S. Mutturi, V. Sahai, S. Sharma, and V.S. Bisaria
Abstract
This chapter provides a brief overview of submerged cultivation method- ologies of Pseudomonas-based microbial inoculants, which when deliv- ered as a formulation improves the health of the host plants. The classifications of bio-inoculant delivery systems in various agronomical applications are discussed in the initial section of the chapter. Among various modules in the supply chain of bio-inoculant development, the chapter deals with medium development and cultivation strategies for successful production of active ingredients. The chapter specifically explores the mass propagation strategies of two potential Pseudomonas strains in submerged cultivation with emphasis on fed-batch mode of cultivation.
Keywords
Pseudomonas • High-density cultivation • Microbial inoculants • Agronomy
10.1 Introduction
Microbial inoculants/biofertilizers are consid- ered as a step towards achieving sustainable agri- culture systems. Bio-inoculants are ready to use live formulates of beneficial microorganisms, which when applied to soil, roots or seeds enhance the availability of different nutrients to the plant by their inherent metabolic activities (Bashan 1998). These delivery systems to host plants essentially consist of plant growth- promoting rhizobacteria (PGPR); the term coined S. Mutturi (*)
Microbiology and Fermentation Technology Department, Central Food Technology Research Institute, Mysuru 570 020, India
V. Sahai • S. Sharma • V.S. Bisaria
Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110 016, India
e-mail: [email protected]
by Kloepper and Schroth (1981) encompasses those bacteria that are able to colonize plant root systems and promote plant growth. It includes all bacteria of rhizosphere origin that promote plant growth. However, PGPR were further subclassi- fied into two groups based on their mechanisms of action according to Bashan and Holguin (1998):
Plant growth-promoting bacteria (PGPB): These are bacteria whose metabolites or their precursors are used as growth regulators.
Biocontrol PGPB: The metabolites released from these bacteria are involved in imparting antagonistic action against microorganisms that are detrimental to plants by means of different direct and indirect modes.
The above definition does not include fungi and class of bacteria that are not essentially rhi- zobacteria. In the last decade, several fungi have been reported to exert beneficial mode of action on host plants in improving their health. Hence, the definition has been updated to plant growth- promoting microorganisms (PGPM) according to Owen et al. (2015). As this term seems to be more generic, it will be used in this chapter for referring to PGPR. PGPM are classified as fol- lows (Owen et al. 2015):
PGPM bacteria: Further classified as intracellu- lar (belonging to genera of Rhizobium,
Azorhizobium, Bradyrhizobium,
Allorhizobium, Mesorhizobium,
Sinorhizobium, etc.) and extracellular (belong- ing to genera of Bacillus, Burkholderia, Paenibacillus, Erwinia, Stenotrophomonas, Micrococcus, Flavobacterium, Streptomyces, Serratia, Azospirillum, Agrobacterium, Actinomycetes, Pseudomonas, Arthrobacter, etc.). Intracellular bacteria, primarily all rhi- zobium species, function as nutrition suppli- ers, whereas extracellular bacteria play the roles of bio-stimulants, bio- protection and bioremediation apart from being nutrient suppliers.
PGPM fungi: These are classified as root- associated fungi (RAF) and mycorrhizae. RAF includes Aspergillus, Trichoderma, Penicillium, Saccharomycetes, etc. Although
Piriformospora indica (Varma et al. 1999) has not been mentioned in Owen et al. (2015), this potential endophytic fungus seems to be fit- ting in RAF category for its PGP activities. Mycorrhizae are subclassified as (EcM) ecto- mycorrhiza (Thelephora, Pisolithus, Rhizopogon and Scleroderma) and (AM) arbuscular mycorrhiza (Rhizophagus, Glomus, Funneliformis, Claroideoglomus, Gigaspora and Scutellospora). RAF and mycorrhizae also span all the functional abilities as extra- cellular bacteria except that mycorrhizae do not act as bio-stimulants.
