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Optimum Temperature and pH for Bacillis subtillis

 

Bacteria species typically thrive best in pH neutral environments. Optimum pH for B. subtillis is 6.5 – 7.0. Optimum temperature is between 40– 470C. However, as noted with Trichoderma spp., always maintain optimum temperatures and pH for optimal plant growth in hydro systems. I.e. pH 5.5 – 6.0 and nutrient/media temp of 20 – 220C (68 – 71.6 °F)

 

Food for Bacteria

 

Bacteria thrives in a high carbon environment. Molasses has been shown to be a cost efficient source of providing this carbon.14

Other sources of food are humates (fulvic and humic acid), kelp, and hydrolysed fish (fish emulsion).

 

Beneficial Bacteria and Fungi Product Quality

 

Benefical microbe products are generally formulated as wettable powders (WPs), dusts, granules and aqueous or oil based liquid products using different mineral and organic carriers.

 

Beneficial microbe products are sold and used, with or without legal registration, for the control of plant diseases. Bio inoculants are either marketed as standalone products or formulated as mixtures with other beneficial bacteria or fungi. Some products with bio inoculant properties may not be registered, and are sold instead as plant strengtheners or growth promoters without any specific claims regarding disease control.15

 

There are several reputable companies that manufacture government registered products. Government registration ensures that products have been subject to trials and scrutiny where claims made about their efficiency are measured and proven. However, for the most part products sold through the agricultural sector largely remain unregistered or are registered with organic bodies (e.g. OMRI) where products aren’t subject to the same levels of scrutiny .16 Among other reasons that many products remain unregistered are cost of registration and the time required to have the registration approved.

 

High quality bio innoculants depend on having high concentrations of the microorganism(s), long shelf-life and a formulation appropriate to their use.

 

Variable results have been reported for all types of microbial products, whether liquid or dry, with variation in their effectiveness attributed to three main causes: (1) presence of an already satisfactory level of the beneficial microbes prior to inoculation; (2) poor survival of the beneficial microbes in their environment; and (3) low quality of the inoculant. Low quality inoculants can be defined as containing insufficient viable cells of the beneficial microbe/s, high numbers of contaminating micro- organisms, or both.17

 

Olsen et al. (1995) found that only 1 of the 40 commercial North American beneficial products prepared from non-sterile peat contained more benefical microorganisms than contaminants. Contaminant microorganisms in non-sterile conditions often out-compete the beneficial organisms for space and nutrients and may also produce allelopathic (toxic) compounds. Manufacturers also risk incorporating pathogenic organisms into formulations when non-sterile carriers are used.18

 

To manufacture a high quality microbial product, it is essential (among other things) that the carrier material is sufficiently sterilised. This allows for non-competitive multiplication and maintenance of the microorganisms in a nutrient rich environment.

 

Molasses and other microbial carriers commonly used for producing liquid beneficial products and peat granule, traditionally used for creating dry micro products, are unique in that they have a high initial ‘bio-burden’ (I.e. high number of contaminating microbes).

 

These factors dictate the use of autoclaving, in the case of liquids, and irradiation in the case of dry products, to achieve carrier sterility prior to introduction of the beneficial microbes to the carrier. However, while autoclaving or irradiation must be sufficient enough to achieve total “kill” of any contaminating microorganisms, it must not cause substrate/carrier breakdown, the creation of toxins, or adversely affect the carrier’s physical properties in another way. Only through “complete” sterilization can it be guaranteed that unwanted competitive microbes are eliminated from the carrier.19

 

Some of the beneficial bacteria and fungi that are government registered in various countries

 

Bacteria

 

Burkholderia cepacia    –     Soil-borne fungi, nematodes

Pseudomonas fluorescens     –    Soil-borne fungi

P. syringae ESC-10, ESC-11 – Post-harvest fungi

P. chlororaphis   –     Soil-borne fungi

Bacillus subtilis   –    Soil-borne fungi

B. subtilis FZB24   –    Soil-borne

B. subtilis KBC 1010 – Gray mold

B. subtilis GB03 – Soil-borne and wilt

B. subtilis GB07   –     Soil-borne fungi

Rhizobium sp. KR181 – Bio fungicide

Streptomyces griseoviridis K61    –    Various fungi

 

