Industrial Biotechnology of Bio-fertilizers: Prospects and Problems

At farm level, IPNS (Integrated plant nutrient system) is the efficient management of plant nutrition sources passing through the soil/crop/livestock systems during crop rotation to optimize the crop productivity. It increases both the reserves in soil and plant nutrients in the system. The IPNS is a key component of agricultural production in order to meet growing demand of food and fiber by the size of farm that is decreasing. If system property is chalked out and tailored to the needs of the farmers it will empower small-scale farmers to increase their technical know-how and decision-making capacities to make adequate changes in plant nutrition systems to support agricultural intensification. The use of organic fertilizer and biofertilizer has increased crop productivity. Therefore the farm practices are to be changed in this direction. Bio-fertilizers [BF] are microbial inoculants that are carrier-based preparations containing beneficial microorganisms in a viable state and designed to improve soil fertility. BF are of agronomic value in nitrogen fixation, phosphate solubilisation, and release of plant growth promoters. BF are intended for seed or soil application. BF help plant growth by increasing the number and biological activity of the desired

microorganisms in the root environment. The microbial classes utilized in BF are listed in Table1 below. Rhizobia are most effective in converting atmospheric nitrogen to ammonia in symbiosis with legumes. Azospirilla enhance root biomass and fix nitrogen in associative symbiosis with cereals, sugarcane and cotton. BF restore natural soil properties.

1 2 3 4 5 6

Rhizobium Azotobacter Azospirillum Cyanobacteria Phosphate solubilizing bacteria VAM fungi

Although general awareness, production and use of BF started in the eighties, BF is yet to become an important component of IPNS in farming and forestry along with chemical fertilizers and organic manures. Popularizing BF to augment N and P supply, promoting balanced use of chemical fertilizers [NPK] based on soil testing and correlation of secondary and micronutrient deficiencies in soil, water and nutrient management and other agronomic practices that maximize nutrient utilization should be paid full attention.
2

The available data indicate that the current production [in 2002] of BF is around 5000 tonnes. According to an estimate of the potential for BF usage in India based on different agricultural and horticultural crops, the requirements for BF in 2005 AD would be 2.5 lakh tones. Unlike the manufacture of chemical fertilizers which is a well established large scale industrial practice, the industrial biotechnology of producing bio-fertilizers is relatively smaller in scale and needs quality control and soil quality and plant species specific factors to be established and standardized.

INDUSTRIAL BIOTECHNOLOGY OF BIOFERTILIZERS: PROBLEMS & PROSPECTS

The dawn of the new millennium poses tough challenges to the farming community. Producing more food to feed the burgeoning population from shrinking land and less water, without eroding ecological foundation, will be an uphill task. The surest means to tide over this challenge is through environmentally sustainable farming methods, which are economically rewarding and intellectually stimulating. Microorganisms play an important role in various chemical transformations in soils and thus, influence the availability of major nutrients like nitrogen, phosphorus, potassium and sulphur to the plants. A few microorganisms such as

nitrogen fixing bacteria and phosphate solubilisers can be utilized to partially augment the supply of major nutrients. Rhizobium, Azotobacter, Azospirillum, Blue -green algae and phosphate solubilizing bacteria can be used as Biofertilizer to increase the crop production. Using a

judicious combination of chemical fertilizers, organic manures and Biofertilizer evolves a strategy for integrated nutrient supply.

Farmers knew legumes effect for ages.

Inclusion of

legumes in cropping system was, therefore, a common practice rather than an exception, but the reason of soil fertility improvement due to inclusion of legume was unknown. After discovery of Rhizobium in 1886, it was revealed that it was the Rhizobium which lived in the root of legume fixed N from the atmosphere. Since then addition of Rhizobium from external source came into practice to increase the native population to enhance the quantum of biological nitrogen fixation. Subsequently, other groups of N-fixers and phosphate solubilisers were discovered, and inoculation of these microorganisms also came in vogue Vesicular Arbuscular Mycorrhiza (VAM) occurence in the nature is more of a rule than an exception and has symbiotic relationship across the cultivated plants-agricultural,

