INDIAN JOURNAL OF APPLIED MICROBIOLOGY
Copyright © 2014 Indian Association of Applied Microbiologists, Chennai, India
Volume 17 Number 1 January-June 2014, pp. 23-31.
Humic Acid, Protein Hydrolysate & Microorganisms as a
Mixed Consortium in Plant Growth
Ramya Dinesh E*, Pushparani A1, Rajendran P2, Raju S3,
Sriranjani S1, Sruthi I4
*Department of Microbiology, Sri Ramachandra Medical College &
Research Institute, Chennai - 600116
1
Department of Microbiology, Valliammal College for Women, Chennai - 600102
2
Department of Microbiology, Madha Medical College & Hospital, Chennai - 600122
3
Microbiology Laboratory, Directorate of Public Health and Preventive Medicine, Chennai - 600006
4
Department of Biotechnology, Dwaraka Doss Goverdhan Doss Vaishnav College, Chennai - 600106
Abstract: In recent decades, many farmers are using chemical fertilizers to cultivate the crop for
fast growth, which culminates in an unhealthy and chemically contaminated food material.
Instead of that, the humic acid, groundnut oil cake, protein hydrolysate and microorganisms were
used as a biofertilizer to improve the crop production without any side effects. Three groups of 6
pots and one control were prepared by filling the above ingredients in different concentrations
with green gram and black gram seeds to check the plant growth and plant height is measured and
compared with other pots. Microbial density in soil was examined for this study. Level of protein
hydrolysate is estimated by Lowry’s method. Finally each ingredients were stimulated the plant
growth and crop production. While the green gram showed good growth in low concentrated
ingredients filled pot [S6(a)], the black gram showed good growth in medium concentrated
ingredients filled pot [S6(b)]. This suggest that, mixed consortium of this ingredients are effective
for the improvement of growth of green gram (Vigna radiate) and black gram (Vigna mungo) and
for stimulating the increase of microorganisms in soil.
Key words: Humic acid, Biofertilizer, Protein hydrolysate, plant growth
*Author for Correspondence. E-mail: e.ramya@live.com
24 RAMYA DINESH
ET AL
Farmers have adapted the strategy of increasing crop yield by applying large amount of chemical
bio-fertilizers. Consequently, the negative effects of heavy use of chemical bio-fertilizers resulted
in human health disorders, inferior quality of yield and adverse environmental conditions (Mishra
et al., 2011). Therefore, the ultimate goal of sustainable agriculture is to develop farming systems
with high yields, profit and conservation of energy and natural resources such as soil and water,
which would ensures food safety and quality (Winget & Gold, 2007).
For instance microbial fertilizers facilitate increase yield and quality of crops with appropriate
investment of money and labor. It can clean the environment, generate plant nutrients like
nitrogen, phosphorus, potassium through their activities in the soil or rhizosphere and make
available to plants in a gradual manner (Muraleedharan et al., 2010).
The fertilizers and bio-fertilizers are different from each other. Some may not be able to produce
or supplement a specific nutrient to the plant. However plant nutrients are very essential for the
production of crops and healthy food for the worlds expanding population (Chen, 2006).
Naggar (2006) suggested that the Nitrogen (N) is very important for the plant growth and to the
crop production. But nitrogen fertilizers are available in small quantities at high prices as means
of supplementing the soil or plant during growth. For supplementing plant nutrient like Nitrogen
rich bio-fertilizers, Jingura (2010) used Jatropha oil cake to produce protein hydrolysate. In this
regard Govindaraju (2003) worked with soy bean, ground nut flour and oil cake to estimate the
amount of protein present by using enzymatic hydrolysis method. Ashwanikumar et al., (2008)
concluded that Jatropha oil cake can be used to produce a high yield of crop. Therefore, ground
nut protein can also be used as a plant nutrient and packing materials to the microorganisms at
low prices.
All the bio-fertilizers do not have capacity to hold the water and stimulate the dead cells but
humic acid helps to break up the compacted soils and assists in transferring micronutrients to the
plants, enhances water retention and increases seed germination (Stevention, 1994). Humic acid is
referred to the combination of humic and fulvic acid content naturally occurring in the soil and
agricultural waste deposits (Vallini et al., 1997). It can stimulate development of micro flora in
soil and has the ability to stimulate the dead cells (Wang et al., 1995). Humic acid has the ability
to hold seven times their volume in water than soil clays & it provides a carrier medium for
nutrients required by soil organisms and plant roots (Pettit, 2004).