Fungi

 

Trichoderma polysporum, T. harzianum     –      Soil-borne fungi

T. harzianum KRL-AG2    –       Soil-borne fungi

T. harzianum     –       Foliar fungi

T. harzianum, T. viride    –      Various

T. viride     –       Various

T. lignorum    –     Vascular wilt

Trichoderma spp     –     Soil-borne fungi

Ampelomyces quisqualis M-10   –    Powdered mildew

Talaromyces flavus V117b   –    Soil-borne fungi

Gliocladium virens GL-21  –    Soil-borne fungi

G. catenulatum     –     Soil-borne fungi

Fusarium oxysporum   –     non-pathogenic Pathogenic Fusarium

 

A number of companies are developing new products that are in the process of registration.

 

Spore Count and CFU (Colony Forming Units)

 

Spore count and cfu (colony forming units) are used to quantify the microbial content of a liquid or dry beneficial product. Spore count is used with fungi and cfu is used with bacteria. These units indicate the levels of microbes that are present in a given product.

 

A beneficial bacteria and/or fungi product should contain a level of bacteria or fungi sufficient to inoculate plants and produce gains. The required level of bacteria and/or fungi required cannot be established as a general standard because it varies from one species to another. While it is possible to undersupply beneficial microbes it is not possible to oversupply them. For this reason the higher the cfu and/or spore count the higher the quality of the product (put simply).

 

Other factors such as bacteria and fungi species (suitability) and contaminants must also be considered in the quality equation.

 

A quality beneficial bacteria and/or fungi product should list a guaranteed analysis of the cfu and/or spore count. Other things to look for are a use by date and actives.

Species Choice

While some species of fungi and bacteria are demonstrated to produce positive results in both hydroponics and soils (e.g. Trichoderma harzianum, Bacillis subtillis), other species (e.g. Mycorrhizae) may not produce such ubiquitous results. By understanding the science of beneficial bacteria and fungi in hydroponics one can more easily make informed purchasing decisions re products that will produce consistent results (given other factors are in check).

 

Liquids Products and Sporulation (suspended spores in solution)

 

Some years ago (2002) I wrote about beneficial bacteria and fungi and the potential they showed for disease control in hydro settings. One point I made was that liquid beneficial microbe products sold through the hydro industry should be avoided and preference should be given to dry micro products instead. This information was oversimplified and was largely based on the quality of liquid products available through the hydro retail sector at that time.

 

The science….

 

Bacteria must obtain nutrient materials necessary for their metabolic processes and cell reproduction from their environment. Thus in order microbes to thrive in their environment adequate food and oxygen must be present in solution and media.

 

A diverse group of gram positive bacteria (e.g. Bacillis spp.) and species of fungi (e.g. Trichoderma) are capable of sporulation as a means of surviving adverse conditions. These specialized bacteria and fungi are able to become dormant under stress and form spores which are resistant to many chemical as well as physical antibacterial measures. Bacterial spores are extremely stable, and resistant to heat, drying, light, disinfectants and other harmful agents than the original living bacterial organism. Spores may survive for many years.

 

When more suitable conditions present themselves, the spore germinates and again develops a similar cell to the one that originally formed the spore. This new cell, under favourable conditions of moisture, temperature, oxygen, pH and food supply, begins reproduction, antibiotic and enzyme production.

 

However, other species (gram negative) do not sporulate and their populations typically die out or are significantly reduced when faced with nutritional or oxygen stress.

 

Put simply – this means is if live sporulating microbes are added to a liquid product they will hibernate if and when faced with nutritional or oxygen stress. The suspended spores can then be regenerated (germinated) when placed in an environment such as a hydroponics system that provides a viable source of oxygen and food.

 

Other than this, quality liquid products are typically produced using spores that remain suspended due to the presence of antimicrobial preservatives (e.g. Kathon™) that while capable of killing live bacteria and fungi do not harm the more resistant spores.

 

While different microbes will germinate more readily than others a basic reference is that Bacillis subtillis will germinate within 24 hours of being added to the nutrient solution (fungi will typically germinate sooner).