horticultural and forest species. It is fast acquiring popularity as a bio fertilizer. The fungi grow from the cortex of the host plant root and send thread -like hyphae out into the soil, which facilitate the roots in the uptake of mineral nutrients. Phosphorus is essential plant nutrient but making it available to plant is critical because of very low solubility of phosphate. The application of mineral P-fertilizer is also not much encouraging due to its rapid fixation into insoluble
5

phosphorus compounds and its consumption in India is also not adequate. Large deposits of rock phosphate in India are still remained unused as a fertilizer for its low grade. Based on these circumstances, emphasis has been given to use Psolubilising Biofertilizer, which have been found useful in making phosphorus available to plants through solubilisation, converting low-grade rock phosphate as fertilizer and increasing yield of different crops. The demand for this

particular Biofertilizer is increasing gradually and many Biofertilizer units have started its production commercially. The status of P-Biofertilizer depends on its mode of action, strain isolation and production technique, constraints and remedies. At present, al India capacity for production of Biofertilizer is estimated to be around 8000 tonnes as against the current production of about 5100 tonnes. The potential requirements for bio-fertilizers for various crops, trees and horticulture can be as high as 2.5 lakh tones. Unlike the chemical fertilizer production, for biofertilizers scaling up to produce in large continuous process factories would be a tough task. Integrated nutrient management: Integrated

nutrient management (INM) is emerging as an economic

6

and environment friendly system for ensuring sustainable agriculture. In INM, biofertilizer component has not

received adequate attention in the past. Keeping this in view, a National Network Project was launched in 1999 for 3 years on the Role of biofertilizers in INM embracing 17 centers throughout the country. The objective was to establish useful biofertilizer packages for yield

optimization of some major crops in various cropping systems in different regions. In addition to generating field data on the biofertilizers used in integration with reduced levels of chemical fertilizers, advanced molecular tools were employed for genotypic characterization of microbial strains for their identification and performance evaluation in the field. Use of biofertilizers in INM under actual

conditions of farming was the basic objective of the project. The important outcome is summarized here. Poplar-eucalyptus-based cropping system Studies at TERI, New Delhi have shown that soil biological health improves with the inoculation of suitable microorganisms resulting in the yield and biomass. The maximum costbenefit ratio of application of biofertilizer was observed in the agroforestry model. However, there is still need to undertake multi-location validation trials.
7

Mungbean-wheat-bajra-based cropping system The seed treatment of Rhizobium ( R )+ phosphate solubilising bacteria (PSB or PSM)+ organic matter (OM) @ 5t/ha has produced significantly higher yield in green gram at Akola, Maharashtra. The residual effect was prominent on the succeeding wheat crop. The grain yield of wheat crop was at par with 25% recommended dose of freshly applied chemical fertilizers. Seed treatment in bajra crop with

biofertilizers enhanced the grain yield significantly. PSB+OM @ 2.5/ha + Azospirillum was found most effective. Its residual effect on the succeeding chickpea crop was pronounced in the same treatment. When the two crops were clubbed together to test the significance of the treatments in bajra-chickpea cropping system, seed treatment of Azospirillum +2.5t O.M./ha found to be better. Application of Zn+PSB was the best treatment in mungbean. The seed treatment with Zn+Mo+R+PSB was beneficial in getting higher grain yield of rajmash. There is definite relationship of varieties and rhizobial isolates in growth parameters and grain yield of rajmash. Variety VL6d3 gave more response to rhizobial isolate AKRR-3 and AKRR-4. Frontline demonstrations also revealed that

combined application of biofertilizers is more beneficial than single inoculation. Rice-wheat-based cropping system Yield of paddy and wheat was significantly higher at IARI New Delhi with inoculation of blue green algae (BGA), PSB, and mycorrhiza (AM) with 30 kg P2O5 as rock phosphate (RP), as compared with the application of 60 kg P2O5 as single super phosphate (SSP), as well as over control. Soil health improved in terms of carbon buildup, residual soil nitrogen and soil phosphorus. The inoculated organisms established well and enhanced the total microbial population. These results have clearly demonstrated that 30 kg P2O5 as SSP can safely be replaced with 30 kg P2O5 as RP in presence of BGA, AM and PSB as inoculant. An additional yield of paddy (1.91 q ha-1) and wheat (2.31 q ha-1) resulted with a net saving of Rs. 2907 as phosphatic fertilizer input cost. Rice-based cropping system Experiments conducted at Nimpeeth, West Bengal indicated that Azolla alone could replace 25% of the chemical nitrogen (20 kg ha-1) in rice cultivation during the kharif season, leaving significant residual effects for subsequent mustard, potato, cotton and lentil crops. Effect of Azotobacter application was