Therefore the purpose of this work is to observe the “Effects of humic acid, microorganisms and
the protein hydrolysate from ground nut oil cake on the growth of green gram, fenugreek, black
gram plant and on the microbial density in the soil.
Material and Methods
Soil samples were collected in sterile polythene bags from the three different agricultural fields
such as paddy, ground nut and banana fields. All samples were serially diluted and the
microorganisms were isolated and identified by standard procedure.
INDIAN JOURNAL OF APPLIED MICROBIOLOGY
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Humic Acid, Protein Hydrolysate & Microorganisms as a Mixed Consortium in Plant Growth
25
Production and estimation of protein hydrolysate
Protein hydrolysate production was carried out as per the procedure of Pittom (1914). In this
procedure the ground nut (oil) cake was ground to powder. Then it was suspended in the 10%
papine enzyme solution (10ml of papine enzyme mixed with 90ml of distilled water).Then it was
kept in stirrer for 1 hour at 60⁰C. Then the protein hydrolysate was controlled at temperature
100⁰C in water bath for 20 minutes. After that, the supernatant (protein hydrolysate) was
separated by centrifugation at 8000rpm for 10 minutes. The maximum yield of protein
hydrolysate was estimated by Lowery et al (1951) method.
Optimization of Plant Growth in Pots
Artificial soil was prepared for this study. It was sterilized at 1800C for 1 hour. Twenty five cm
diameter pots were filled with compressed 150 gram of sterilized soil to within 1cm of the top.
Soil in all pots was damp but not saturated. Pots were divided into 3 groups of 6 pots each. One
pot was the negative control with only water added throughout the study. The percentage of
concentrations was calculated based on the soil taken. The 3 sets of pots named as S1 – S6 and
each set contains 6 pots and its concentrations were 1%, 2% & 3%respectively. In 3 pots of S1
Ground nut oil cake were added in the amount of 1.5g, 3gm & 4.5gm according to their
concentration 1%, 2% & 3% respectively. Likewise it is followed for other set of pots S2-S5
(namely humic acid, isolated Bacterial culture, Protein Hydrolysate & Nitrogen fertilizer).
S6 pots were divided as S6(a), S6(b) & S6(c) according to the concentration and the amount of
ingredients added in the pot. In this particular pot the amount of ingredients taken varies because
it’s all mixed together. In S6(a) the concentration 1% and the amount of ingredients are 0.35gm of
groundnut oil cake, o.35ml of bacterial culture (isolated 3 strains of bacterial culture was diluted
in water), 0.35ml of Protein hydrolysate and 0.35gm of humic acid were added. In S6(b)
concentration was 2% and the amount of ingredients are 0.75gm of groundnut oil cake, 0.75ml of
bacterial culture 0.75ml of Protein hydrolysate and 0.75gm of humic acid was added. In S6(c) the
concentration was 3% and the amount of ingredients were 1.12gm of groundnut oil cake, 1.12ml
of bacterial culture, 1.12ml of Protein hydrolysate and 1.12gm of humic acid was added.
Black gram (Vigna mungo) and green gram (Vigna radiate) seeds were added in all pots and
growth rate (Stem length in cm) was measured by using the scale.
Enumeration of Colony Forming Unit before and after inoculation of soil sample
The microbial density within the soil was measured on the day adding the culture to the pots and
after 7 days of growth of the plants as per the method of Winget & Gold (2007)
Antibacterial activity of Humic Acid
Antimicrobial activity was estimated for humic acid by disc preparation kept in Luria bertani
medium separated with microorganisms, such as Staphylococcus aureus (ATCC 29213),
Salmonella typhimurium (ATCC 29629), Escherichia coli (ATCC 25922), Clostridium
perfringens (ATCC 3626) and isolated soil organisms B1, B2 & B3 strains and kept in incubator
INDIAN JOURNAL OF APPLIED MICROBIOLOGY
Vol. 17 No. 1 Jan.-Jun. 2014
26 RAMYA DINESH
ET AL
at 370C for 24 hours. This method was done to prove that humic acid can stimulate the activity of
microorganisms.
