The ability to ‘scale up’ larger cultures from an initial purchase is one of the highest criteria I have for evaluating the worth of any product advertised as ‘live.’ Truly efficacious probiotic products should be shelf stable enough so that the purchased culture is ready to replicate when favorable conditions are present. They must also be uncontaminated so as to ensure the customer is receiving the species, and only the species, advertised on the packaging. The rewards of probiotic products which can deliver on these criteria are the biggest ‘bang for the buck’ that any hobbyist or commercial aquaculturist could ask for: The ability to grow your own, and scale up to fit use. I am happy here to report my experiences working with Hydrospace’s PNS HomeGro™ kit!
Purple non-sulfur bacteria provide a myriad of benefits to the home aquariums, commercial aquaculture farms, aquaponics farms and terrestrial farms alike. These benefits can range from wastewater mitigation to biofiltration to stabilizing phosphate/nitrate levels in soil to the suppression of Vibrio sp. It is therefore understandable that those who enjoy the product will enjoy learning about how to grow it on-site and apply the probiotic on a much larger scale.
With my Hydrospace’s PNS Grow Kit, I received the following:
1x 4-gallon growth vessel (flexible plastic)
1x spout for growth vessel (like a lemonade dispenser)
1x bag of special Hydrospace PNS growth media
1x 6-oz inoculant bottle of Rhodobacter capsulatus.
To start the inoculation process, I assembled/purchased the following additional items:
5% bleach (no splashless, scented or other nonsense)
90% rubbing alcohol (to cleanse all sins)
Clean cooking pot
I also saw the need to purchase several gallons of sterilized spring water, as the tap in Orange county, Florida is often rich in organic matter, high TSS and contains the spores of various cyanobacteria (Anabaena, Microcystis etc.).
Staying sterile at home is a challenge but not impossible in practical terms. Late at night, I donned surgical gloves and began cooking my witch's brew. I first sterilized a freshly dishwashed cooking pot with rubbing alcohol, and then poured in four gallons of bottled spring water, put the burner on high. I left the covered pot to boil and then added my special Hydrospace PNS Growth Media…the solution turned a delightful yellow and smelled of a strange and benevolent aroma. While the solution reached boiling temperature, I switched my attention to the flexible plastic growth vessel. As per the instructions, I placed a capful of 5% bleach into a cup of hot water and poured the hot water into the vessel. The heat soon facilitated the expansion of the vessel allowing me to unfold it to its true volume while violently shaking the bleach solution around every inch of exposed surface area. After I was convinced all surfaces had made contact with the bleach solution, I poured as much of it out as I could…do NOT rinse the vessel to remove leftover bleach…whatever residual left behind is far less risk than the chance of bacterial infection…though distilled water could be used those needing to rinse.
After letting the media solution boil for 4 minutes, I allowed it to cool for 2 hours (it is ideally poured into the culture vessel at around 170F). There is a cautious window between being too hot and melting the vessel and too cool as to risk contamination…this could be avoided in a glass vessel which could withstand boiling heat. Rubbing alcohol was used to sterilize the funnel and the outside of the growth vessel and the semi-hot media solution was carefully poured in…expanding the vessel further. Once the vessel was nearly full, I used alcohol to sterilize the cap again before gently squeezing the vessel so as to make any residual air escape. Rhodobacter capsulatus grows fastest under anaerobic conditions and air is to be avoided in excess. The growth vessel was sealed and allowed to cool overnight…no need to cook all the bacteria after all that work sterilizing!
Inoculation Achieved! Once the sterilized media broth cools to the same temperature as the inoculant bottle, both vessel caps received a saturating spray with rubbing alcohol. The transfer was done in under 5 seconds and the culture vessel was promptly sealed.
The vitamin-B12 yellow media was immediately stained a prominent red as the bacteria fused into their new domain. Quickly massaged the bag to encourage complete mixing and done with a long work week. Now time to wait for microbial reward! Wanted to get a better shot of the two liquids mixing but was difficult to achieve without compromising sterility-- alas, next time!
Temperature and Lighting. These are the questions. I initially thought I should keep my cultures in an outside work shed to enjoy Florida's warmth. The goal is a stable 30C (86F). Stable. After the first night, I decided that I should bring the cultures indoors where I did not have to worry about excess heat in the day. Decided on using a human-grade heat pad (without auto shut off). We use these for reptiles and they come with many layers of protective insulation so as to not melt the culture vessel. The pad has settings 1 through 8 so I have it on 2 now and will keep reading temp till we close to a stable 86F.