significant in potato and cotton as was that of Rhizobium in

9

lentil. The residual effect of a combination of Azolla and Azotobacter or Rhizobium could replace 50% of the chemical nitrogen (100 Kg ha-1 in potato and 40 Kg ha-2 in cotton and 10 Kg ha-1 in case of lentil) A biofertilizer-based INM package (OM @1-3t/ha dry weight basis, Azospirillum amazonense A10, Bacillus megaterium P5, P2O5 @ 10 kg/ha as RP and K2O @ 10-50 kg/ha) for rice-legume-rice and rice-rice cropping systems has been developed at AAU, Jorhat. It was demonstrated that the biofertilizer established itself in the rice rhizosphere on the evidence of protein profile, antibiotic resistance profile and the use of antibiotic resistant mutants of the strains. The input cost of the INM package was lower than that of the recommended dose of fertilizer. The suitability of this package was demonstrated in farmers fields at 17 sites during 2000-2002. Sugarcane-based cropping system INM approaches were also effective in terai soil of Uttaranchal specifically with biofertilizer + farm yard manure (FYM) with 50% inorganics in terms of nutrient efficiency. It was found that 66% inorganic fertilizer + biofertilizer, or 50% inorganic fertilizer with biofertilizer +FYM was beneficial in terms of microbial dynamics and soil health. Yield of sugarcane
10

was maximum in biofertilizer amendment while ratoon was comparable to inorganics. Yield of lentil was similar to that in inorganic treatment in terms of cost. Better oil

yields were obtained from the subsequent mentha crop supplemented with 59% inorganics with biofertilizer + FYM. Biofertilizer with 66% of recommended dose of inorganic fertilizer gave similar oil: herb ratio as in 100% inorganics- supplemented crop. Among all cropping

systems, sugarcane-ratoon-lentil was observed best in terms of soil health, yield, nutrient efficacy and INM practices. The experiments conducted at IISR, Lucknow indicates that there is significant increase in crop yield when Acetobacter is used alone or in combination with Azospirillum. Azotobacter is individually used without affecting the sugarcane juice quality. The improvement in the cane yield was mainly due to increased tillering and improved average cane weight. Effect of bio-inoculants was more at 75 kg N/ha than at the recommended dose of 150 kg N/ha. Overall, there was a gain of 5-8 tones/ha due to inoculation of Acetobacter alone and also in combination of Azotobacter and Azospirillum. Rice-wheat-pulse-based cropping system At IARI, New Delhi, inoculation of BGA and Azolla alone or in
11

combination increased the tiller number and plant height of rice with increase in root weight over control in all

biofertilizer treatments irrespective of nitrogenous fertilizer levels. Grain yield and straw yield showed a significant enhancement with increase in nitrogen fertilization. Treatment of BGA and Azolla in combination alongwith 60 kg Nitrogen (N60) per hectare of urea resulted in the maximum percent increase in grain yield and with 30 kg N per hectare of urea in straw yield. The Azolla

multiplication also increased with increasing levels of nitrogen with highest biomass observed at N90. nitrogen fixation activity of inoculated Azolla. The

nitrogen level also seems to have no adverse effect on A

significant increase was observed in population of BGA in plots inoculated with BGA alone or in combination with Azolla. Nitrogen fixation by BGA + Azolla as observed in soil core was maximum at N30 +BGA + Azolla followed by N60+BGA+Azolla. Saline area cropping system Over 200 PSM strains of bacteria, mycorrhizal fungi and yeasts were isolated from saline soils of Gujarat. Among these, four cultures were selected on the basis of salt tolerance and phosphate solubilising activity with different inorganic phosphates
12

and native forms of phosphates. The organisms showed increasing phosphate Solubilising (PS) activity with increase in NaCl concentration in liquid medium. The cultures also showed significant results with different sources of P-fertilizers against respective controls for groundnut and wheat plants under green house condition. The use of these four cultures in the field experiments saved 50% P-fertilizer with more grain yield of groundnut, pearl millet and wheat. Molecular methods for evaluation of microbial

inoculants: At SPIC Science Foundation, Chennai, GUStagged Rhizobium and, Bradyrhizobium were used to study nodule occupancy in legumes. Similarly, GUS tagged

