Microbial characterization of cow pat pit and biodynamic preparations used in biodynamic agriculture

Abstract

As of today, biodynamic agriculture is practised across 60 countries of the world. Cow pat pit (CPP) and biodynamic preparations (BD) are the key components of biodynamic agriculture. However, there a is dearth of scientific explanation on account of their mode of action. CPP and BD preparations are very effective in compost production, soil health management and eventually, enhancing the yield and quality of crop produce. Efficacies of these preparations were evaluated with isolation and characterization of beneficial microbes. Study revealed that CPP contained maximum gram positive and gram negative bacteria (184 ± 14 × 10 5 cfu/g), (225 ± 9 × 10 5 cfu/g) and Rhizobium (310 ± 24 × 10 7 cfu/g), while BD-507 contained the highest number of actinomycetes (792 ± 194 × 10 6 cfu/g) and Azotobacter (201 ± 14 × 10 5 cfu/g) among the all preparations. Actinomycetes isolated from CPP and BD-507, showed ammonia, indole acetic acid (IAA), siderophore and HCN producing activities. Out of total isolated microbes from CPP and BD preparations, 9 isolates showed high ammonia, 23 IAA, 18 siderophore and 12 HCN producing activities. Based on the study, it may be suggested that CPP and BD preparations may be used as bio-inoculants and combined with compost in organic production of various crops.

Full text

42
1Principal Scientist (Horticulture) (e mail: ra.ram@icar.gov.
in), 2Scientist (Agricultural Microbiology) (e mail: singha.atul@
gmail.com), 3Student (e mail: ak291289@gmail.com)
Indian Journal of Agricultural Sciences 89 (2): 210–4, February 2019/Article
Microbial characterization of cow pat pit and biodynamic preparations used in
biodynamic agriculture
R A RAM1, ATUL SINGHA2 and A KUMAR3
ICAR- Central Institute for Subtropical Horticulture, Lucknow, Uttar Pradesh 226 101
Received: 15 March 2018; Accepted: 04 September 2018
ABSTRACT
As of today, biodynamic agriculture is practised across 60 countries of the world. Cow pat pit (CPP) and biodynamic
preparations (BD) are the key components of biodynamic agriculture. However, there a is dearth of scientic explanation
on account of their mode of action. CPP and BD preparations are very effective in compost production, soil health
management and eventually, enhancing the yield and quality of crop produce. Efcacies of these preparations were
evaluated with isolation and characterization of benecial microbes. Study revealed that CPP contained maximum
gram positive and gram negative bacteria (184 ± 14 × 105cfu/g), (225 ± 9 × 105cfu/g) and Rhizobium (310 ± 24 ×
107cfu/g), while BD-507 contained the highest number of actinomycetes (792 ± 194 × 106cfu/g) and Azotobacter
(201 ± 14 × 105cfu/g) among the all preparations. Actinomycetes isolated from CPP and BD-507, showed ammonia,
indole acetic acid (IAA), siderophore and HCN producing activities. Out of total isolated microbes from CPP and
BD preparations, 9 isolates showed high ammonia, 23 IAA, 18 siderophore and 12 HCN producing activities. Based
on the study, it may be suggested that CPP and BD preparations may be used as bio-inoculants and combined with
compost in organic production of various crops.
Key words: Actinomycetes, Azotobacter, Azospirillum, BD-500, Cow pat pit, Plant growth promoting
activities
Rudolf Steiner initially developed the biodynamic
agriculture in the year 1920 wherein all the inputs required
for crop production were produced in the farm itself. Steiner
(1997) dened that biodynamic agriculture is a system of
“systematic and synergistic harnessing of energies from
Cosmos, Earth, Plants and Cow for sustainable production”.