There are several bio-inoculant products sold across the globe, which contain either products (including viable cells) derived from pure culture or consortium of different inoculants marketed by companies. Some of the trademark names with active ingredient (ai) composition are listed in Table 10.1.
As the chapter focuses on fluorescent pseudo- monads, the microbial description of traits for plant growth promotion is described here. Pseudomonas is the most important genus in the
order Pseudomonadales, family
Pseudomonadaceae. A group of bacteria among genus Pseudomonas, which produces yellow- green fluorescent water-soluble pigments, is termed as fluorescent pseudomonads. The exhaustive list of mechanisms and role of PGPM in phyto-promotional activities is well detailed in reviews by Lugtenberg and Kamilova (2009) and Podile and Kishore (2006). Figure10.1 depicts some of the direct and indirect mechanisms exhibited by fluorescent pseudomonads for improving the plant health.
The culture broth of the pseudomonad or other PGPM containing potential metabolites, which form the active ingredients listed in Fig.10.1, is usually formulated into a deliverable form (either liquid or carrier-based formulations) that is applied to host plants to exert growth promotion. The entire supply chain of such bio-inoculant development process is depicted in Fig.10.2. In brief, an organism of interest is screened from the isolates obtained from the rhizosphere of a certain plant as a first step. Later, the screened organisms
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Table 10.1 A few commercially available bio-inoculant productsa
S. No. Product name Company Active ingredient (ai) PGPM category
1 Promot JH Biotech Inc,
USA
30 cfu/g Pisolithus
tinctorius
Ectomycorrhiza
2 Mycormax JH Biotech Inc,
USA
25 cfu/g Glomus
intraradices
AMF 25 cfu/g Glomus mosseae 15,300 cfu/g Pisolithus
tinctorius
3 Endomycorrhizal Inoculant (BEI)
BioOrganics, USA Glomus aggregatum, G.
etunicatum, G. clarum, G.
deserticola, G.
intraradices, G.
monosporus, G. mosseae,
Gigaspora margarita and
Paraglomus brasilianum
AMF
4 Viva Roots AgBio Inc, USA Endomycorrhizae mix Mycorrhiza 5 Bactofil A 10 Agro. Bio Hungary Azospirillum brasilense,
Azotobacter vinelandii,
Bacillus megaterium,
Bacillus polymyxa,
Pseudomonas fluorescens,
Streptomyces albus as well as other agents. 4.3 × 109
cells/mL
Bacteria (extracellular)
6 Bactvipe International
Panaacea Ltd., India
Pseudomonas fluorescens Bacteria (extracellular) 7 Microbion UNC Syn-Bio-Tech Ltd,
Hungary Azotobacter vinelandii-B 1795, Bacillus megaterium B1091, Clostridium pasteurianum, Azospirillum sp., Bacillus subtilis, Rhodobacter sp., Lactobacillus sp., Trichoderma reesei, Saccharomyces cerevisiae, Streptomycessp. 4.0 × 1010 cells/g Bacteria (extracellular)
8 Nodulator XL BASF Rhizobium leguminosarum
biovar viceae
Bacteria (intracellular)