Azospirillum and Acetobacter were used to study plant colonization and to check their quality. PCR-DNA fingerprinting as well as PCR-RFLP analysis were standardized to distinguish and classify microbial strains using specific and random primers. In another experiment conducted at MKU, molecular for metabolic characterization and plant growth promoting

fingerprinting,

rhizobacteria (PGPR) pseudomonads were classified on the three major plant growth promoting characteristics such as IAA production, phosphate solubilization and antifungal
13

activity. Antifungal principle (siderophores or antibiotics) of PGPR-pseudomonads was confirmed by the pattern of inhibition of fungal growth, with and without iron supplementation. The siderophore produced by the PGPR Pseudomonads was confirmed as hydroxymate type. PCRRAPD analysis and construction of similarity dendrogram facilitated differentiating the PGPR-pseudomonads within the species. Specific RAPD marker of PGPR-

pseudomonads suggested the use of PCR analysis of 16S and 23S rRNA genes for direct selection of pseudomonads at the genus level. Fingerprints of PGPR generated by ARDRA analysis gave a reliable method of differentiation of the PGPR-pseudomonads within the species. A method was developed for extraction of soil DNA for PCR reaction. Research and development projects: At TERI, Delhi, it has been seen that composting of various organic wastes takes 9-10 weeks. Diverse microorganisms inhabit the

compost at different intervals; all mature composts are good sources of cellulose-degrading microorganisms, particularly vegetable compost and sewage sludge. Both these composts have also been shown to be rich sources of protein-degrading microbes. These functional properties
14

were taken into account in the development of mixed consortia of microbes that can be used as inoculants for decomposition. It was also observed that the composts were ready when the C:N ratio narrows down from 10:1 to 15:1. The end product of all the degraded organic wastes had a C: N ratio ranging from 8:1 to 14:1. Among all the composts, sewage sludge and poultry manure had the highest available phosphorus concentrations. Poultry manure is

found to be a rich source of potassium which can be used as a fertilizer in crops requiring high potassium amendments. For vermicomposting, leaf litter and semi-degraded cow dung were found to be the best substrates. The worms fed on these substrates showed the maximum increase in population. Vermicomposting in the cement pots took

longer time than when done on the polyhouse slabs (four weeks) indicating that moisture and aeration affect the vermicomposting process. Twenty seven teak plantations in Kerala were studied by KFRI, Thrissur to collect information on their AM fungal root infection (mycorrhizal status), AM fungal diversity, physical and chemical characteristics for rhizosphere soils. Teak plants raised in different eco-climatic zones and belonging to different age groups (4 to 50 years-old)
15

showed a range of 7 to 59% and a mean of 33.66 percent in AM fungal root infection. Physical and chemical factors of the soil were found influencing the root infection. AM fungi showed high diversity in teak rhizosphere soils and distribution, species composition, as well as spore density depending on various edaphic and environmental factors. All the six genera of glomalean fungi were encountered and among these Glomus predominant ones. Development of transgenic biofertilizers: A network project involving 11 centres was launched during the year for development of transgenic biofertilizers with better nitrogen fixing and phosphate solubilization ability. Rhizobium The role of the dicarboxylic acid transport system in the Mesorhizobium ciceri-Chickpea symbiosis is being investigated at IARI, Delhi. Transposon Tn5 mutagenesis was employed to generate mutants defective in C-4 dicarboxylate transport system. Five mutants and Acaulospora were the most

designated SD-1, SD-2, SD-3, SD-4 and SD-5 that are unable to utilize succinate, fumarate and malate were taken as an initial indication of a defect in the dicarboxylic acid transport system as all three dicarboxylic acids are known to share a common transport system in other bacteria.
16