Result:
Three bacterial strains were isolated from three rhizospheric soil samples and identified by
standard methods as Micrococcus sp, Pseudomonas sp & Azotobacter sp only upto genus level.
Estimation of Protein in Protein hydrolysate prepared from Groundnut Oil cake:
Protein concentration (in %) of ground nut oil cake (Lowry’s Method, 1951) was estimated as
57%.
Optimization of plant growth Rate in Green Gram
In green gram (S6 pot) the growth was high in 1% pot (S6a) concentration. It showed less growth
rate in 2% (S6b) & 3 % (S6c) concentration. (Table 1; Fig 1)
Table 1: Showing the effect of growth of green gram in different growth substitutes
(Plant Stem in cm within 7 days)
Particulars/Concentrations
1% Conc
2% Conc
3% Conc
Control
4cm
4cm
4cm
ground nut oil cake (S1)
4.2cm
4.3cm
4.5cm
Humic acid (S2)
5.3cm
5cm
5cm
Bacterial culture (S3)
5.1cm
4.7cm
3.9cm
Protein hydrolysate (S4)
6cm
6.2cm
6.3cm
fertilizer (S5)
4.2cm
5.3cm
4.2cm
GOC+HA+B+Prtn Hydro (S6)
9cm (S6a)
8.2cm (S6b)
4.2cm (S6c)
INDIAN JOURNAL OF APPLIED MICROBIOLOGY
Vol. 17 No. 1 Jan.-Jun. 2014
Measurement of plant stem in cm
Humic Acid, Protein Hydrolysate & Microorganisms as a Mixed Consortium in Plant Growth
10
9
8
7
6
5
4
3
2
1
0
27
1% Conc
2% Conc
3% Conc
Particulars (Samples)
Fig 1: Showing High growth rate in 1% concentration of prepared biofertilizer
Optimization of plant Growth Rate in Black Gram
Black gram showed high growth rate in 2% concentration in S6 pot (S6b) and it showed less
growth rate in 2% (S6a) & 3% (S6b) concentrations (Table 2; Fig 2)
Table 2: Showing the effect of growth of black gram in different growth substitutes
(Plant Stem in cm within 7 days)
Particulars/Concentrations
1% Conc
2% Conc
3% Conc
Control
3 cm
3cm
3cm
ground nut oil cake (S1)
4cm
4.2m
4.9cm
Humic acid (S2)
5.5cm
5.9cm
4.2cm
Bacterial culture (S3)
4.5cm
4.3cm
4cm
Protein hydrolysate (S4)
5.6cm
5.9cm
6cm
fertilizer (S5)
4cm
3.9cm
3.7cm
GOC+HA+B+Prtn Hydro (S6)
7.1cm
7.9cm
6.2cm
INDIAN JOURNAL OF APPLIED MICROBIOLOGY
Vol. 17 No. 1 Jan.-Jun. 2014
Measurement of plant stem in cm
28 RAMYA DINESH
ET AL
9
8
7
6
5
4
3
2
1
0
1%
% Conc
2%
% Conc
3%
% Conc
Particularrs (Samples)
Fig 2:
2 Showing Highh growth rate in 2% concentratioon of prepared biofertilizer
b
Enumeration of CFU beforee and after in
noculation off soil samplees
Thee Microbial dennsity of the add
ded microorgannisms were graadually increasses their growthh.
An
ntimicrobial activity
a
of hu
umic acid
Theere is no zone of
o inhibition inn the Luria Berrtani medium containing
c
miccroorganisms indicating
thatt the humic aciid is not an antiimicrobial agennt.
Disscussion:
Thee isolated micrroorganisms lik
ke Micrococcuus spp., Azotobbacteria Spp., and
a Pseudomoonas spp.,
are Phosphate Sollubilizing Bactteria. These thrree bacterial strrains are free lliving bacteria it can fix
mospheric nitroggen in cereal crops
c
without aany damage to the environmeent (Chen 2006
6). Due to
atm
the release of plaant growth promoting substtances by thesse organisms they
t
increase the plant
wth. Also connverts the prottein hydrolysatte prepared frrom ground nuut oil cake to Nitrogen
grow
sou
urce for the plannt growth.
