Bacterial Analysis.

[ShaunW]

I am going to need these references, so to make my life easier I am posting here in this thread for later use.

Significance of pelagic bacteria as a trophic resource in a coral reef lagoon, One Tree Island, Great Barrier Reef
E. A. S. Linley and K. Koop
Marine Biology
Volume 92, Number 4
September, 1986


Abstract

Bacterial numbers and frequency of dividing cells (FDC) were estimated in surface waters at seven stations along a transeet across One Tree lagoon and reefs at 2 h intervals over one tidal cycle in May 1984 and once a day on 8 d in December 1984/January 1985. Cell numbers ranged between 1.24 and 13.10x105 ml-1, with an overall mean value of 4.81x105. There was a progressive depletion in standing stock from windward to leeward stations across the lagoon, also reflected in the FDC, which generally showed similar dynamic patterns to cell numbers throughout the study. Predator-free seawater (filtered through 2 m pore-size filters) and mucus-enriched incubations were also used to establish the relationship between the growth rate () and FDC for coral-reef populations. Average growth rates predicted from FDC values ranged from 0.062 to 0.174 h-1, which is equivalent to doubling times of 5.74 to 16.00 h, with an overall mean value of 11.5 h. These fast doubling times suggest that bacteria respond rapidly to pulses of enrichment as they float over the lagoon and reefs, characteristically achieving about one doubling per tidal cycle. This probably ensures that the bulk of labile organic matter and dissolved nutrients is conserved within the immediate coral-reef environment. Estimates of bacterial production also suggest that they may contribute up to 40% of total picoplankton production and about 25% of total microplankton production. Pelagic bacteria are therefore potentially a major food resource for benthic filter-feeders, especially for those mainly dependent on the smaller (pico) components of the plankton.

Source
http://www.springerlink.com/content/h628k70015088250/

 

[ShaunW]

another revelant article

Biomass of suspended bacteria over coral reefs
D. J. W. Moriarty
Marine Biology
Volume 53, Number 2
July, 1979


Abstract

The biomass of bacteria suspended in water flowing over coral reefs at Lizard Island and Yonge Reef (Northern Great Barrier Reef) was estimated by measurement of muramic acid. Values ranged from 20 mg C m-3 in the open water up to about 60 mg C m-3 over the reef flat. Direct counts of total numbers of free bacteria were made for comparison. Values of around 2.0x109 cells g-1 muramic acid showed that there was a good agreement between direct counts and muramic acid content of free bacteria in the open water. In samples containing suspended particulate matter, ratios of direct counts to muramic acid concentration were lower, because bacteria on particles could not be counted. Thus, these ratios were used to indicate the proportions of bacteria attached to particles. Changes in the biomass and numbers of bacteria were determined in water masses identified either by a drogue or fluorescein, as they moved across the reefs. In the zone on the outside of the reef, the number of free bacteria decreased compared to open sea water, but total biomass increased, showing that particulate matter containing bacteria was thrown up into suspension. About 50% of bacteria were attached to particles. Water flowing over the reef flats contained much particulate material with bacteria attached. Bacteria constituted between about 5 and 20% of particulate organic carbon.

Source
http://www.springerlink.com/content/w462282873n22q0k/

 

[ShaunW]

yet another.

Increase in the proportion of metabolically active bacteria along gradients of enrichment in freshwater and marine plankton: implications for estimates of bacterial growth and production rates
Paul A.del Giorgio and Gregg Scarborough
Journal of Plankton Research
Vol. 17, No. 10, pp. 1905-1924
1995


Abstract

It is generally recognized that a fraction of all bacterioplankton cells enumerated using conventional epifluorescence techniques is neither growing, dividing nor metabolically active, but the variation in the proportion of active cells among aquatic systems is not well understood. Here, we hypothesize that the proportion of metabolically active cells increases systematically along gradients of enrichment, and to test this hypothesis the number and proportion of metabolically active planktonic bacteria were investigated during the summer in a set of 24 temperate lakes, which span a considerable range in productivity. The tetrazolium salt 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was used as an indicator of cells with an active electron transport system. The total number of bacteria ranged from 1.88x106 to 7.70x106 ml–1, whereas the number of active cells was more variable and ranged from 0.37x106 to 2.18x106 ml–1 in the study lakes. The proportion of metabolically active cells ranged from 15 to 33%, and tended to increase with nutrient and chlorophyll concentrations, but not with dissolved organic carbon (DOC). Data on the number of total and active bacteria culled from the literature for marine, estuarine and freshwater systems show that the trends we measured in lakes are valid for pelagic systems in general. Over a broad range of aquatic systems, the total number of bacteria varied by three orders of magnitude, whereas the number of active bacteria varied by four orders of magnitude as system productivity increased. The proportion of active cells increased from ultraoligotrophic open ocean areas (<5%) to highly productive estuaries (>50%). Our results suggest that in most aquatic systems there is a pool of rapidly growing cells, embedded in a usually larger matrix of inactive bacteria, and that the relative size of the active and inactive pools varies systematically among bacterial populations along gradients of enrichment.

