AP302A

AP302A - Production of trichoderma harzianum (T77)

Info

Publication number: AP302A
Application number: APAP/P/1992/000398A
Authority: AP
Inventors: Mary Jennipher Toet; Amanda Somers
Original assignee: Agricura Private Ltd
Priority date: 1991-07-01
Filing date: 1992-06-09
Publication date: 1994-01-24
Also published as: AU650147B2; US5330912A; AU1856692A; AP9200398A0; ZA924416B

Abstract

The invention provides a process for production of trichoderma harzianum strain T77 on a cemmercial scale. Process comprises loading receptacles with culture medium; sterilisation of the medium in situ; inoculation with spores of T77; incubation for 21 days at 29 degrees centigrade; cooling and packaging in ambient conditions.

Description

This invention relates to the bulk production of Trichoderma Harzianum and more particularly to T Harzianum T77 strain.

The T77 strain of T Harzianum is known from Zimbabwe Patent no 104/89 granted to the Tobacco Research Board of Zimbabwe and its pathology has been described herein.

It is an object of this invention to produce the product of Patent No 104/89 on a commercial scale for general use.

According to the invention, a process for the production of strain T77 of Trichoderma Harzianum includes the steps of preparing a culture medium comprising spent grain, water and molasses in a ratio from about 1 to 1 to 0.06 of spent grain to water to molasses; sterilising the culture by known means;

placing the sterilised culture medium in suitable receptacles;

inoculating the sterilised culture medium with T77 spores;

BAD ORIGINAL ft

2.

inclubating at about 29 degrees centigrade for 21 days; drying the resultant growth for five to seven days at ambient temperatures in sterile conditions and packaging the product for sale.

Spent grain may be substituted by other carriers such as crushed maise cobs; bran; cellulose such as coffee waste; bagasse, peat or brick dust provided sugar is added to 4.2 percent and the PH is kept between 4 and 5.

Also according to the invention, the culture medium may be sterilised by the Tyndalisation procedure wherein culture medium is spread over trays of presently preferred dimensions being height 16 cms, breadth 50 cms and length 60 cms, which trays are then placed in a heating chamber and subjected to heated air at 130-140 degrees centigrade for 24 hours followed by a cooling period of 24 hours and a re-heat at 130-140 degrees centigrade for a further 24 hours; the sterile culture medium in the trays then being inoculated by known means with T77 spores followed by incubation for 21 days at about 29 degrees centigrade whereafter the product ie dried under ambient conditions for from 5 to 7 days and packaged.

In order to illustrate the nature of the invention non-limiting examples will be described hereunder but without limitation of the inventive concept.

AP Ο Ο Ο 3 Ο 2

3.

EXAMPLE 1

In a first example of possible commercial manufacture, glass tomato sauce bottles of 750 mm size hexagonal shape were procured and filled with culture medium made up in 5 kilogram batches consisting of spent grain, water and molasses in the ratio 5 kilograms to 5 litres to 282 grams respectively. The bottles were then plugged with cotton wool and covered with a grease proof paper whereafter they were sterilised for twenty minutes at 121 degrees centigrade and allowed to cool before being inoculated with Trichoderma T77 spores.

The spores were harvested from a Sabouraud Dextrose agar plate by washing the plate with 20 mililitres of sterile distilled water.

Each bottle was inoculated with 2 millimitres of the inoculum using a sterile plastic syringe. The spore count was in excess of one times ten to the power six spores per millimitre.

After inoculation, the bottles were put in an incubator which in this instance was a modro (mobile tobacco bulk curer) made by Modro Manufacturers (Private) Limited in which incubator the temperature was controlled by an air conditioner and maintained at 29 degrees centigrade, plus or minus 1 degree centigrade, for twenty one days covering the growth period.

The bottles were then emptied onto a plastic sheet in the modro and dried for five to seven days under ambient conditions.

^νίΜ

4.

Problems were encountered with this method in that it was labour intensive and produced rather a low yield, each bottle harvesting approximately 150 to 200 grains of dried Trichoderma. In addition it was found that the bottles began to break after being autoclaved twice and the very limitation of autoclaving a number of bottles in a small apparatus limited the production capacity of this particular method.

EXAMPLE II

In this example, both aluminium and steel trays were manufactured to a dimesion of 16 cms in height by 50cms breadth and Θ0 cms length and these trays were filled with non-sterile culture medium in the ratio used in Example I. After charging the trays with medium, sterilisation was initiated by Tyndalisation within the modro where the temperature was raised to 150 degrees centigrade for twelve hours; allowed to cool down for 24 hours and then re-heated to 150 degrees centigrade for a further twelve hours. The sterile medium was then inoculated with Trichoderma T77 spores harvested from a Sabouraud Dextrose Agar, plate system using a plastic sterile syringe to get a good distribution of the spores over the tray. The trays were left in the air conditioned modro at 29 degrees, plus or minus 1 degree centigrade, for 21 days to cover the growth period.

