PNS PROBIO as food source vs yellow sno

reefluvrr · Oct 7, 2023

Hi @Kenneth Wingerter

I listened to your talk at Reefbum this week and am still trying to digest all the information given.

I would like to pick your brain about the use and benefit as a food source.
I googled up how shrimp farms are using Purple Non-Sulfur Bacteria and linked the article here:

Probiotic Effects of a Marine Purple Non-Sulfur Bacterium, Rhodovulum sulfidophilum KKMI01, on Kuruma Shrimp (Marsupenaeus japonicus)

Purple non-sulfur bacteria (PNSB) are used as probiotics in shrimp aquaculture; however, no studies have examined the probiotic effects of PNSB in shrimp at the gene expression level. In this study, we examined the effects of a marine PNSB, Rhodovulum sulfidophilum KKMI01, on the gene expression...

www.mdpi.com

Probiotic Effects of a Marine Purple Non-Sulfur Bacterium, Rhodovulum sulfidophilum KKMI01, on Kuruma Shrimp (Marsupenaeus japonicus)

Figure 5. Effect of R. sulfidophilum KKMI01 on the growth of kuruma shrimp (M. japonicus) in long-term outdoor 200-ton aquaria experiment. (a) Average body weights of control (n = 20) and PNSB-treated (n = 30) shrimp 70 days after the start of the experiments. Values represent means ± S.D. Asterisk (***) indicates significant difference with control at p < 0.001. (b) Photograph of control and PNSB-treated shrimp.

That is a significant difference in growth size for shrimps.
However, I wonder how one can attain similar results from corals. I mean there are other independent variables such as non ideal tank parameters. IF given ideal tank parameters though, can you correct what I think is occurring with PNS bacteria and our corals?

I have better success at reducing my PO4 level using Tropic Marin Carbon Source as opposed to acetic acid even while using PNS Probio as my bacteria.
I admit I would seed my tank about once a week with PNS Probio and Yellow Sno as Vitamin B source for the PNS Probio bacteria. Therefore I never really gave the PNS Probio a chance to prove itself as a constant food supply for the corals.

1. On the podcast you mentioned we have to regularly seed the bacteria in our tank because the bacteria grows best in anaerobic condition even though it can survive in aerobic conditions. Just out of curiosity, do you have an idea of how much better your PNS grows in anaerboic vs aerobic conditions? I am thinking of seeding brand new biocubes/biospheres with PNS bacteria. I am hoping this will create the PNS rossetts biofilm you mentioned from the slides. I plan to put 20 biocubes with sterile fresh water in a sterile jar seeding it with PNS bacteria as well as Yellow Sno. What do you think of this method of seeding the biocubes ? How many days should I let it sit in the sterile container?

2. I was surprised Yellow Sno was not mentioned in the podcast. Can just seeding with PNS Probio bacteria in our tank grow the bacteria without Vitamin B?

3. Just like how you mentioned PNS bacteria grows best in anaerobic condition. You mentioned PNS bacteria does not need Nitrogen because they can fix their own nitrogen from the air.
What do you think will make these bacteria grow best though? Lou Ekus from Tropic Marin mentioned corals prefer Ammonium over Nitrate. Fish excrete urea/ammonia. Does PNS grow better using ammonium vs having to Nitrogen fix from the air? The reason I am asking this is that if my reef tank is a "closed system", I don't think I can bring much Nitrogen gas into the anaerobic PNS bacteria laden biocubes. I have very low Nitrate levels

4. I understand how PNS bacteria lives and help feed in a SPS coral polyp. However can you help explain how these bacteria will feed my mushroom/soft corals? Do they reside somewhere in their soft tissue? Where does the PNS bacteria reside in an LPS coral? Does LPS grow well from bacterioplanktons as well?

As always thank you for helping me out with your products.

Kenneth Wingerter · Oct 14, 2023

reefluvrr said:
Hi @Kenneth Wingerter

I listened to your talk at Reefbum this week and am still trying to digest all the information given.