Source
http://plankt.oxfordjournals.org/cgi...urcetype=HWCIT

 

[ShaunW]

Christine, so after some initial testing of my tank water, my tank is starved for planktonic bacteria by 10-100 orders of magnitude depending on what natural reef we are comparing it to, .

I am now extremely interested to test the other tanks to contrast and compare.

 

[Spracklcat]

You starved-tank SPS keepers....LOL.

Yes, it will be, and I will offer my own tank water as an example of a way-overfed, underskimmed DSB system to see if there is a difference.

What method are you using to enumerate bacterial counts, what counts did you use, and does your method distinguish between live/dead/nonmetabolizing undead cells?

I have a huge number of articles to send you, less dealing with pelagic and more about coral surface bacteria, which are not pertinent to this phase of the study but are important and interesting nonetheless.

How do your corals grow (isn't that a nursery rhyme? oh wait never mind)? Are they SPS that will depend on plankton, or do you think that they are mainly photosynthetic?

Would be neat to set up isolated systems of organisms which "eat" plankton:

Tank 1: well lit, low bioload water
Tank 2: poor light low bioload
Tank 3 well lit high bioload
Tank 4 poor light high bioload

to see which is rate-limiting in growth, if either.

 

[ShaunW]

You starved-tank SPS keepers....LOL.

I guess so.

Yes, it will be, and I will offer my own tank water as an example of a way-overfed, underskimmed DSB system to see if there is a difference.

Cool. The more tanks the better. Fill 15mL sterile tubes with water twice a day (lights on and off). Do this twice a week for two weeks.

What method are you using to enumerate bacterial counts, what counts did you use, and does your method distinguish between live/dead/nonmetabolizing undead cells?

I am centrifuging at 4,000 rpm 15mL water samples. Removing the supernatant and counting the concentrated sample using a Petroff Hausser bacterial cell counter on the Phase contrast microscope.

My counts so far are on average: 50,000 bacterial cells/mL.

I have a huge number of articles to send you, less dealing with pelagic and more about coral surface bacteria, which are not pertinent to this phase of the study but are important and interesting nonetheless.

Please send them to me or add them to the thread here, just add the abstract I can find the full text.

How do your corals grow (isn't that a nursery rhyme? oh wait never mind)? Are they SPS that will depend on plankton, or do you think that they are mainly photosynthetic?

Would be neat to set up isolated systems of organisms which "eat" plankton:

Tank 1: well lit, low bioload water
Tank 2: poor light low bioload
Tank 3 well lit high bioload
Tank 4 poor light high bioload

to see which is rate-limiting in growth, if either.

I am of the firm believe that all SPS eat, and that their consumption will provide amino acids that they are unable to synthesize by photosynthesis alone. An example of one amino acid that is unable to be made directly via photosynthesis is aspartic acid.

 

[ShaunW]

Interactions between zooplankton feeding, photosynthesis and skeletal growth in the scleractinian coral Stylophora pistillata.
Fanny Houlbr?que, Eric Tambutt?, Denis Allemand and Christine Ferrier-Pag?s