Thee ground nut oiil cake containn minimum of 36-50%
3
of cru
ude protein (Naaggar, 2006; Rahman &
Iderresit, 2011). Jingura et al
a (2010) saidd that Jatropha oil cake contain Proteeins and
Carrbohydrates. Moreover
M
Jatrobbha oil cake iss used in indu
ustry for preparration of green
n manure
andd fertilizers, foo
od, soap, pestiicides, charcoaal and medicinaal uses (Ashwani kumar & Satyawati
S
Shaarma, 2008). Suumitra et al (20007) said that ground
g
nut oil cake contain pprotein that cann be used
as a protein supp
plement for media
m
preparatiion. Likewise,, this study shhowed that the protein
hyddrolysate prepaared from Groound nut oil ccake increasess the plant groowth. And thiis protein
hyddrolysate can also
a be used ass a packing maaterial of this biofertilizer,
b
beecause it supplementing
IND
DIAN JOURNAL
L OF APPLIED MICROBIOLO
OGY
Vol. 17 No. 1 Jan.-JJun. 2014
Humic Acid, Protein Hydrolysate & Microorganisms as a Mixed Consortium in Plant Growth
29
the food as nitrogen for both plant and microorganisms. Therefore the plant grows well in the pot
containing only ground nut oil cake indicating that these plants can grow well with or without
protein hydrolysate and microorganisms etc. At the same time addition of ground nut oil cake
extract and its protein hydrolysate in increased concentration facilitates the growth of plants.
In this study humic acid was used to stimulate the plant growth and microorganisms present in the
soil. This was proved by antimicrobial activity testing of Humic Acid and Enumeration of CFU
from the pots which contain humic acid. The addition of humic acid to the both plant resulting in
increase of plant nutrient uptake. In black gram the large amount (3%) of humic acid containing
pots (S2 – S5) showed less plant growth when compared to the other concentrations 1% and 2% of
humic acid containing pots. But it shows good result in medium concentration (2%) of humic
acid containing pots (S2 – S5). Likewise, in Green Gram lower amount of humic acid containing
pot S6 (a) at 1% concentration shows high growth rate when compared to medium concentration
(2%) and high concentration (3%) of humic acid containing pots. Because, excess amount of
humic acid can destroy or decrease the microbial density in the soil (Fagbernro et al 1993). For
this reason the microbial density of all pots containing humic acid and bacterial culture were
carried out. It shows gradually increase their microbial density in lower concentration and
decreasing result in higher concentration of humic acid.
Finally in this study, green gram showed good growth in the S6 (a) sample where the ingredients
are added in the lower concentration (1%) and it consists of ground nut oil cake, protein
hydrolysate, Bacterial culture & humic acid. It shows all the ingredients are valuable and friendly
with each other. Lower growth rate in S6 (b) and S6(c) (ie., 2% & 3% concentration of this
biofertilizer).
Likewise, In black gram it showed good growth in the S6(b) sample where the ingredients are
added in the medium concentration (2%) it consist of ground nut oil cake, protein hydrolysate,
Bacterial culture & humic acid. Lower growth rate in S6 (a) & S6(c) (ie. 1% & 3% concentration
of this biofertilizer).
Conclusion:
Although all the ingredients are useful and stimulate the plant growth, adding microorganisms and
nutrient will facilitate the plant growth better when compared to chemical fertilizer. Ground nut
oil cake and humic acid prepared from decomposed plant materials can improved the growth of
plants. Therefore, the farmers can apply these ingredients for good production which are
economically cheap and better for plant growth. Moreover, these ingredients are ecofriendly and
helps in improving the soil fertility. Using biofertilizers we can keep the agricultural land
unpolluted and helps in producing healthy products with high yield.
INDIAN JOURNAL OF APPLIED MICROBIOLOGY
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30 RAMYA DINESH
ET AL
References:
1.
Ashwani Kumar & Satyawati Sharma (2008), An evaluation of multipurpose oil seed crop for
industrial uses (Jatropha curcus L): A review, Industrial crops and Products, INDCRO5087:1-10.
2.