I would like to pick your brain about the use and benefit as a food source.
I googled up how shrimp farms are using Purple Non-Sulfur Bacteria and linked the article here:

Probiotic Effects of a Marine Purple Non-Sulfur Bacterium, Rhodovulum sulfidophilum KKMI01, on Kuruma Shrimp (Marsupenaeus japonicus)

Purple non-sulfur bacteria (PNSB) are used as probiotics in shrimp aquaculture; however, no studies have examined the probiotic effects of PNSB in shrimp at the gene expression level. In this study, we examined the effects of a marine PNSB, Rhodovulum sulfidophilum KKMI01, on the gene expression...

www.mdpi.com

Probiotic Effects of a Marine Purple Non-Sulfur Bacterium, Rhodovulum sulfidophilum KKMI01, on Kuruma Shrimp (Marsupenaeus japonicus)

Figure 5. Effect of R. sulfidophilum KKMI01 on the growth of kuruma shrimp (M. japonicus) in long-term outdoor 200-ton aquaria experiment. (a) Average body weights of control (n = 20) and PNSB-treated (n = 30) shrimp 70 days after the start of the experiments. Values represent means ± S.D. Asterisk (***) indicates significant difference with control at p < 0.001. (b) Photograph of control and PNSB-treated shrimp.

That is a significant difference in growth size for shrimps.
However, I wonder how one can attain similar results from corals. I mean there are other independent variables such as non ideal tank parameters. IF given ideal tank parameters though, can you correct what I think is occurring with PNS bacteria and our corals?

I have better success at reducing my PO4 level using Tropic Marin Carbon Source as opposed to acetic acid even while using PNS Probio as my bacteria.
I admit I would seed my tank about once a week with PNS Probio and Yellow Sno as Vitamin B source for the PNS Probio bacteria. Therefore I never really gave the PNS Probio a chance to prove itself as a constant food supply for the corals.

1. On the podcast you mentioned we have to regularly seed the bacteria in our tank because the bacteria grows best in anaerobic condition even though it can survive in aerobic conditions. Just out of curiosity, do you have an idea of how much better your PNS grows in anaerboic vs aerobic conditions? I am thinking of seeding brand new biocubes/biospheres with PNS bacteria. I am hoping this will create the PNS rossetts biofilm you mentioned from the slides. I plan to put 20 biocubes with sterile fresh water in a sterile jar seeding it with PNS bacteria as well as Yellow Sno. What do you think of this method of seeding the biocubes ? How many days should I let it sit in the sterile container?

2. I was surprised Yellow Sno was not mentioned in the podcast. Can just seeding with PNS Probio bacteria in our tank grow the bacteria without Vitamin B?

3. Just like how you mentioned PNS bacteria grows best in anaerobic condition. You mentioned PNS bacteria does not need Nitrogen because they can fix their own nitrogen from the air.
What do you think will make these bacteria grow best though? Lou Ekus from Tropic Marin mentioned corals prefer Ammonium over Nitrate. Fish excrete urea/ammonia. Does PNS grow better using ammonium vs having to Nitrogen fix from the air? The reason I am asking this is that if my reef tank is a "closed system", I don't think I can bring much Nitrogen gas into the anaerobic PNS bacteria laden biocubes. I have very low Nitrate levels

4. I understand how PNS bacteria lives and help feed in a SPS coral polyp. However can you help explain how these bacteria will feed my mushroom/soft corals? Do they reside somewhere in their soft tissue? Where does the PNS bacteria reside in an LPS coral? Does LPS grow well from bacterioplanktons as well?

As always thank you for helping me out with your products.