 

Liquid products that contain sporulating bacteria and/or fungi are therefore very viable products when they are produced correctly.

 

Just some of the microbes that sporulate

• Trichoderma harzianum
• Trichoderma viride
• Trichoderma koningii
• Trichoderma polysporum
• Bacillus subtilis
• Bacillus laterosporus
• Bacillus licheniformus
• Bacillus megaterium
• Bacillus pumilus
• Arthrobotrys oligospora
• Hirsutella rhossiliensis
• Acremonium butyri

 

Dry Products – Storage

 

Dry products consist of spores (inactive bacteria and/or fungi) and a carrier medium. Dry products should be stored out of direct sunlight at between 4oC and 10oC in an airtight container. If the product becomes damp from air moisture the spores will become active and then die out when conditions are not suitable. This will greatly reduce product quality. Packets of bacteria/fungi should not be opened until they are ready to be used.

 

Water Quality and Beneficial Microbe Viability

 

Often mains water is treated with monochloramine to kill off undesirable microorganisms. The problem is that monochloramine does not discriminate between undesirable and beneficial microorganisms and therefore residual monochloramine in your tap water supply may also disrupt the viability of beneficial microbes in your hydroponic system (if you are using mains water – this does not apply to RO treated water where carbon filtration is incorporated). Monochloramine is reasonably stable and therefore can persist in tap water for some time. Varying levels of monochloramine can be present in tap water ranging from as low 0.2ppm to much higher; the EPA lists the maximum allowable limit as 4ppm while the World Health Organisation maximum allowable limit is 3ppm.

 

While both chlorine and monochloramine residuals decrease with time, monochloramine decreases more slowly than chlorine. Chlorine may take days to dissipate in a jug left on a counter and it will take much longer for monochloramine to dissipate. The decomposition rate will be faster when the water is exposed to air and sunlight. Chloramine, like chlorine, will eventually dissipate completely over time but this could take many days.

 

The easiest way to remove monochloramine from tap water is to first run it through an activated carbon filter. The activated carbon filter can reduce chloramine concentrations of 1 to 2 ppm to less than 0.1 ppm.

 

Which brings us to our next point… Water Sterilzation methods …..

 

Sterilization versus Beneficial Micros in Hydroponics

Sterilization is another means to prevent root disease in hydroponics.

 

Sterilization works by creating an entirely bio-devoid system. This means, sterilization eradicates microflora from the hydro system completely. I.e. by sterilising the nutrient you are killing both beneficial and pathogenic bacteria. UV, ozone, monochloramine, chlorine and hydrogen peroxide are commonly/widely used methods of sterilization in hydroponics.

 

Products such as Hydrogen Peroxide (Oxy Plus, Hy-Gen Peroxide), or Monochloramine (Pythoff) are useful sterilising agents. Because Pythium is a living organism, sterilization will kill the Pythium spores before they have a chance to enter the plant’s root zone.

 

It is important to note that both monochloramine and hydrogen peroxide act only as root disease preventatives. I.e. once root disease is present in the crop they are ineffective and other means for controlling the disease should be sort.

 

WARNING – DO NOT USE OXIDANTS WITH ORGANIC MEDIAS OR ORGANIC ADDITIVES: monochloramine, chlorine and hydrogen peroxide are not suitable for use where organic media (e.g. coco substrate) or organic additives are used. These products are oxidants and oxidants break down organic matter.

 

Monochloramine in Hydroponics

 

Inorganic chloramines such as monochloramine are formed when chlorine and ammonia are combined in water. One of the key uses for monochloramine is it is used for disinfecting mains water supplies.