According to recent data, biodynamic agriculture is being
practised in 161,074 ha land in across 60 countries of the
world. Germany accounts for 45% of the global biodynamic
agriculture; the remainder average is 1750 ha per country
(Shankaraswamy et al. 2017). A few BD preparations are
used in minute quantities but show remarkable effects on
plant growth, yield and quality (Reganold et al. 1993,
Droogers and Bouma, 1996). BD-500, BD-501, CPP and
BD-502-507 are used for improving soil fertility, biotic,
abiotic stress management and compost production. These
BD preparations are produced with cow dung and other
herbal plants and fermented for a specic period. Deffune
and Scoleld (1995) found that humic acids extracted from
BD-500 and other BD preparations (505 and 507) caused
positive growth response in wheat seedlings relative to
the control. However, scientic explanations are lacking.
Reganold (1995) also reported improvement in soil quality
and protability after use of BD preparations. It is, therefore,
imperative to study these preparations from microbiological
point of view.
MATERIALS AND METHODS
Cow pat pit, a eld preparation, is also called as ‘soil
shampoo’. It is prepared with fresh cow dung collected
from lactating and pasture going cows and fermented
along with crushed egg shells powder and basalt/bentonite
(clay) dust duly mixed and placed in a pit sized of 3’ × 2’
× 1.5' in shed. Two sets of BD-502-507 are incorporated
for catalyzing the composting process. Compost gets ready
in 90-120 days at 30-40°C atmospheric temperature and
60-70% humidity. Ready compost was stored with 50-60%
moisture in earthen pot. BD-500 was produced in cleaned
cow horns lled with fresh cow dung like CPP and buried at
30 cm depth in the soil in root free zone during descending
period of Moon in the months of October-November.
After 6 months of incubation, horns were taken out during
descending period of Moon in the months of March-April.
Properly decomposed compost was stored at cool place
with 50-60% moisture in earthen pot. After taking out of
BD-500, same horns were thoroughly cleaned with water,
lled with silica powder paste and buried in same pit

211February 2019]
43
MICRBIOAL CHARACTERIZATION OF COW PAT PIT
crystal violate agar (Goud et al. 1985), Pikovskaya’s agar
(Pikovskaya 1948), yeast extract mannitol agar with congo
red (CRYEMA, Fred et al., 1932), modied Jenson’s agar
(Jensen 1954, Norris and Chapman 1968) and N-free malate
medium (Okon et al. 1977), respectively.
The compositions of the selective media were:- Nutrient
agar (Hi-Media): Beef extract-3.0 g, peptone-5.0 g, NaCl-
5.0 g, agar-15.0 g, distilled water-1 l., pH-7.2; RBCA
(Hi-Media): mycological peptone-5.0 g, dextrose-10.0
g, monopotassium phosphate-1.0 g, MgSO4-0.5 g, rose
bengal-0.05 g, chloramphenicol -0.1 g, agar-15.5 g, nal
pH (at 25°C) 7.2 ± 0.2; actinomycetes isolation agar
(Hi-Media): Sodium caseinate-2.0 g, L-Asparagine-0.1
g, sodium propionate,-4.0 g, dipotassium phosphate-0.5
g, magnesium sulphate-0.1 g, ferrous sulphate-0.001 g,
agar 15.000, distilled water-1 l. pH-8.1 ± 0.2; King’s
B: protease peptone-20 g, glycerol-10.0 g, K2HPO4-1.5
g, MgSO4, 7 H2O-1.5 g, agar-15.0g, distilled water-1 l,
pH-7.2; methyl red agar: beef extract-3.0 g, peptone-5.0
g, methyl red-0.15 g, agar-15.0 g, distilled water-1 l, pH-
7.0; crystal violate agar: beef extract-3.0 g, peptone-5.0
g, crystal violet-4 ml (stock solution of crystal violet 0.05
w/v), agar-15.0 g, distilled water-1 l, pH-7.0; Pikovskaya’s
agar (Hi-Media): yeast extract-0.5 g, dextrose-10.0 g,
calcium phosphate-5.0 g, ammonium sulphate-0.5 g, KCl-
0.2 g, MgSO4-0.1 g, manganese sulphate 0.0001 g, ferrous
sulphate 0.0001 g, agar 15.0 g, distilled water-1 l., pH-7.5;
CRYEMA (Hi-Media): yeast extract 1.0 g, mannitol 10.0
g, dipotassium phosphate 0.5 g, magnesium sulphate 0.2
where cow horns were buried for the preparation of BD-
500 during the ascending period of Moon in the months
of March-April. After 6 months of incubation, horns were
taken out in October-November during ascending period of
Moon. Light yellowish silica powder was taken out from
the horn and stored in glass jars (Pathak and Ram, 2003).