Kenneth Wingerter · Oct 14, 2023

I'll tackle these individually, from 1. to 4. Regarding 1., that would depend upon a number of factors. For example, to survive in an aerobic environment, these bacteria (Rhodopseudomonas palustris) are especially dependent upon a rich source of carbon in a form that they highly prefer. But remember that they are at a huge disadvantage in aerobic conditions, as they cannot photosynthesize in the presence of oxygen, nor can they perform denitrification or nitrogen fixation. In other words, they function just like any other chemoheterotroph that one might target with vinegar, vodka, etc. (except that their aerobic counterparts will generally outcompete them in an aerobic environment). Like most (if not all) purple non-sulfur bacteria, R. palustris can perform all four major modes of metabolism but is most competitive where it can perform photoheterotrophy (in other words, utilizing organic carbon as a source while building biomass, while utilizing light (i.e., photosynthesis) as a source of energy). In nature, such habitats may include the area just under the surface of the substrate where DO is low/absent but light is still penetrating. Or, say, within the microaerophilic regions in the gastrovascular cavities of coral polyps. You certainly could provide a suitable habitat using biomedia, and encourage them to immobilize on the medium by providing an adequate food source. YelloSno definitely would work as a food source (not just in terms of carbon and nutrients, but also for required trace elements). In terms of duration, the process would likely take place within hours rather than days; not sure of the material used to make the media you refer to, but when using a glass pumice material (such as MarinePure, I believe) complete immobilization may occur within as little as one hour. Finally, I'd use saltwater rather than freshwater during the seeding process; this will ensure that the bacteria are fully adapted to marine conditions as soon as the media are placed in the system (thereby preventing competitors from successfully invading the space while the PNSB are 'stunned').

Kenneth Wingerter · Oct 14, 2023

Regarding 2., I know right? Would have mentioned YelloSno in that discussion for sure, as I'm actually most proud of that product (yes, our bread and butter is the live PNS bacteria, but I'm hardly the first to promote their use in aquaculture, while on the other hand, PNS YelloSno is our own unique formulation and has gotten a lot of great reviews). But yeah, in that podcast we really got focused on the live products and the 1.5 hours blew right by haha. I'd love to do an episode somewhere/sometime just devoted to YelloSno and to marine snow generally. To your main question, different PNSB have different vitamin requirements. In the case of R. palustis, the specific requirements may differ between strains; our strain seems capable of growth while restricted to vitamin B10 ( which is technically more like a B vitamin precursor), but definitely grows better when afforded a complete B complex supplement. As an aside, I'd point out that the main benefit of YelloSno in terms of holistic reef nutrition is actually B12, which is required in the diets of both corals and zooxanthellae for their survival (though neither can synthesize their own). Even some bacteria cannot synthesize B12, including some nitrifying bacteria (Nitrobacter, for example). But I digress... Yes, the bacteria in PNS ProBio can survive in any aquarium environment so long as their basic vitamin requirements are met, whether those vitamins are supplied specifically by YelloSno or through some other food or more 'naturally' through production by some other microbe.

Kenneth Wingerter · Oct 14, 2023

Regarding 3., these bacteria undoubtedly prefer ammonia to nitrate. They definitely can use nitrate as a nitrogen source, and additionally benefit from nitrate as a source of metabolic energy since they're capable of performing denitrification. But you'll see overall fastest growth rates using ammonia as opposed to nitrate (or an organic nitrogen source, for that matter). And yes, they most definitely prefer having a source of ammonia at hand in the environment, as opposed to relying on nitrogen fixation. The short explanation for this is that nitrogen fixation is an energetically expensive process. That's why they have an 'ammonia switch-off' system in place, which halts nitrogen fixation if ammonia is already present in their environment. Similar regulation is observed in the other two PNSB species we offer, Rhodospirillum rubrum and Rhodobacter sphaeroides. Here's an interesting paradox to consider though... Even though these bacteria prefer an environment rich in ammonia/nitrate, they tend to flourish in nitrogen-limited environments such as normal, healthy, unpolluted coral reefs. This is because they have a distinct and critical advantage over their competitors in such conditions (i.e., their ability to make their own ammonia out of thin air). By the way, no need to worry about your closed system, they'd have plenty of access to nitrogen gas, as your tank is exposed to air which is composed of like 80% nitrogen gas. It'll find its way to your biomedia for sure. These porous biomedia for aquaria are a lot like coral rubble chunks, which actually provide an excellent anaerobic habitat for anaerobes. Some studies even suggest that this material is comparable or even better than a deep bed of fine sand due to the unrestricted movement of waters (and subsequent exchange of nutrients/wastes) between the chunks (the anaerobes live deep within the anaerobic core of the chunk). To be clear though, you'd have to be pretty close to completely bottoming out on NH4/NO3 before these guys would switch to N fixation. This is one reason why I think Rhodopseudomonas and other PNSB appears to be scarce in the core aquarium microbiome, despite being so abundant in normal, healthy, unpolluted coral reef habitats (see Fig. 1); while the aquarium environment is quite hospitable to them, the typical excess of NH4/NO3 promotes the growth of their competitors, which apparently wipe them out. That being said, I guess I misspoke on the show when I said something like 'these bacteria almost never show up in microbiome analyses.' Oregon State University professor Dr. Bouwma (who previously appeared on the show with Dr. Meyer of Aquabiomics) reached out to me to correct me on that, saying that Dr. Meyer does indeed find R. palustris in samples with some regularity, and believes those are usually systems where my products have been used.