However, they all grew normally on glucose, ribose, pyruvate, citrate as the sole carbon source. To analyze the stability and the randomness of transposon insertion as well as for the localization of the DNA region responsible for the C-4 dicarboxylic acid transport system, all the five putative mutants were selected and analyzed by Southern hybridization. Hybridization results indicated that signals were obtained for all the mutants and the transposon Tn5 was identified on 17.5 and 11.0 kb EcoR1 fragments. In addition microscopic examination of roots from plants inoculated with the wild type strain of chickpea rhizobia revealed large pink color nodules. However, the nodules present on the roots of plants inoculated with mutant strains were small and white, characteristics typical of ineffective nodules and anatomical results confirmed this. Screening of strains was carried out to identify

Bradyrhizobium sp. (Vigna) strain which can produce bacteriocin. Of the 50 strains screened, four (M-2-97, CP 20, CP 34 and K6) were found to produce bacteriocins. Antibiotic resistance profile for all the four strains was developed. Of the four strains tested for nodule occupancies, one exhibited superior nodulation

competitiveness against the native population and could

17

occupy 45% of the nodules. Of the 57 native rhizobial isolates tested on green gram, 40 were inhibited by M-2-97, thereby indicating that it produces a potent

bacteriocin/toxin that allows the strain to occupy more nodules on the roots of the host. Phosphate-solubilising fluorescent bacteria were Fifty isolated strains from of the

pseudomonads

rhizosphere of rice and sugarcane at MKU, Madurai. The identities of these were confirmed by biochemical tests and PCR with rRNA gene-specific primers. Among them, 94% of fluorescent pseudomonads isolated from the sugarcane rhizosphere were PSBs. To assign molecular markers for these genetic relatedness among the PSBs and non-PSBs, PGPR strains were studied by RAPD with three different primers and ARDRA fingerprints from HaeIII digestion of 16S rDNA. Azospirillum Phosphoenol pyruvate carboxylase (pqq) synthase gene has been transferred to Azospirillum at UAS, Dharwad. It was found that the transconjugant Azospirillum now possess mineral phosphate solubilizing (MPS) ability. No change was observed in the nitrogen fixation of these transconjugants. Later pqq synthase gene was isolated

from Burkholderia cepacia via PCR amplification of

18

primers designed using the conserved sequence across the gene reported in different species and cloned into E coli. The E coli containing the pqq synthase gene now showed MPS activity. These results indicate the possibility of

developing transgenic bacteria by cloning pqq synthase genes into certain gram-negative rhizobacteria. The gene has also been electroporated into Rhizobium and

Azospirillium ACD 15 and 20 and they have shown MPS activity. Mineral phosphate-solubilising bacteria Glucose

dehydrogenase gene (gdh) of Bacillus licheniformis has been successfully amplified at the University of Hyderabad using gene-specific primers. The amplicon (802bp) was then cloned into pTZ57. The clone was confirmed as gdh gene by sequencing. Primers have been designed for the gdh sequences of Serratia marcescens and Ppseodomonas aeruginosa and the genes would be amplified using specific primers. Pure Genomic DNA has been isolated using Attempts are being made to construct a

QIAGEN kit.

library in pUC18. The library would be screened for the gdh gene clone based on the zone of solubilization. Selected recombinants would be probed for the presence of the gene using heterologous gdh probes.
19

Hyphal fusion in mycorrhiza Conditions of in vitro growth of AMF species Glomus intraradices have been optimized at TERI, New Delhi. Clonal variation of hosts on symbiosis was studied using four clonal cultures of carrot and one of clover. A working system for Initial

compartmentalization was designed and tested.

efforts of hyphal fusion and repairing was successful in case of G.intraradices and currently, compatible species selection among various AMFs such as Gigaspora margarita, G. gigantean and two species of Glomus in vitro is underway. Besides this several monosporal cultures of these are successfully raised and hyphal fusion to develop new isolates. Transfer of technology and launch of product: Mycorrhiza, colonise 85% of land plants, offers 50 percent saving in chemical phosphorus fertilizer, higher resistance to soil-and-root borne pathogens, and helps in wasteland reclamation by providing extended arm to plant root system. A broad-spectrum mycorrhizal biofertilizer has will be used for

been launched in the market by two industries (M/s Cadila Pharamaceutical Ltd., Ahemdabad, and KCP Sugar and Industries (Pvt.) Ltd., Chennai) to whom DBT-TERI, New Delhis technology was transferred. KCP Suger Industries
20

also received the All India Biotechnology Association Award for production and marketing of the mycorrhizal biofertiliser.

21