Production of Azotobacter chroococcum (Biofertilizer)

A.  Purpose:

The soil  having poor organic carbon and microbes therefore, need of application of Azotobacter chroococcum is needed to enhance soil fertility.

B.  Scope:

      Biofertilizer has been identified as an alternative to chemical fertilizer to increase soil fertility and crop production in sustainable farming. The use of biofertilizer is steadily increased in agriculture and offers an attractive way to replace chemical fertilizers, pesticides, and supplements. The main objective of this study is to evaluate the effect of Azotobacter chroococcum as nitrogen-biofertilizer on growth and yield of Cucumis sativus (cucumber) under greenhouse conditions. The study was done by planting 210 cucumber seeds distributed into seven treatments which were used in our study as follows: control (without treatment), biofertilizer only, organic fertilizer only, chemical fertilizer only, organic fertilizer + biofertilizer, 20% chemical fertilizer + biofertilizer, and biofertilizer, (two dose). After 3 months and through cucumber growth criteria, (shoot length, root length, shoot wet and dry weight, root wet and dry weight, number of leaves, number of branches), yield parameters, mineral content (N%) of cucumber were measured. In the green house experiment, growth parameters of cucumber showed that the productivity of cucumber increased. Seed inoculation with A. chroococcum increased yield about 6%, compared to control. The increase of biofertilizer treated plants in dry root weight were 31%, 18% in wet root weight, 11% in dry whole plant weight, 13% in wet whole plant weight, 14% in whole plant length, 10% in number of branches, 27% in number of leaves over control. The increase in shoot nitrogen percentage was 15% in biofertilizer treated plants, where it was 40% in biofertilizer + 20% chemical over control. The increase in root nitrogen percentage was 18% in biofertilizer treated plants, where it was 22% in biofertilizer + 20% chemical over control. Our results provided a proof of the efficiency of Azotobacter chroococcum as an important biofertilizer in yield of Cucumis sativus (cucumber).  

C.  Preparation of PDA media:

Culture media are essential for isolation of fungi and maintaining them in a pure culture either in test tubes or in Petri plates. The fungal cultures may be grown in liquid media (broths) or on solid agar media. (Agar agar is a solidifying material obtained from sea seed weed like Geladium sp., which melts on boiling and solidifies upon cooling).

Potato dextrose agar (BAM Media M127 and potato dextrose broth are common microbiological growth media made from potato infusion, and dextrose. Potato dextrose agar (abbreviated "PDA") is the most widely used medium for growing fungi and bacteria.

Potato dextrose agar (PDA): 

Potato	200 g (sliced washed unpeeled)
Dextrose	20 g
Agar agar	20 g
Streptomycin	0.05 g
Distilled water	~ 1000 ml. (pH : 7)

  

Method of making PDA

1.      Wash 200 g potato, peel off the skin, and slice them into small pieces

2.      Cook the sliced potato in 500 ml. water for 30 minutes in an open vessel or pressure cooker for 20 minutes

3.      Collect the potato extract by filter through cheesecloth, saving effluent, which is potato infusion (or use commercial dehydrated form)

4.      Mix with Dextrose, Agar and make the volume to 1 litre with distilled water and boil to dissolve

5.      Autoclave 15 min at 121.6°C

6.      After solidifying, the tubes / Petridishs / flasks are arranged in a wire basket and stored in a clean room for further use

7.      It can store in at 5-25 °C for a month

Procedure for checking pH:  Stir the medium with a glass rod and take a small bit of pH indicator paper and dip it in the medium. Take it out and compare the colour development with the colour chart given on the cover sheet of the filter paper book. If pH is above 7, add a few drops of 0.1N Hydrochloric acid (HCl), stir well and again check the pH for neutral. Contrastingly, if the pH of the medium is below 7, add a few drops of 0.1 N Sodium hydroxide (NaOH) to increase

D.  Sterilization (microbiology):

1. Steam sterilizationInvented by Charles Chamberland in 1880Steam sterilization exposes each item to direct stream contact at required temperature and pressure for the specified time.Steam sterilization is primary used for heat-stable materials such as – Glassware, surgical instrument & different type of biological Medias 

There are two types of stream sterilizers

•           Gravity displacement Autoclave
•          High speed prevacuum sterilizers

The recommendations for sterilization in an autoclave are 15 minutes at 121-124 °C (200 kPa). The temperature should be used to control and monitor the process; the pressure is mainly used to obtain the required steam temperature. Alternative conditions, with different combinations of time and temperature, are given below.

Temperature (°C)	Approximate corresponding pressure (kPa)	Minimum sterilization time (min)
126-129	250 (~2.5 atm)	10
134-138	300 (~3.0 atm)	5

2. Dry heat sterilization

Dry heat sterilization is one of the earliest forms of sterilization practiceIt used as product that may be degraded when exposed to stream or moisture but which can with stand high temperatures
Examples of items sterilized by dry heat sterilization: Mental surgical instrument, needles, glassware, powders, oils, etc

Preparations to be sterilized by dry heat are filled in units that are either sealed or temporarily closed for sterilization. The entire content of each container is maintained in the oven for the time and at the temperature given in the table below. Other conditions may be necessary for different preparations to ensure the effective elimination of all undesirable microorganisms.

Temperature (°C)	Minimum sterilization time (min)
160	180
170	60
180	30

E.   Procedure for rearing of Beau+Meta: 

Maintaining nucleus culture

 

1. Isolation of Beau+Meta spp from soil of that area with help of serial-dilution method        on selected media
2. Purification of available strain by sub culturing
3. Purified fungi maintained nucleus culture on regular interval sub culturing

Mass culturing of Beau+Meta

1.      It can be mass cultured by liquid media (broth) or cereals grains but we have used waste of Corcyra boxes.

2.      Make 200 gm waste of Corcyra boxes in plastic bag and plug with nonabsorbent cotton

3.      Steam Sterilized all pkts ar desired pressure & temperature

4.      Sterilized pkt keep in inoculation room for inoculation

5.      Before inoculation laminar air flow surface sterilized by sprit or expose of UV lights for 15-20 min.

6.   Sterilized pkts pore with Beau+Meta desired species @ 1 nucleus culture plate pore 6 pkts

7.    Inoculated Beau+Meta pkts keep in culture room for development of fungus at 20-25 °C temperature.

Commercial culturing of Beau+Meta

1.    We can be make commercial form of Beau+Meta in Talcum powder, Charcol, Pressmud, liquid form or any other organic martial

2.     Collect pressmud keep in heap (1 fit height, 1 mt wide & desirable length) and broken all lamps and removed all unwanted material

3.     Pressmud moist with water

4.     Add Corcyra maize @ 0.5 bag/qt of pressmud

5.   Mixed mass culture pkt @ 20 pkts (10 of each sp) in 1 qt of pressmud before mixing make peats with help of mixer grander

6.      Fungal suspension spore sprayed on pressmud mix thoroughly

7.  Leave for 7-10 days for fungal devolvement churning of culture every 2^nd day for increasing aeration

8.      Packing of culture in yellow bags @ 2 kg / bag

9.      It can be store up to 60 days at room temperature

Precaution

      1.      Purification of species is most important activity
2.      Maintaining of nucleus culture
3.      Avoiding contamination during inoculation
4.      Adding water in pressmud is not run-off