Kenneth Wingerter · Oct 14, 2023

Regarding 4., there are three distinct parts of a coral where beneficial bacteria may reside: The mucus, the tissues, and the skeleton. Purple non-sulfur bacteria are most abundant in the tissues (interestingly, the other important nitrogen fixers on a reef, planktonic cyanobacteria, are most abundant elsewhere, the mucus)--more about that in my CORAL Magazine article on nitrogen fixation. Both hard and soft corals as well as mushroom anemones feed on these and other bacteria via their mucus. So, doesn't matter whether or not there are even tentacles, or how large the mouth is (yes, LPS feed in the same manner via mucus). For additional info on that, as well as the importance of bacterioplankton in the diets of corals in nature, check out this REALLY good article by Eric Borneman, who was way ahead of his peers on this subject.

reefluvrr · Oct 15, 2023

Kenneth Wingerter said:
In terms of duration, the process would likely take place within hours rather than days; not sure of the material used to make the media you refer to, but when using a glass pumice material (such as MarinePure, I believe) complete immobilization may occur within as little as one hour.

I went ahead and inoculated with PNS YellowSno and PNS Probio along with Tropic Marin Carbon source onto my new Maxspect Bio Balls. I let them simmer in a closed lid to make it an anaerobic environment in front of my window to try and grow the PNS rossetts biofilm. After a 6 hours, I placed them into the back of my sump with light and flow to hopefully keep the rossetts colony from collapsing. What do you think?

reefluvrr · Oct 15, 2023

Also thank you so very much for taking time from your busy schedule to help write well thought out and thorough responses to all my questions!

Kenneth Wingerter · Oct 15, 2023

reefluvrr said:
I went ahead and inoculated with PNS YellowSno and PNS Probio along with Tropic Marin Carbon source onto my new Maxspect Bio Balls. I let them simmer in a closed lid to make it an anaerobic environment in front of my window to try and grow the PNS rossetts biofilm. After a 6 hours, I placed them into the back of my sump with light and flow to hopefully keep the rossetts colony from collapsing. What do you think?

Can't see why this wouldn't work great. The remaining question now is whether or not the colonies are able to resist competitors. A swab of the media for an Aquabiomics test in, say, 6 months would be interesting.

Kenneth Wingerter · Oct 15, 2023

reefluvrr said:
Also thank you so very much for taking time from your busy schedule to help write well thought out and thorough responses to all my questions!

Thank you for your interest in our products, and also for your willingness to be adventurous and experiment with new applications.

NanPoriferan · Oct 30, 2023

@Kenneth Wingerter I have my PNS HomeGro Kit all ready for dosing after 1 month of culturing! Please see attached pic. I am happy to see the deep purple/red color. I have 1 question: how do I store it now? I would hate for the bacteria to slowly die. They are not under lights anymore and kept under room temp now.

I will also prevent contamination by wiping the inside of the tap with a q-tip with rubbing alcohol and letting that dry out before opening the tap, each time. But I am thinking to pour out some portions in disinfected bottles and cap-off, and use these before loading more from the main receptacle.