This preparation is called as BD-501. BD preparation set
(BD- 502-507) was purchased from M/S Supa Biotech (P)
Ltd, Nainital (Organic certied), Uttarakhand, India and
were used for compost, CPP, biodynamic liquid pesticides
production. Main ingredients of BD preparations are as
follows: BD-502 contains fermented owers of yarrow
grass (Achillea millefolium), BD-503- fermented chamomile
owers (Matricaria recutita), BD-504- air dried leaves of
stinging nettle (Urtica dioca) fermented in the soil, BD-
505 - fermented oak bark (Ouercus sp), BD-506 - fermented
ower of dandelion (Taraxacum ofcinalis) and BD-507-
valerian plant (Valeriana ofcinalis) extract. one g compost
of each and 10 ml of BD-507 were added in compost heap,
cow pat pit and biodynamic liquid pesticides to catalyse
the fermentation process (Koepf et al. 1990, Steiner 1993).
Enumeration of different benecial microbial populations,
viz. bacteria, fungi, actinomycetes, Pseudomonas, gram
positive and negative bacteria, p-solubilizing bacteria,
Rhizobium, Azotobacter and Azospirillum were carried
out by using dilution plate count method using selective
media, viz. Nutrient agar, Rose Bengal Chloramphenicol
Agar (RBCA), actinomycetes isolation agar, King’s B (King
et al. 1954), methyl red agar (Hagedorn and Holt 1954),
Table 1 Different microbial populations in cow pat pit and biodynamic preparations
Type of microbe Multiplication
factor
Microbial population (cfu/g) (Mean±sd)
Cow
Pat Pit
Biodynamic preparations
500 501 502 503 504 505 506 507
Bacteria 10816.7 ±
0.91
3.80 ±
0.67
1.80 ±
0.31
2.40 ±
0.46
85.50 ±
7.45
8.50 ±
1.62
110.3 ±
8.92
12.20 ±
1.49
2.60 ±
0.27
Fungi 1058.30 ±
0.96
6.90 ±
1.00
11.30
±1.55
11.97
± 1.50
28.33 ±
7.55
33.53 ±
3.18
13.27 ±
2.00
35.07 ±
5.61
5.10 ±
0.60
Actinomycetes 10612.7 ±
3.2
15.9 ±
3.7
3.1 ±
1.5
24.9 ±
5.5
14.8 ±
4.3
68.0 ±
12.7
20.3 ±
4.8
207.2 ±
71.3
792.0 ±
194.2
Gram positive
bacteria
108184.1 ±
14.21
0.55 ±
0.21
0.10 ±
0.05
0.89 ±
0.35
06.57 ±
0.85
2.85
±0.21
12.30 ±
2.91
1.47 ±
0.35
Gram negative
bacteria
107225.1 ±
9.75
0.02 ±
0.01
0.02 ±
0.01
1.58 ±
0.21
0.03 ±
0.01
15.63 ±
2.15
0.63
±0.15
11.23 ±
1.05
15.43 ±
2.76
Pseudomonas 1066.47 ±
0.67
1.38 ±
0.04
4.73 ±
0.61
0.65 ±
0.25
1.85 ±
0.50
21.73 ±
7.29
2.78
±0.43
7.03 ±
0.59
12.17 ±
3.21
P-solubilizing
microbes
1058.30 ±
0.56
3.93 ±
0.67
25.43 ±
3.95
39.63
± 3.71
24.17 ±
1.10
0.69 ±
0.17
95.13
±7.20
21.70 ±
7.33
10.00 ±
2.41
Azotobacter 10528.37 ±
2.85
23.57 ±
2.42
46.5 ±
11.53
34.30
± 0.95
77.43 ±
7.03
28.60 ±
3.85
26.89
±3.03
53.07 ±
12.83
201.4 ±
14.91