Summary

We investigated the effect of zooplankton feeding on tissue and skeletal growth of the scleractinian coral Stylophora pistillata. Microcolonies were divided into two groups: starved corals (SC), which were not fed during the experiment, and fed corals (FC), which were abundantly fed with Artemia salina nauplii and freshly collected zooplankton. Changes in tissue growth, photosynthesis and calcification rates were measured after 3 and 8 weeks of incubation. Calcification is the deposition of both an organic matrix and a calcium carbonate layer, so we measured the effect of feeding on both these parameters, using incorporation of 14C-aspartic acid and 45Ca, respectively. Aspartic acid is one of the major components of the organic matrix in scleractinian corals. For both sampling times, protein concentrations were twice as high in FC than in SC (0.73 vs 0.42 mg P?1 cm?2 skeleton) and chlorophyll c2 concentrations were 3?4 times higher in fed corals (2.1?0.3 ?g cm?2). Cell specific density (CSD), which corresponds to the number of algal cells inside a host cell, was also significantly higher in FC (1.416?0.028) than in SC (1.316?0.015). Fed corals therefore displayed a higher rate of photosynthesis per unit area (Pgmax= 570?60 nmol O2 cm?2 h?1 and Ik=403?27 ?mol photons m?2 s?1). After 8 weeks, both light and dark calcification rates were twofold greater in FC (3323?508 and 416?58 nmol Ca2+ 2 h?1 g?1 dry skeletal mass) compared to SC (1560?217 and 225?35 nmol Ca2+ 2 h?1 g?1 dry skeletal mass, respectively, under light and dark conditions). Aspartic acid incorporation rates were also significantly higher in FC (10.44?0.69 and 1.36? 0.26%RAV 2 h?1 g?1 dry skeletal mass, where RAV is total radioactivity initially present in the external medium) than in SC (6.51?0.45 and 0.44?0.02%RAV 2 h?1 g?1 dry skeletal mass under dark and light conditions, respectively). Rates of dark aspartic acid incorporation were lower than the rates measured in the light. Our results suggest that the increase in the rates of calcification in fed corals might be induced by a feeding-stimulation of organic matrix synthesis.

Extracted from:
Journal of Experimental Biology 207, 1461-1469 (2004)

If you read the paper there is a treasure trove of information within.

For example:

Feeding has also been shown to enhance sketetal growth, suggesting that corals allocate a high proportion of the energy brought by food to calcification processes. It is important to note that calcification is also a dual process, involving the secretion of an organic matrix and the deposition of a CaCO3 fraction. The presence of an organic matrix in coral skeletons is widely documented and is considered an essential prerequisite in the formation of a biomineral structure. This matrix potentially plays key roles in various processes such as crystal nucleation and growth, crystal size and orientation and regulation of skeletal formation. Cuif et al (1999) demonstrated that the composition of the matrix was different between symbiotic and asymbiotic corals, and Allemand et al. (1998) suggested that heterotrophy is a source of aspartic acid, once of the major components of the coral matrix.

From another part of the paper.

..... Allemand et al. (1998) also showed that no aspartic acid pool was present inside the coral tissue, suggesting the need for a constant supply from an exogenous source. By using 14C-aspartic acid as a precursor for organic matrix synthesis, we measured a higher incorporation of this amino acid into the organic matrix of fed corals.............

Feeding might therefore have enhanced the construction of the organic matrix by (i) supplying additional input of energy, espercially for the dark processes. Under high plankton concentrations...........uptake of organic carbon (and hence energy) may be significant and could provide some energy for calcium/proton exchange at night. Alternatively, the larger biomass of fed corals may have provided larger energy stores for dark processes. Thus, feeding might have (ii) directly provided the necessary 'external' amino acids and/or (iii) indirectly increased photosynthesis and therefore the supply of 'autotrophic' amino acids.

So in conclusion from the thoughts above, we have a internally synthesized pool of amino acids created via photosynthesis and secondary metabolism and an amino acid that is naturally limited in coral tissue (no storage) but is also the most abundant within the coral organic matrix.

And this amino acid must be supplied externally.

 

[sanjay]

If you are interested I can send you water samples from the Penn State tank.. its a mostly SPS dominated reef with some softies that has been running for 7 years.

sanjay.

 

[ShaunW]

If you are interested I can send you water samples from the Penn State tank.. its a mostly SPS dominated reef with some softies that has been running for 7 years.

sanjay.

Sanjay, sure that would be great! Would you be able to obtain sterile 15mL tubes from any of the labs at Penn State? or would you like me to send them to you?

I think a total of eight samples over the course of two weeks would be good for now, .
Shaun.

 

[Spracklcat]

Sanjay/Shaun,

I have the blessing of my company to collect samples and have them post paid for return (as long as you sign a silly legal waver) so if you both like I can mail Sanjay and whomever else "sample packets" containing tubes and instructions and post-paid return mailers, and have them split the samples, taking two at a time and sending one of each time point to you, Shaun, and to me as well.