I am using this for my sponge tank so will be dosing this heavily. It's a 32g biocube. What would you consider a heavy dosing schedule?

My sponges are growing steadily from my bioplastics reactor which produces a lot of bacteria, but could definitely use the extra feeding from the PNSB.

Please let me know your thoughts!

Kenneth Wingerter · Oct 31, 2023

Looks good!

Is there any reason you can't keep it under the lights? That probably extends shelf life, as it provides a constant input of energy. Additionally, it may also inhibit certain competitors.

With or without light, however, your new culture should remain viable for 8 months (probably longer).

Room temp is just fine (after the culture has been fully grown out, of course).

Alcohol is alright for a disinfectant, but I recommend peroxide. Peroxide kills spores, whereas alcohol is mostly ineffective against spores. 70% alcohol, given enough contact time, is pretty lethal. Ironically, higher content, like in the 90s%, is actually less effective because it doesn't contain enough water to optimally denature proteins. So yeah, the household stuff (if you're going to use alcohol) is better than the stronger stuff.

One thing I like about alcohol, though, is that it is useful for washing peroxide or bleach off of items that may come into contact with your culture. (A little of bleach or peroxide is okay, but too much can adversely impact your culture.) For example, you can sterilize your bottles/caps with peroxide and then use alcohol as a sterile rinse. Q-tips would work, BTW, but a spray bottle can be really handy if you're needing to disinfect often. One thing I like about bottling as you described is that you can manage a lot of the handling away from the cube; in other words, there is less risk of contamination in the bulk source, the cube (because you open the valve only once every week or so, rather than every day).

I think the 'max' dose of 1.25 ml/gal is pretty heavy if done every day, but you certainly can safely feed more heavily if desired (particularly if you have a large population of bacterioplanktivores like sponges in the tank). I've personally seen sponges do extremely well on a diet based on PNS ProBio (alongside a little PNS YelloSno and OceanMagik phyto). Unfortunately I wasn't very consistent in the frequency or size of the ProBio dosage, but from my recollection of that system it was definitely a bit more on average than the recommended 1.25 ml/gal.

I agree, the bacteria from your bioplastics reactor might provide some nutrition, but the sponges could definitely use the extra feeding from the PNSB on account of its rich carotenoid content and probiotic properties. The fact that they may also perform denitrification and consume tough organics such as yellowing compounds and detritus could also make them desirable for a generously fed NPS reef system.

MBruun · Oct 31, 2023

As input for @reefluvrr question 1.
I have 12 liter of porous media (Seachem matrix) in my sump, seeded with PNS Substrate sauce. Added more than six months ago.
The media are in two large mediabags (large masks bags for Koi) covering most of the bottom of the sump as a 5cm layer.
I have no filtersocks and no fleece filter, but I see allmost no detritus nor on or in between the media (or in the tank). Don't know for sure what role the PNSB have in this, but before adding the seeded media chunks detritus use to build up in my sump and then I even used a fleece roller.
My tank is 700 liter sps dominated well stocked tank with heavy bioload. I added the seeded media like 6 months ago along with two bottled of PNS Probio (all I could get in Europe) and I add B-vitamins regularly hoping the PNSB to sustain a culture.

NanPoriferan · Oct 31, 2023

Kenneth Wingerter said:
Looks good!

Is there any reason you can't keep it under the lights? That probably extends shelf life, as it provides a constant input of energy. Additionally, it may also inhibit certain competitors.

With or without light, however, your new culture should remain viable for 8 months (probably longer).

Room temp is just fine (after the culture has been fully grown out, of course).

Alcohol is alright for a disinfectant, but I recommend peroxide. Peroxide kills spores, whereas alcohol is mostly ineffective against spores. 70% alcohol, given enough contact time, is pretty lethal. Ironically, higher content, like in the 90s%, is actually less effective because it doesn't contain enough water to optimally denature proteins. So yeah, the household stuff (if you're going to use alcohol) is better than the stronger stuff.