Azospirillum 105224.3 ±
30.01
9.80 ±
1.28
0.67 ±
0.15
54.40
± 6.62
96.10 ±
9.22
76.47 ±
8.27
0.53
±0.23
528.8 ±
77.52
830.3 ±
94.38
Rhizobium 107310.8 ±
24.85
6.00 ±
1.51
4.80 ±
0.95
6.17 ±
0.83
10.83 ±
2.61
18.10 ±
2.95
2.10
±0.53
6.07 ±
0.50
0.02 ±
0.01

212 [Indian Journal of Agricultural Sciences 89 (2)
44
RAM ET AL.
g, sodium chloride 0.1 g, congo red 0.025 g, agar 20.0 g,
distilled water-1 l., nal pH (at 25°C) 6.8±0.2; modied
Jenson’s agar: sucrose-20.0 g, K2HPO4-1.0 g, MgSO4-0.5
g, Na2MoO4-0.001 g, FeSO4, 7H2O-0.01 g, CaCO3-2.0
g, agar-18.0 g, distilled water-1 l., pH-7.2.; N-free malate
medium: malic acid-5.0 g, K2HPO4-0.5 g, KOH-4.0 g,
MgSO4-0.1 g, NaCl-0.02 g, CaCl2-0.01 g, FeSO4-0.05 g,
Na2MoO4-0.002 g, MnSO4-0.01 g, bromothymol blue-0.002
g, agar-18.0 g, pH-9.6-7.3, distilled water-1 l. Petri dishes
were prepared by pouring each specic solid medium. Then
10 ml of each preparation sample was diluted with 90 ml
sterile water and that was considered being 10-1 dilution
factor. Transferring of 1 ml of 10-1 dilution to 9 ml sterilized
water with the help of a sterilized pipettes yielded 10-2
dilution. In this way, a series of up to 10-8 dilutions were
prepared under aseptic condition. Point one ml (0.1ml) of
the suspension from required dilution (e.g. 10-8) was taken
and poured into the respective agar media on petri dish
and spread with L-spreader with the help of Plate Master
(Hi-Media). Then plates were incubated at 28±2°C for 3-5
days. The number of visible colonies were counted. The
total count was obtained by multiplying number of visible
colonies on the plate by the dilution factor. The individual
selected colonies were streaked on a new perti dish with
respective solid medium for two consecutive times to purify
the microbial cultures. The puried cultures were stored on
agar slants in a refrigerator for further use. Observations
were statistically analysed for mean and standard deviation
and presented in the tables (Panse and Sukhatme 1976).
This study was undertaken during 2016-17.
Puried microbial isolates were evaluated for different
plant growth promoting attributes, viz. ammonia, IAA,
siderophore and HCN producing activities. Ammonia, indole
acetic acid (Bric et al. 1991) and siderophore producing
activity was determined by growth in chrome azurol S (CAS)
medium after 48–72 hr growth at 28°C. HCN producing
activity was estimated by change in the colour of lter paper
from yellow to brown (Bakker and Schippers 1987). The
test results were denoted as (+): positive, (-): negative; (+):
Low and (++): High.