Up to you guys.

Christine

 

[sanjay]

Sanjay/Shaun,

I have the blessing of my company to collect samples and have them post paid for return (as long as you sign a silly legal waver) so if you both like I can mail Sanjay and whomever else "sample packets" containing tubes and instructions and post-paid return mailers, and have them split the samples, taking two at a time and sending one of each time point to you, Shaun, and to me as well.

Up to you guys.

Christine

That will be great. save me the trouble of chasing down people in other labs to get the sterile tubes.

I also have a 55G soft coral tank that has been running now for 13 years with minimal waterchanges and no skimmer for almost 7 years. It has no SPS in it, mostly soft corals and anemones with one small frogspawn and its completely full of corals that have been growing in there for 10+ years or more. I can get you samples of this tank too if you like.


sanjay.

 

[ShaunW]

That will be great. save me the trouble of chasing down people in other labs to get the sterile tubes.

I also have a 55G soft coral tank that has been running now for 13 years with minimal waterchanges and no skimmer for almost 7 years. It has no SPS in it, mostly soft corals and anemones with one small frogspawn and its completely full of corals that have been growing in there for 10+ years or more. I can get you samples of this tank too if you like.


sanjay.

I would definately like samples of the 55g, .

Christine, that's great on your company providing the postage.

At this point I think I have enough tanks to test, a wide range of mature tanks. Alot of work is waiting for me, :lol: .

So let's leave it at this number of tanks for now, pending what the test results provide.

 

[Spracklcat]

Sounds good. If everyone that is collecting samples can PM me addresses, I will mail out collection kits. Again, I will warn you there is a legal disclaimer a very annoying company person made me put in there, I apologize. Each person just let me know how many tanks are being sampled so I know what to mail.

Shaun/Everyone:

Another thing I was thinking about yesterday. the amount of pelagic bacteria is going to be affected by the quantity, frequency, and timing of water changes. We need to know from everyone their water change schedule, and ideally the samples should be taken BEFOREwater changes at the point where hypothetically the bacterial load is the strongest and most stable.

Christine

 

[ShaunW]

Shaun/Everyone:

Another thing I was thinking about yesterday. the amount of pelagic bacteria is going to be affected by the quantity, frequency, and timing of water changes. We need to know from everyone their water change schedule, and ideally the samples should be taken BEFOREwater changes at the point where hypothetically the bacterial load is the strongest and most stable.

Christine

Christine, why do you think this? just wondering? if there is any evidence of such bacteria fluctuations due to water changes. There is plenty of evidence in the scientific literature that the bacterial load fluctuates the most with nutrient availability.

But I do agree that at some point we would need a description of each tank in the study, and the husbandry of each.

 

[Spracklcat]

I think it will somewhat directly after a water change. If you take a smaple, then replace 50% of the water in the tank with new ASW, then sample again i would expect the bacterial load to be 50% of pre-change levels, or slightly higher. After some time though, the pelagic bacterial load would return to its stable level from benthic organisms entering the water or from replication of remaining pelagic organisms. In the wild, there is a continuous flow--there is not influx of "pure" water with no bacteria.

Does this make sense? Do you agree? And yes, I would agree of course that levels will change with nutrient level, assuming no large influx of new water.

 

[ShaunW]

I think it will somewhat directly after a water change. If you take a smaple, then replace 50% of the water in the tank with new ASW, then sample again i would expect the bacterial load to be 50% of pre-change levels, or slightly higher. After some time though, the pelagic bacterial load would return to its stable level from benthic organisms entering the water or from replication of remaining pelagic organisms. In the wild, there is a continuous flow--there is not influx of "pure" water with no bacteria.

Does this make sense? Do you agree? And yes, I would agree of course that levels will change with nutrient level, assuming no large influx of new water.

Oh...., OK, yes I would agree.

 

[Spracklcat]

Randy/Prattreef:

Can you PM me your address so we can get you on board too?

Thanks

 

[cali_reef]

Christine, did the containers go out yet?

 

[ShaunW]

Christine, did the containers go out yet?

Not yet. Behind the scenes we have been trying to come to a unified procedure to get the most "bang for our bacteria", :lol: .

Christine, we really should post up our behind the scenes conversations, just to document were we are. Is that OK with you if I do it?

 

[Spracklcat]

No Shaun, I'll pout if you do that (LOL).

Of course it is ok.