 

Monochloramine is an oxidant. It kills bacteria by penetration of the cell wall and blockage of the metabolism. Monochloramine is considered to have moderate biocidal activity against bacteria.1 While there are more effective products available for eradicating bacteria (e.g. chlorine) these have been deemed unsuitable for use in treating mains water supplies due to the byproducts they form when interacting with organic matter.2 Monochloramine hydrolyses (breaks down) slowly in aqueous solutions, producing hypochlorite (at alkaline pH) or hypochlorous acid (at acid pH).3

 

Research into the use of monochloramine in other areas suggests that where bacteria are able to attach to surfaces this provides a primary means for bacteria to survive disinfection. Research of K. pneumoniae grown in a high-nutrient medium attached to glass microscope slides demonstrated a 150-fold increase in disinfection resistance.4

 

Similar findings have been demonstrated in nursery fertigation systems where organic debris or particles prevented direct contact of chlorine (not to be confused monochloramine) with fungal propagules (Phytophthora spp.) and as a result reduced chlorine efficiency.5


This may have implications in hydroponic systems where hosing, pipes, pots, drip emitters and, for that matter, media may offer pathogens protection.

Because monochloramine hydrolyzes (dissipates) slowly careful use is advised. I.e. monochloramine is an oxidant and overuse can result in build up, resulting in fine root hair burning, which will reduce nutrient uptake.

 

Use of Chlorine (not to be confused with monochloramine) in Hydroponics

 

A handy tip. Monochloramine sold through the hydro industry can be replaced by chlorine at greatly reduced cost.

 

Chlorine (Cl) is demonstrated to be a more effective sterilizing agent than monochloramine.1

 

Research has demonstrated that 0.5ppm (780 mV) of chlorine in greenhouse irrigation systems at pH 6.0 eliminated Phytophthora sp., Fusarium sp. and bacteria within 0.5 minutes of contact time.2 Chlorine efficiency is pH dependent and efficiency at pH 6.0 – 7.5 has been shown to be the ideal (maximum efficiency of chlorine is 6.5). Below pH 6.0 and toxic chlorine gas will be released. Because optimum pH in hydroponics is pH 5.8 – 6.0 this makes chlorine ideal as an effective and low cost sterilizing agent.

 

Products such as sodium hypochlorite (liquid typically 12.5% chlorine), calcium hypochlorite (bleaching powder/pool chlorine = approx 65% Cl), and chlorine dioxide are cheap sources of chlorine. Take for example calcium hypochlorite at 65% available chlorine. To achieve 0.5ppm chlorine in 100L of solution 0.08 grams would be required. This would mean that 250 grams of sodium hypochlorite would be good for 3125 treatments. The cost of 250grams of sodium hypochlorite is approximately £12.00 in the UK (in small volume purchased online – far cheaper in volume) or less than $20 USD. Now consider this; you would use only 14.6 grams a year to treat 100L every two days, so 17 years of chlorine treatment would cost less than $20.00 USD. When you consider that a 1L monochloramine product is sold through UK hydroponic stores for £30.00 – £34.99 ($49.00 -$57.00 USD) and is used at 0.2mL/L (50 treatments of 100L) the use of chlorine over monochloramine represents massive savings.

 

Chlorine – Potential Toxicity to Plants

Chlorine obviously has some potential toxicity (phytotoxicity) issues associated to plants, if used at excessive levels – as does monochloramine and hydrogen peroxide. Sensitive plants such as lettuce may be detrimentally affected if chlorine is present in solution at even 1ppm. Less sensitive plants will be tolerant to higher levels.

Research has demonstrated that 2ppm of chlorine at riser outlet, in fertigation systems poses little or no risk of toxicity to the majority of ornamental crops.3 This indicates that treatment with 0.5ppm of chlorine poses very little risk to even sensitive plants.

 

Treatment

Chlorine hydrolyzes (dissipates) more quickly than monochloramine and, therefore, treatment should take place every two days. Directly after treatment the chlorinated nutrient should be cycled through the growing system to ensure pipes, pots, channels and media are adequately sterilized.

 

Measuring Chlorine in Solution

 

ORP Meters


It’s important to note that simply adding oxidants such monochloramine, chlorine and hydrogen peroxide to solution and hoping for the best can only be described as entering the realms of hydro cowboy country. Numerous factors will influence the levels of oxidant (e.g. temp, pH, ionic strength, organic content, dissipation rates, and existing chlorine or monochloramine in the water supply).

 

The most efficient means of accurately monitoring oxidant levels is through the use of an ORP meter.