It was found that growth appeared to be best in those trays where the medium was deepest, but a good average depth was about 16 cms .

AP Ο Ο Ο 3 Ο 2

5.

Certain problems were experienced, notably by way of contamination of the trays by other species of fungus, particularly around the edges of the tray and at the corners which may well not have been too well formed or sealed during manufacture. During this experiment, the trays had been covered by a sheet of glass and some sealing had been effected by sticky tape, but it was found in subsequent repeats of this particular example that Trichoderma T77 does not require light for growth so that future trays will be provided with a metal lid which can be clamped down into a better sealing relationship with the body of the tray.

A further problem encountered in experimental trays made of galvanised steel seems to indicate that there might be an attack upon the material of the trays during the growth period.

EXAMPLE III

Trays were manufacutred from stainless steel provided with airtight sliding stainless lids. Contamination was reduced to negligible proportions and yields increased to near theoretical being about 3 tons per modro per cycle period.

Based upon the Examples a full-scale plant was constructed and a plan of this plant is depicted in Figure I of the accompanying drawings.

. , » .· « Ί.

6.

*

In use, raw growth medium is drawn from the bulk store and passed to the washing and filling area where the medium is mixed by mechanical means. Trays are filled and loaded, 48 to a trolley to make up a six trolley batch. One batch of six trolleys with trays moves through the system daily.

Four sterilisation chambers are provided, each capable of accepting one batch, and a four day tyndalisation regimen is observed. Heat, for the process is derived from a boiler and ducting assembly capable of servicing the chambers on a continuous basis. Heating takes place in the chambers for an initial 24 hour period at temperatures of 130-140 degrees centigrade followed by cooling periods of 24 hours, which cooling is expedited by means of heat-exchangers in each chamber heat-transfer gain as well as effective The first heating/cooling cycle is followed by a second 24 hour/24 hour period whereafter the batch progresses to an inoculation chamber which is under positive air pressure during use. , resulting a tyndalisation.

Inoculation comprises removal of the trays from the trolleys onto a conveyor and removal of the tray lids. An automatic metered pumping system of conventional design then ensures that the sterile medium in each tray is sprayed with spores of T77. Trays are re-covered with their lids and replaced in the trolleys whereafter the batch is transferred to the incubation room which is also maintained under positive air pressure when in use.

AP Ο Ο Ο 3 Ο 2

7.

Incubation takes place under strictly controlled conditions of temperature. The optimum temperature is 29 degrees centigrade but variations of 1 degree centigrade each side of the optimum may be tolerated. The period of incubation is 21 days per batch whereafer the batch will have progressed through the bays in the incubation room and are then transferred to the cooling and drying room where trays are uncovered and the contents emptied into drying racks. Drying takes place in a cool stream of sterile air derived in conventional manner from ambient.

After the T77 has cooled and dried over a period of five to seven days it is bagged and stored at temperatures below 20 degrees centigrade until required for use in the field.

Claims

1. A proceee for the production of strain T77 of Trichoderma Harzianum including the steps of preparing a culture medium comprising spent grain, water and molassep in a ratio from about 1 to 1 to 0.06 of grain, water and molasses; sterilising the culture by known means; placing the sterilised culture medium into receptacles; inoculating the sterilised culture medium with T77 spores; incubating at about 29 degrees centigrade for 21 days; drying the resultant growth for five days to seven days at ambient temperatures in sterile conditions and packaging the product for sale.

2. A process as claimed in Claim 1 wherein spent grain is substituted by other carriers such as crushed maize cobs; bran; cellulose such as coffee waste; bagasse; peat or brick dust provided raw sugar is added to these substituted carriers at a rate of about 4,2 per cent by weight and the pH is maintained between 4 and 5.

3. A process as claimed in Claim 1 or Claim 2 wherein the culture medium is sterilised by the Tyndalisation procedure wherein culture medium is spread over trays of presently preferred dimensions being height 16 cms, breadth 50 cms and length 80 cms, which trays are then placed in a heating chamber and subjected to heated air at 130-140 degrees centigrade for 24 hours followed by

AP Ο Ο Ο 3 Ο 2

9.

a cooling period of 24 hours and a re-heat at 130-140 degrees centigrade for a further 24 hours; the sterile culture medium in the trays then being inoculated by known means with T77 spores followed by incubation for 21 days at about 29 degrees centigrade

whereafter the product is dried under conditions for from 5 to 7 days and packaged.

4. A process as claimed in any one of Claims 1 to 3 the receptacles are constructed from stainless steel and are fitted with airtight lids.

5. A process as claimed in any one of Claims 1 to 4 wherein 48 receptacles are grouped into a batch and are processed * ~~