9 Vault SP BASF Bradyrhizobium sp.
(Arachis), 2.0 × 109cells/g
Bacteria (intracellular) 10 Rhizo-Flo BASF Consortium of rhizobium Bacteria (intracellular)
11 Primo Verdesian Life
Sciences
High load of Rhizobium Bacteria (intracellular) 12 Accolade-L Verdesian Life
Sciences
Azospirillum brasilense
strains
Bacteria (extracellular) 13 Kodiak HB Chemtura Bacillus subtilis, 6.0 × 109
spores/g
Bacteria (extracellular)
14 Poncho/Votivo Bayer Bacillus
firmus + Clothianidin, 2.0 × 109cfu/ml
Bacteria (extracellular)
(continued) 10 Strategies for High-Density Cultivation of Bio-inoculants in Submerged Culture…
are established by microbiological and genetic tools for their plant growth-promoting character- istics. Usually the secondary metabolites whose traits exert growth promotional activities (direct or indirect) in the host plant are qualitatively screened by analyzing the gene sequence respon- sible for their synthesis in the metabolic path- ways (Gaur et al. 2004). In the process
development stage, nutritional medium is designed for cultivation of the organism. Here in this stage, efforts should be made to design a syn- thetic medium instead of complex medium as complex sources would act as bait for contamina- tion if any residual components are present dur- ing formulation and storage (Saharan et al. 2011). Fluorescent pseudomonads enjoy the advantage
Table 10.1 (continued)
S. No. Product name Company Active ingredient (ai) PGPM category
15 Grandevo Marrone Bio
Innovations
Chromobacterium subtsugae strain PRAA4-1 + spent medium
Bacteria (extracellular)
16 Jumpstart Novozymes BioAg Penicillium bilaiae, 7.2 × 108cfu/g
RAF (extracellular) 17 TagTeam MultiAction Novozymes BioAg Penicillium bilaiae,
3.7 × 106cfu/g
RAF (bacteria consortium)
Rhizobium leguminosarum, 7.4 × 108cfu/g
18 BlightBan A506 Nufarm, Australia Pseudomonas fluorescens
A506, 71 % of ai
Bacteria (extracellular)
aData collected from Owen et al. (2015), product lists of various companies and other web sources such as www.seed-
quest.com
Fig. 10.1 Various mechanisms by which fluorescent
pseudomonads influence plant health (Adapted from Podile and Kishore2006). ISR induced systemic resis-
tance, LPS lipopolysaccharide, DAPG diacetylphloroglu- cinol, ACC deaminase 1-aminocylopropane-1-carboxylate deaminase
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of assimilating glycerol as the primary carbon source, which is selective to some extent. Once the medium is designed, process conditions for submerged cultivation need to be standardized in a bench-scale reactor, and later the process is scaled up to higher volumes. The culture broth obtained is subsequently formulated for storage and delivery to host plants. Finally the supply chain ends with enumeration of target growth parameters in controlled or open field conditions and also their possible detection from the test plant rhizosphere using molecular tools (Mathimaran et al.2008).
The chapter focuses on methodologies used for development of nutritional medium for culti- vation of bio-inoculants (the upstream compo- nent of Fig.10.2), and the process strategies that are used for their mass multiplication in bioreac- tors with a view to get their high colony-forming units (cfu) count along with the desired metabo- lites having PGP properties (the bioreactor engi- neering component of Fig. 10.2) with special reference to Pseudomonas spp.
10.2 Medium Development
and Cultivation Strategies
for Pseudomonas-Based
Bio-inoculant Production
10.2.1 Medium Development
Development of medium for cultivating potential PGPM is critical during production and formula- tion with respect to contamination. In most cases, the obtained bio-inoculant broth post submerged cultivation is formulated into a delivery system and later stored under shelf. This gestation during storage may allow room for contaminants to pro- liferate on nutritional components present in the residual cultivation medium. Especially when complex sources such as peptone and yeast extract are used, such contamination risk would be pronounced. Hence, a simple synthetic medium should be designed and optimized for submerged cultivation. One of the functional needs for optimizing medium components is to improve the production levels of biocontrol
Microbiology component Isolation of microorganism from rhizosphere Delivery to host plant Screening for PGP properties Formulation (Liquid or carrier- based) Efficacy enumeration and molecular detection Large-scale cultivation Bench scale cultivation Genomic studies for characterizing PGP properties Development of nutritional medium for maximizing PGP properties Upstream component Molecular component
Downstream component Bioreactor engineering component
Fig. 10.2 Supply chain for bio-inoculant production and delivery to host plant
agents in the active ingredient so that they not only keep check on microbial contaminants dur- ing shelf storage but also initiate defence mecha- nisms even before the colonization by the strain of specific root niches. For instance, siderophore and 2,4-diacetylphloroglucinol (DAPG) are the compounds released by fluorescent pseudomo- nads contributing to their defensive mode of action against antagonists (Saharan et al. 2011). If the synthesis level of these compounds is increased during submerged cultivation by manipulating the medium components, then it would serve its intended purpose.