One thing I like about alcohol, though, is that it is useful for washing peroxide or bleach off of items that may come into contact with your culture. (A little of bleach or peroxide is okay, but too much can adversely impact your culture.) For example, you can sterilize your bottles/caps with peroxide and then use alcohol as a sterile rinse. Q-tips would work, BTW, but a spray bottle can be really handy if you're needing to disinfect often. One thing I like about bottling as you described is that you can manage a lot of the handling away from the cube; in other words, there is less risk of contamination in the bulk source, the cube (because you open the valve only once every week or so, rather than every day).

I think the 'max' dose of 1.25 ml/gal is pretty heavy if done every day, but you certainly can safely feed more heavily if desired (particularly if you have a large population of bacterioplanktivores like sponges in the tank). I've personally seen sponges do extremely well on a diet based on PNS ProBio (alongside a little PNS YelloSno and OceanMagik phyto). Unfortunately I wasn't very consistent in the frequency or size of the ProBio dosage, but from my recollection of that system it was definitely a bit more on average than the recommended 1.25 ml/gal.

I agree, the bacteria from your bioplastics reactor might provide some nutrition, but the sponges could definitely use the extra feeding from the PNSB on account of its rich carotenoid content and probiotic properties. The fact that they may also perform denitrification and consume tough organics such as yellowing compounds and detritus could also make them desirable for a generously fed NPS reef system.

@Kenneth Wingerter thanks for the fantastic reply!

Agree, limiting potential contamination of the main culture cube is definitely avoided by having dispensing bottle(s) prefilled. Great breakdown of when to use peroxide/alcohol also.

I will keep the culture under the daylight LEDs i use for my plants while indoors for the Winter, which is around 10hrs of daylight per day.

And the dosing, based on what you said I've decided I will dose @ 35 - 40ml daily for a month and see what results I get.

NanPoriferan · Oct 31, 2023

pics of some of the sponges I have in the tank. will post a month later so we can see the growth.

reefluvrr · Oct 31, 2023

MBruun said:
As input for @reefluvrr question 1.
I have 12 liter of porous media (Seachem matrix) in my sump, seeded with PNS Substrate sauce. Added more than six months ago.
The media are in two large mediabags (large masks bags for Koi) covering most of the bottom of the sump as a 5cm layer.
I have no filtersocks and no fleece filter, but I see allmost no detritus nor on or in between the media (or in the tank). Don't know for sure what role the PNSB have in this, but before adding the seeded media chunks detritus use to build up in my sump and then I even used a fleece roller.
My tank is 700 liter sps dominated well stocked tank with heavy bioload. I added the seeded media like 6 months ago along with two bottled of PNS Probio (all I could get in Europe) and I add B-vitamins regularly hoping the PNSB to sustain a culture.

This is so fascinating! Can you share with us how your tank is doing ever since using the mediabags with 12 L of media for the past 6 months? Are you seeing better colors on SPS, faster growth, lower pH from more metabolic activity of bacteria?, do you see stable nutrients PO4 and NO3 levels?

Thank you for sharing!

MBruun · Nov 1, 2023

Ny tank is doing a lot better now.
Better growth and color on the sps corals , but I cannot say for sure that it's only related to the media in the sump and PNS bacteria, as I have made other changes too, but a definitly think it play a major part. The tank is maturing after the tank almost crashed in summer 2022 due to 3 weeks of overheating - 80% of my corals died and everyting got covered in algeas for months. Think caused by microfauna got disrupted. NO3 became way over 100 and PO4 around 0.35. It took several months to get back on track. I have stabilised PH and also had to get a new light fixture. Two months ago I implemented a DND (Donovans Nitrate Destroyer) as my NO3 slowely rised to 40 so now my NO3 is stabile around 5 ppm .
I feed the 18 fish very well, detritus is almost not existing and i clean the glass like once a week, even with no filtersocks or roller filter. I can only imagine the PNSB is a major part of that, and I'll add more as soon as I get some.