Temperature and Lighting. These are the questions...had a great assortment of lights from past aquariums and algae experiments...back up north. Alas, only brought a single LED unit from my girlfriend’s old freshwater tank. This has an all blue feature and mixed white feature. For this process, I will be running the ladder. This mixed white feature is composed of alternating "white" diodes (6500K?) and blue diodes. No red or uv. Has a few diodes out but will be placing right up against culture so hopefully delivering between 100 and 200 PAR. Light also provides a modest, but not insignificant, source of localized heat to the culture.
Snug as a bug...except not quite...not quite up to temp at 84F. Adjusted electric heat pad to setting 3 and will check again in a few hours…
Giving this Bacteria Baby a New Crib! Placed the culture vessel in a nice cooler given to me by my girlfriend. This insulation is sorely needed to ensure that the Rhodobacter can metabolize at peak efficiency! One week in and the culture is a notable pinker (diluted by blue light). Cannot wait till we get to a dark red!
Agitation. Another important factor when culturing aquatic microbes. Agitating the vessel mixes the water and more uniformly distributes nutrients, waste and living biomass. Don't be afraid to give your bag a daily shake & smack!
Snug as a Bug! With the insulation of the cooler, the light alone can heat the culture to 88F. This is much closer to to the target and have opened the cooler door in an effort to cool an additional 2 degrees. I would suggest anyone buying the kit first see if they can accomplish target temp with their lighting alone as a heat pad may prove to be too much.
The culture is growing nicely, requiring more frequent agitation. An earthy aroma can be whiffed from the vessel and briefly in the cooler if enclosed for long periods. It is the faint but unmistakable aroma of beneficial microbial respiration! Much like the same 'sweetness' of well-aged garden soil, a productive trout stream or a beautifully stable aquarium. It's the smell of concentrated good times.
Been busy with work and various projects and been slacking on the bacteria log. Culture temp has remained mostly stable at 84-86F. This has been done by keeping the heat pad at setting 1 and shutting off if I leave and turn the AC down to 80F for the day. Agitation has been my greatest errand, as I am convinced culture would be darker if I could somehow rotate the vessel continuously. I have been on top of agitating vessel at least three times a day and have noticed darker coloration, though the culture is still pink and not the dark rose red of the inoculant. Still at almost 30 days, the culture is still usable and I am in the process of designing three experiments to evaluate Rhodobacter capsulatus as an aquarium and garden probiotic…
…and then Hurricane Ian arrived…
The bacterial culture survived Hurricane Ian just fine...once my street is no longer a river, can begin making preparations to test out the probiotic! Evaluations will be primarily based around marine reef aquarium applications…though I may certainly include applications pertaining to freshwater aquariums, home gardening and/or wastewater management. With a jug full of live Rhodobacter capsulatus, the opportunities for experimentation and cultivation are limitless!
From the time of receiving the Grow Your Own Kit from Hydrospace LLC to the time I ended up getting a chance to play with it, more than three months had passed. It was an incredibly busy time for me moving down from Connecticut to my current residence in Florida. In that time, the Rhodobacter capsulatus starter culture I received required NO maintenance and was still incredibly potent by the time I was ready to inoculate. I was genuinely impressed by this shelf-life and persistent product efficacy without refrigeration, light or oxygen. I found the initial stages of inoculation to be easy enough for anyone with a home kitchen and a basic grasp on sterile technique…I do wish that the kit came with a inexpensive cooler or insulated box of some kind as it is a careful balance trying to find a heating setup that will not melt the plastic culture vessel. I could have used a thermostat…but that is one more added capital cost to the experience though I would certainly recommend it if you want faster growth/higher harvest density out of your PNS home culture. I would also like it if Hydrospace offered an accessory light that could be purchased with the kit, so that the user knows exactly what spectrum and intensity they need and that they have it.
Overall, though, the cultivation process requires minimal daily attention and the reward is gallons of photosynthetic probiotic that can be applied to an immense array of challenges facing the gardener, agriculturist, aquaculturist and home aquarist alike…more details and examples of these specific benefits in the next article!