Most of the medium development strategies for extracellular PGPM rely on existing synthetic medium that has been used for some purpose other than that of bio-inoculant production. This would perhaps be the starting point for medium design, and the challenge would be the inclusion of nutritional components for production of com- pounds which exert the effects of bio-stimulation, bioremediation and bio-protection. The follow- ing section will focus on medium development for two such as PGP traits of fluorescent pseudo- monads, namely, siderophore (iron-chelating agent) and DAPG (antifungal polyketide).
The production of siderophores by strains of Pseudomonas spp. depends on several nutritional and environmental factors as detailed by Elena and de Villegas (2007): ferric ion concentration (Meyer and Abdallah1978; Budzikiewicz1993; Laine et al. 1996; de Villegas et al.2002), carbon and nitrogen source (Albesa et al. 1985; Park et al. 1988; Duffy and Défago1999) and phos- phate concentration (Barbahaiya and Rao1985; Défago and Haas1990). The critical factor among these is the concentration of ferric ion in the cul- ture medium. The concentration of iron in the vicinity of 10μM is considered good enough to yield biomass with modest levels of siderophores (Neilands1984). In the strain Pseudomonas fluo- rescens 94, the siderophore levels were high even at 50μM of Fe+3concentration (Manninen and Mattila-Sandholm1994). Co+2, fructose, mannitol and glucose increased in vitro production of pyo- chelin by P. fluorescens, while NH4Mo+2, glycerol and glucose increased the production of its pre- cursor salicylic acid (Duffy and Défago 1999).
Bultreys and Gheysen (2000) found that the strains of Pseudomonas syringae produced pro- nounced levels of siderophore when amino acids were used as the sole source of both carbon and nitrogen. In studies conducted by Meyer and Abdallah (1978), citric and succinic acids were used as sole carbon sources for producing sidero- phore in Pseudomonas fluorescens strains. As described earlier, glycerol has been widely used as the carbon source for Pseudomonas spp. in dif- ferent media, including the standard King’s B medium (Nowak-Thompson and Gould 1994). The effect of nature of carbon source is remark- able on growth during mass multiplication of the Pseudomonas strains. Sugars like glucose and sucrose caused digression of pH from near neutral to below 6.4, while organic acids like citric or suc- cinic acid cause upward pH digression to 7.4 and above, while the use of glycerol causes relatively small digressions (Saharan et al. 2010). The extreme digressions slow down the growth rate considerably leading to low productivities. During inoculum development in shake flask, such digres- sions in pH may result in very low optical density and larger lag periods, thereby necessitating the use of a larger inoculum size. In addition to car- bon source, nitrogen source also causes digres- sion in pH substantially upwards or downwards. A synthetic medium was developed (Saharan et al. 2010) using glycerol (as a carbon source) and urea and ammonium sulphate (as dual nitro- gen sources) which was able to contain pH digressions within 7.0 ± 0.2 during fermentation. The use of such a medium is desirable in shake- flask cultures where pH control is not possible. Glycerol metabolizes very slowly due to very low level of key enzymes, glycerol kinase and glyc- erol phosphate dehydrogenase, involved in its catabolism. The presence of citrate or succinate at low levels (0.05 %) in the medium increased activity of the glycerol kinase almost 15-fold causing rapid utilization of glycerol (Saharan et al. 2010).
In submerged cultivation, antibiotic produc- tion by many organisms is influenced by the type and abundance of carbon and nitrogen sources. Phosphate, iron and micronutrients modulate antibiotic production (Weinberg 1977; Slininger