Ahmed El-Naggar, Ahmed El-Araby, Andreas de Neergaard, Henning Høgh-Jensen (2008),
Crop responses to 15 N-labelled organic and inorganic nitrogen sources Nutrient Cycling in
Agro ecosystems, 80(1):49-60. DOI: 10.1007/s10705-007-9120-8.
3.
Chen Y P, Rekha P D, Arun A-B, Shen F T, Lai W-A, Young C C (2006), Phosphate
solubilizing bacteria from subtropical soil and their tricalcium phosphate solubilizing
abilities, Journal of Applied soil Ecology, 34:33-41.
4.
Jen-hshuan Chen (2006), The combined use of chemical and organic fertilizers and/or
Biofertilizer for crop growth and soil fertility, International Workshop on Sustained
Management of the Soil-Rhizosphere System for efficient Crop Production and Fertilizer
Use, Land Development Department, Bangkok, 10900 Thailand.
5.
Fagbernro J A & Agboola A A(1993), Effect of different levels of Humic acid o the growth
& nutrient uptake to teak seedlings. Journal of Plant nutrition, 16(8):1465-1483.
6.
Govindaraju K (2003), Studies on the Preparation and Characterization of Protein
Hydrolysates from Groundnut and Soybean isolates A. PhD thesis, Central Food
Technological Research Institute, Mysore.
7.
Hari Muraleedharan, Seshadri S and Perumal K (2010), Booklet on Biofertilizer
(Phosphobacteria), Shri AMM Murugappa chettiar research centre, Chennai.
8.
Lowry O H, Rosebrough N J, Farr AL, Randall R J (1951), Protein measurement with the
Folin phenol reagent. Journal of Biological Chemistry, 193(1):265-75.
9.
Nishant Mishra, Chandra Pal Singh and Mishra U S, (2011) Effect of Bio-fertilizers on Bionutrients, Nitrogen, Total Protein, Extractable Lipid and Mineral Contents of Cultivated
Variety of Fenugreek (Trigonella foenum graecum Linn.) Journal of Phytology, 3(8): 15-17
ISSN: 2075-6240.
10. Pettit R E (2004). Organic matter, humus, humate, humic acid, fulvic acid and humin: Their
importance
in
soil
fertility
and
plant
health
[Online].
Available
at
www.humate.info/mainpage.htm.
11. Rahman Akinoso and Idaresit VyaiEkaete (2011), Influence of Natural additives on Ground
Nut Oil yield and Cake quality, international Journal of science and Advanced Technology,
1(8):162-166, ISSN:2221-8386.
12. Rapel M Jingura, Downmore Musademba and Rutendo Metengaifa (2010), An evaluation of
utility of Jatropha curcas L as a source of multiple energy carriers, International Journal of
Engineering, Science & Technology, 2(7):115-122.
INDIAN JOURNAL OF APPLIED MICROBIOLOGY
Vol. 17 No. 1 Jan.-Jun. 2014
Humic Acid, Protein Hydrolysate & Microorganisms as a Mixed Consortium in Plant Growth
31
13. Stevenson F J (1994), Humus Chemistry; Genesis, Composition, Reactions, 2nd Edition, John
Wiley & sons, pg: 13,236-256.
14. Sumitra Ramachandran, Sudheer Kumar Singh, Christian Larroche, Carlos Ricardo Soccol,
Ashok Pandey (2007), Oil cakes and their biotechnological applications – A review,
Bioresource Technology 98: 2000–2009.
15. Vallini G, Pera A, Agnolucci M and Valdrighi M (1997), humic acids stimulate growth and
activity of invitro tested axenic cultures of soil autotrophic nitrifying bacteria. Journal of
Biology & Fertilizers of Soil, 24(3): 243-248.
16. Wang X P, Q Z & Li S G (1995), The effect of Humic acid on the availability of
Phosphorous fertilizers in alkaline soils, Journal of Soil Use Mange 11(2): 99-102.
17. William Wynn Pratt Pittom (1914), Studies in Protein Hydrolysis, biochemical journal, 8(2):
157-169.
18. Winget and Gold (2007), Effects of effective microorganisms on the growth of Brassica
rapa, Brigham young university of Hawaii. Bio493 Yuka Natano.
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