bad. But other bacteria good.

So I went looking for them...

Originally Posted by Antimicrobial properties of resident coral mucus bacteria of Oculina patagonica
The inhibitory properties of the microbial community of the coral mucus from the Mediterranean coral Oculina patagonica were examined. Out of 156 different colony morphotypes that were isolated from the coral mucus, nine inhibited the growth of Vibrio shiloi, a species previously shown to be a pathogen of this coral. An isolate identified as Pseudoalteromonas sp. was the strongest inhibitor of V. shiloi. Several isolates, especially one identified as Roseobacter sp., also showed a broad spectrum of action against the coral pathogens Vibrio coralliilyticus and Thallassomonas loyana, plus nine other selected Gram-positive and Gram-negative bacteria. Inoculation of a previously established biofilm of the Roseobacter strain with V. shiloi led to a 5-log reduction in the viable count of the pathogen within 3 h, while inoculation of a Pseudoalteromonas biofilm led to complete loss of viability of V. shiloi after 3 h. These results support the concept of a probiotic effect on microbial communities associated with the coral holobiont.

Coral bleaching is caused by disturbance of the mutual symbiotic relationship between algae (zooxanthellae) within the tissues of the coral animal. The symbiosis can be disrupted due to a range of external environmental physical and toxic stressors, which can act either alone or together. In addition to these widely accepted factors in coral bleaching, the hypothesis that bacterial infection may also trigger bleaching was developed as a result of the study of interactions observed since 1997 between the coral Oculina patagonica and the bacterium Vibrio shiloi. Oculina patagonica is an invasive species of the eastern Mediterranean Sea, first recorded in 1993. The infection and bleaching of O. patagonica by V. shiloi was first described by Kushmaro et al. (1996, 1997) and shown to be temperature dependent; it does not occur at 1620C, but is stimulated at temperatures above 25C. ... Once in the coral tissue, the pathogen multiplies and produces extracellular toxins that block photosynthesis, bleach and lyse the zooxanthellae. In addition to the zooxanthellae, the tissue of healthy corals and their secreted mucus layer supports a diverse community of other microorganisms, including bacteria, archaea, fungi, and viruses. Since 2004, it has not been possible to recover V. shiloi from healthy or diseased corals. Ainsworth et al. (2008) confirmed the absence of V. shiloi during the annual bleaching event in 2005. ... A possible explanation for this disappearance was suggested in the Coral Probiotic Hypothesis proposed by Reshef et al. (2006) and developed by Rosenberg et al. (2007). This proposes that the abundance and types of microorganisms associated with corals change in response to global environmental changes such as temperature, allowing the coral to adapt to new conditions by altering its population of specific symbiotic bacteria. ...

Several strains of bacteria cultured from O. patagonica showed antagonistic activities towards a range of marine and terrestrial pathogens, with 5.8% of the isolates active against V. shiloi, the former bleaching pathogen of this coral. Previous studies have shown similar Alpha- and Betaproteobacteria to be present in healthy O. patagonica. Similarly, Ritchie (2006) found that almost 20% of the cultured bacteria from the Acropora palmata coral in the Caribbean displayed antibiotic activity, including towards the causative agent of white pox disease. Not surprisingly, when the antibiotic producers that were isolated from O. patagonica mucus were tested against each other, no inhibition occurred, suggesting that the strains isolated could be resistant to these antagonistic mechanisms, and therefore may be better adapted to life in the mucus. ...

The bacteria had different strengths and spectra of activity against the various test bacteria, suggesting that the microbial interactions in the mucus are diverse and complicated. The Roseobacter isolate had the broadest range of activity and inhibited a range of terrestrial and marine pathogens. There are numerous reports of antibiotic production by bacteria belonging to the Roseobacter clade. Bruhn et al. (2005) and Rao et al. (2005, 2006) demonstrated the selective advantages that members of the Roseobacter clade have in colonizing the surface of algae and outcompeting previously established biofilms. ... The strongest inhibitor of V. shiloi and the other coral pathogens tested was the strain JNM12, identified as Pseudoalteromonas. This genus is known to produce a range of bioactive compounds and strain JNM12 shows red-brown pigmentation of its colonies, which has previously been associated with antibiotic production. ...

Both cells and culture supernatants of Pseudoalteromonas JNM12 and Roseobacter JNM14 inhibited the growth of V. shiloi. ...