RESULTS AND DISCUSSION
Microbial population dynamics in CPP and BD preparations
(BD -500-507)
Microbial population, viz. bacteria, fungi, actinomycetes,
gram positive and gram negative bacteria, Pseudomonas,
P-solubilizing microbes, Azotobacter, Azospirillum and
Rhizobium in cow pat pit and BD-502-07 were enumerated.
Maximum total bacteria (110.3 ± 8.92 × 108cfu/g) were
counted in BD- 505 and minimum (1.80 ± 0.31 × 108cfu/g)
in BD-501. This might be due to oak bark a good source
of tannins which are bioactive, while BD-506 contained
maximum fungi population (35.07 ± 5.61 × 105cfu/g) and
BD-500 (6.90 ± 1.00 × 105 cfu/g) contained minimum.
Maximum actinomycetes (792 ± 194 × 106 cfu/g) were
isolated in BD-507 and minimum (3.1 ± 1.5 × 105 cfu/g) in
BD-501. It might be due to phosphorus utilizing microbes
as BD-507 is a good source of phosphorus. Highest gram
positive and negative bacteria (184 ± 14 × 105 cfu/g), (225
± 9 × 105 cfu/g) were isolated from CPP, respectively.
Increase in bacteria might be due to inoculation of BD-
502-507 during CPP preparation. Maximum Pseudomonas
population (21.73± 7.29 × 106cfu/g) was recorded in BD-504
and p-solubilizing microbes (39.63 ± 3.73 × 105cfu/g) in
BD-502, while highest population of Azotobacter (201 ± 14
× 105 cfu/g) and Azospirillum (830 ± 94 × 105 cfu/g) were
found in BD-507. Microbial studies on this issue are scanty.
Findings of this study therefore, can’t be supported with
sufcient number of references. CPP contained maximum
Table 3 Plant growth promoting activity of actinomycetes isolates
from cow pat pit
PGPR
strains
HCN
production
Ammonia
production
Siderophore
production
IAA
production
CISH-
PGPA
13
++ - + -
CISH-
PGPA
14
- - - -
CISH-
PGPA
15
- + - +
(+): Test positive, (-): Test negative; (+): Low, (++): High
activity.
Table 2 Plant growth promoting activity of bacterial isolates
from cow pat pit
PGPR
Isolate
HCN
Production
Ammonia
production
Siderophore
production
IAA
production
CISH-PGPR
69
- + - +
CISH-PGPR
70
- ++ - +
CISH-PGPR
71
- + - +
CISH-PGPR
72
- + + +
CISH-PGPR
73
+ - - -
CISH-PGPR
74
+ + + -
CISH-PGPR
86
+ + - -
CISH-PGPR
87
- - - -
CISH-PGPR
88
- + - +
(+): Test positive, (-): Test negative; (+): Low, (++): High
activity.

213February 2019]
45
MICRBIOAL CHARACTERIZATION OF COW PAT PIT
Table 4 Multifarious plant growth promoting traits of bacterial cultures isolated from biodynamic preparations
Culture No. HCN production Ammonia production Siderophore production IAA production
CISH-PGPR-BD-500 -1 - ++ + +
CISH-PGPR-BD -500 -2 ++ ++ + +
CISH-PGPR-BD -500 -3 + + - +
CISH-PGPR-BD -500 -4 ++ ++ + +
CISH-PGPR-BD -500 -C ++ ++ + +
CISH-PGPR-BD -501 -I - + - -
CISH-PGPR-BD -501 -II - ++ - -
CISH-PGPR-BD -501 C I - - - +
CISH-PGPR-BD -501 CII - + - -
CISH-PGPR-BD -501 C III - ++ + +
CISH-PGPR-BD -501 C IV - ++ - -
CISH-PGPR-BD -502 I - ++ + +
CISH-PGPR-BD -502 II - ++ - -
CISH-PGPR-BD -503 1MR - ++ - -
CISH-PGPR-BD -503 2MR - ++ - -
CISH-PGPR-BD -503 3MR - ++ - -
CISH-PGPR-BD -503 4MR - ++ - -
CISH-PGPR-BD -503 5MR - ++ - -
CISH-PGPR-BD -503 6MR - ++ - -