 

ORP is a measurement of ‘Oxidation Reduction Potential’ (mV) most commonly used to measure the effectiveness of water disinfection systems using sanitizers such as chlorine, bromine, ozone, peroxyacetic acid, hydrogen peroxide etc. ORP standards have been long established for water sanitation and are recommended over ppm measurements with traditional test kits. ORP meters are relatively inexpensive (a handheld pen meter should set you back approximately $100 -150 USD) and easy to operate and should be an essential piece of equipment for people using a chlorination system in hydroponics. Optimal ORP for Pythium control with chlorine = 780 mV at pH 6.0

 

Desired Chlorine in Solution: 0.5ppm – 780 mV

 

Optimum pH for chlorine treatment in hydroponics: 6.0 (below pH 6.0 will release chlorine gas – above pH 6.0 is less than optimal for nutrient uptake)

 

Treatment: every two days – if using an ORP meter maintain at 780 mV

 

Safety Warnings: Calcium hydrochlorite CAS No: 7778-54-3 has strong oxidizing properties and is a corrosive. Handle with care and store in an airtight, lightproof, sealed container safely out of the reach of children.

 

Use our online calculator to establish chlorine ppm in solution with any given product. https://www.medteknutrients.com.au/calculators/dilution-concentration-calc.php

 

Hydrogen Peroxide (H2O2) In Hydroponics

 

Hydrogen peroxide, as with monochloramine and chlorine, is an oxidant. However, as a benefit it is also a disinfectant. The oxidant and disinfection mechanism of hydrogen peroxide is based on the release of free oxygen radicals:
H2O2 → H2O + O2

 

Free radicals have both oxidising and disinfecting abilities.

 

Unlike monochloramine, hydrogen peroxide does not produce residues. I.e. Hydrogen peroxide is completely water-soluble.

 

Hydrogen peroxide hydrolyzes (dissipates) quickly and, therefore, treatment should take place every 2 days.

 

IMPORTANT NOTES: Sterilizing agents such as monochloramine, chlorine and hydrogen peroxide should not be used in conjunction with beneficial microbes. These compounds do not make a distinction between beneficial and harmful microbes and their use can result in killing off or reducing beneficial microbe numbers. If sterilization is used, it is important to reapply beneficial microbes when the sterilizing agent has completely hydrolyzed/dissipated.

 

WARNING – DO NOT USE OXIDANTS WITH ORGANIC MEDIAS OR ORGANIC ADDITIVES: monochloramine, chlorine and hydrogen peroxide are not suitable for use where organic media (e.g. coco substrate) or organic additives are used. These products are oxidants and oxidants break down organic matter.

 

Monochloramine and hydrogen peroxide should never be used together at the same time. Hydrogen peroxide is one method employed by water treatment experts to chase chlorine from mains water supplies. I.e. each product (monochloramine/hydrogen peroxide) renders the other inert.

 

Pythium – Cure

 

In 2002, in Edition 1 of Integral Hydroponics, I wrote about using Fongarid (active furalaxyl) as a cure for Pythium with:

 

[Quote]

 

“Once you have Pythium, control is not an easy matter. There are off the shelf fungicides that are available in Australia, but they need to be used with caution as they are systemic. I have found that Fongarid – a systemic fungicide that contains active furalaxyl – eradicates Pythium quite successfully. However, if Pythium is able to take hold in the crop this situation may change due to the reproductive cycle of the fungi (genetic mutations and more resistant spore types). For this reason prevention is a far smarter practice than cure.”

 

[End Quote]

 

My advice was based on two things. One, research by the CSIRO conducted in 1998 demonstrated the ability of furalaxyl to eradicate pythium 1 and, two, through my own experiences with the product in hydroponics I had/have found that drenching coco coir with a 20ppm solution of furalaxyl for four hours cured Pythium and regenerated healthy root growth within days. To date I haven’t found a better product, hence, still recommending furalaxyl years later.

 

Warnings: Furalaxyl is systemic fungicide and should never be used past week 3 of flower.

 

Use of furalaxyl will also add sodium (Na) to nutrient solution.

 

For this reason it is recommended that furalaxyl isn’t run constantly through the growing system. I.e. High levels of sodium are undesirable in solution.

 

REFERENCES for BENEFICIAL BACTERIA AND FUNGI AND STERILIZING AGENTS can be found here