Our results support the conclusions of Ritchie (2006) that coral mucus and its associated microorganisms play an important role in promoting beneficial microbial communities. From the experiments performed here, it is clear that different coral bacteria may contribute differently to the protection of the coral. It is unlikely that one or two coral mucus isolates can fully explain the development of immunity to a disease, but a 'cocktail' of bacteria with different antibiotic properties could together prevent infection by a pathogen such as V. shiloi.
Roseobacter?!? That name has come up more than once in association with toxic dinoflagellates...

Originally Posted by Bacteria Associated with Toxic Clonal Cultures of the Dinoflagellate Ostreopsis lenticularis
Aeromonas, Alteromonas, Bacillus, Cytophaga, Flavobacterium, Moraxella, Pseudomonas, Roseobacter, and Vibrio are the bacterial genera most frequently associated with toxic dinoflagellates.
Originally Posted by Phylogenetic and functional diversity of the cultivable bacterial community associated with the paralytic shellfish poisoning dinoflagellate Gymnodinium catenatum
The bacterial flora of G. catenatum generally mirrors that found associated with other dinoflagellates, being dominated by the Alphaproteobacteria (principally the Rhodobacteraceae -- frequently referred to as Roseobacter clade)
...but if they can protect corals, clearly that deserves a closer look.

Originally Posted by Production of Antibacterial Compounds and Biofilm Formation by Roseobacter Species Are Influenced by Culture Conditions
Bacterial communities associated with marine algae are often dominated by members of the Roseobacter clade... Nine of 14 members of the Roseobacter clade, of which half were isolated from cultures of the dinoflagellate Pfiesteria piscicida, produced antibacterial compounds. ... We hypothesize that the ability to produce antibacterial compounds that principally inhibit non-Roseobacter species, combined with an enhancement in biofilm formation, may give members of the Roseobacter clade a selective advantage and help to explain the dominance of members of this clade in association with marine algal microbiota. ...

Several studies have found that members of the Roseobacter clade inhibit other bacteria, and this may contribute to their dominance among alga-associated bacteria. ... Indeed particle-associated members of the Roseobacter clade are 13 times more likely to produce antimicrobial compounds than are free-living members. Furthermore, while growing in a biofilm, a member of the Roseobacter clade was able to prevent the growth of other bacteria on surfaces. ...

None of the Roseobacter clade strains tested were sensitive to filtered culture supernatants containing the antibacterial activity produced by either Silicibacter sp. strain TM1040 [a laboratory strain of roseobacter isolated from the toxic dinoflagellate Pfisteria piscicida] or Phaeobacter strain 27-4 [a roseobacter that produces an antibiotic called tropodithietic acid, or TDA, and is used as a probiotic in marine aquaculture]. This is in contrast to the non-Roseobacter marine species tested, many of which were sensitive to the filtered supernatant. Vibrio anguillarum 90-11-287, Pseudomonas elongate, "Spongiobacter nickelotolerans," environmental and clinical strains of Vibrio cholerae, V. coralliilyticus, V. shiloi, and a Halomonas sp. (all members of the gamma-Proteobacteria) were sensitive to the compound(s). ...

Vibrio coralliilyticus and V. shiloi are important coral pathogens causing coral bleaching, and both were inhibited by Silicibacter sp. strain TM1040 and Phaeobacter strain 27-4. The coral polyp is protected by a mucus layer that is populated by alpha-Proteobacteria group bacteria, and bacteria taxonomically related to Silicibacter sp. strain TM1040 are associated with corals. One may therefore hypothesize that Roseobacter species play a role in preventing coral bleaching, and, indeed, antibacterial activity in coral extracts which inhibit members of the Vibrionaceae has been detected, supporting this hypothesis.
It has been at the back of my mind since Day 1 that the dinos-as-farmers hypothesis needs a villain -- or a hero, from our perspective. That is to say, if the dinos are defending their farms to keep them from being overrun by unfriendly bacteria, what bacteria are they trying to keep out?

Or to put it another way: What bacteria were in Montireef's skimmate when he nuked his dino bloom?

Bacteria that don't seem to get along well with gamma-proteobacteria, especially vibrio, and which are known for invading and outcompeting existing biofilms look like awfully good candidates for the active ingredient in the Montireef Protocol.


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