CISH-PGPR-BD -503 6 - ++ - +
CISH-PGPR-BD -503 N7 - ++ - -
CISH-PGPR-BD -503 C8 - ++ - -
CISH-PGPR-BD -503 8 - ++ +
CISH-PGPR-BD -503 9 - ++ - +
CISH-PGPR-BD -503 10 - ++ - +
CISH-PGPR-BD -503 11 - ++ - +
CISH-PGPR-BD -504 C1 - ++ - -
CISH-PGPR-BD -506 I - ++ - +
CISH-PGPR-BD -506 MRI + + + -
CISH-PGPR-BD -506 MRII - + + -
CISH-PGPR-BD -506 MRIII - + + -
CISH-PGPR-BD -506 MRIV - - + +
CISH-PGPR-BD -507 CRI - ++ - -
CISH-PGPR-BD -507 CR2 - + + -
CISH-PGPR-BD -507 CR3 - ++ - -
CISH-PGPR-BD -507 CR4 - ++ + -
CISH-PGPR-BD -507 CR5 - + + -
CISH-PGPR-BD -507 CR 6 - + - -
CISH-PGPR-BD -507 NI - ++ + +
CISH-PGPR-BD -507 NII - ++ + +
CISH-PGPR-BD -507 MEI + + - -
CISH-PGPR-BD -507 MEII - - - -
CISH-PGPR-BD -501 MRI + + - -
(+): Test positive, (-): Test negative; (+): Low, (++): High activity

214 [Indian Journal of Agricultural Sciences 89 (2)
46
RAM ET AL.
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population of Rhizobium (310 ± 24 x 107cfu/g) (Table
1). This might be due to added natural calcium and cow
dung and bentonite powder. Stalin et al. (2014) have also
enumerated microorganisms in organic and biodynamic
manures, and reported that cow pat pit contained highest
bacterial load (4.8 x 106 cfu/g); in which Bacillus subtilis
was predominant.
Growth promoting activities of microbial isolates
Fifty five microbial isolates (52 bacteria, 3
actinomycetes) were isolated and evaluated for different
plant growth promoting attributes, viz. ammonia, indole
acetic acid, siderophore and HCN producing activities
(Table 2, 3, 4). Among them, 9 bacterial isolates and 3
actinomycetes isolates were from CPP and rest 43 bacterial
isolates from other BD preparations. Actinomycetes
isolated from CPP showed siderophore and HCN producing
activity (CISH-PGPA 13). Similarly, CISH-PGPA 15, an
actinomycete isolated from CPP produced both ammonia
and IAA in the test medium. Radha and Rao (2014) have
also reported presence of actinomycetes in CPP due to
addition of calcium during preparation. The bacterial
isolates from CPP also tested for ammonia, IAA, HCN
and siderophore producing activities. Perumal et al.
(2006) reported plant growth hormones such as Indole
Acetic Acid IAA (28.6 mg/kg), kinetin (7.6 mg/kg) and
gibberellic acid (23.6 mg/kg) in CPP. Possibly for this
reason, CPP stimulates plant growth by providing nutrients,
plant hormones and protects plant’s root zone against
fungal diseases with bacteria and actinomycetes produces
HCN and siderophores. Fifty two bacterial isolates from
BD preparations have shown multifarious plant growth
promoting activities. Nine of them showed high ammonia
and 23 IAA producing activity. Eighteen of them showed
siderophore and 12 HCN producing activity together which
revealed their bio-inoculation potential for either making
compost or spraying directly to the crop plant or soil.
ACKNOWLEDGMENT
Authors are thankful to the Director, ICAR-Central
Institute for Subtropical Horticulture, Lucknow for providing
all necessary facilities to complete the study.
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