1. Introduction
The agriculture sector faces significant new challenges associated with climate change, resistance to biopesticides and soil degradation. An emerging solution towards sustainable agricultural practices is the use of cost-effective, environmentally friendly biofertilisers. Biofertilisers are a suitable alternative to chemical fertilisers and exploit native microorganisms present in the soil, known as plant growth-promoting bacteria (PGPB). Such products are termed ‘bioinoculants’, as they induce rhizospheric colonisation with the target bacterium rather than merely supplying nutrients to the soil as chemical fertilisers do.
The use of PGPB in sustainable agriculture has gained importance in the past decade due to their beneficial effects on soil and crop productivity. PGPB exhibit different mechanisms of action in their interactions with plants, leading to improved productivity through various means. Those mechanisms can be direct (production of phytohormones, nitrogen fixation and phosphate solubilisation) or indirect (production of antibiotics, iron-sequestering siderophores, cell wall degrading enzymes and quorum quenching) [
1,
2]. PGPB can be classified based on their mode of action as biofertilisers, phytostimulators, biopesticides and bio-remediators. Biofertilisers are used to promote growth by supplying or increasing the availability of nutrients. Phytostimulators can regulate the concentration of growth regulators. Biopesticides allow plant growth by killing phytopathogenic agents, usually through the production of antibiotics. Bioremidators exhibit the ability to sequestrate heavy metals [
3]. Most research in biofertilisers focuses on the understanding of the diversity, importance and mechanisms of soil PGPB and their beneficial role in agricultural productivity [
3]. There are a wide range of bacterial species with plant growth-promoting effects (PGPE), including Alcaligenes, Arthrobacter, Azospirillum, Azotobacter, Bacillus, Burkholderia, Enterobacter, Klebsiella, Pseudomonas, and Serratia, among others [
4]. The mode of action of these PGPB varies according to the host plant. However, it is also influenced by several other biotic factors such as the soil composition, the developmental stage of the plant, the diversity of the associated microbial communities in the soil, and the climatic conditions. Those factors define the specificity of PGPB-plant interactions, providing a wide range of potential products. However, the ideal candidate for large scale production would be a PGPB that exhibits pleotropic effects, covering more than one mode of action in more than one host plant.
Successful inoculants that have been reported to be effective biofertilisers have typically been cultivated in shake flasks. This production method presents several inconveniences, starting with the lack of tight control of growth parameters [
5]. Recently, an increasing number of studies have aimed to improve biomass production of a particular PGPB. In most cases, liquid fermentation was studied, and the key focus was to optimise the medium composition. Interestingly, most studies report the use of complex, rich media, despite the issues that complex media introduces (expensive materials, batch to batch variation and little reproducibility) [
6]. Reported media compositions have included different substrates such as glycerol [
7], yeast extract [
8], sweet potato wastewater [
9], chicken manure [
10] and whey. While it is clear that the use of these complex mixtures leads to higher biomass yields, their complexity and batch-to-batch variability make it difficult to generate a standardised product. In this regard, several efforts have been implemented to develop an optimised culture medium composition that yields high cell densities [
6].
The market for PGPB inoculants commercially available is limited, representing less than 5% of the total chemical fertiliser market [
11]. Amongst the available products, liquid formulations are preferred because they are easier to process and imply lower production costs. However, it has been reported that many current commercial products are low-quality bio inoculants. This is associated with low cell density in the product (<10
7 CFU/mL of product), the loss of PGPE due to the cultivation strategy employed [
11], the short shelf life of the product, and even contamination with other species. Those inconsistencies harm the perception of the products and reduce the confidence of consumers. It is possible to develop a high-quality PGPB bioinoculant by selecting adequate fermentation processes and the smart formulation of the final product [
12].
Recently, a PGPB isolate,
Paraburkholderia sp. SOS3, was identified as a promising bioinoculant. Providing multiple benefits to plants, SOS3 has the potential to be implemented in several agronomically important crops such as rice, sugarcane, and pasture [
13,
14,
15]. Still, the potential of SOS3 is restricted by limited cultivation strategies in bioreactors and poor growth in instrumented fermenters. These limitations can be overcome through the implementation of an efficient fermentation bioprocess. Here, we present a scale-up strategy for the cultivation of SOS3 at high cell density in a low-cost semi-defined medium. The bioprocess described here was validated through plant trials, demonstrating that the final product maintains PGPE and is an adequate candidate for large scale production.
2. Materials and Methods
2.1. Bacterial Strains
SOS3 bacterium (NCBI ID: 1926494) was provided by Sustainable Organic Solutions Pty. Ltd. (Brisbane, Australia).
2.2. Media Composition
R2A agar solid media (Difco) was used to grow SOS3 from glycerol stock. Five different liquid media were used for production: nutrient broth, or NB (8 g/L NB granules, Difco); minimal salts medium (MMS) (10 g/L mannitol, 1 g/L glucose, 0.5 g/L arabinose, 1 g/L yeast extract, 0.5 g/L K2HPO4, 0.2 g/L MgSO4·7H2O, 0.1 g/L NaCl, 0.03 g/L Fe(EDTA)); and three MMS variants, namely arabinose-MMS (10 g/L arabinose, 1 g/L yeast extract, 0.5 g/L K2HPO4, 0.2 g/L MgSO4·7H2O, 0.1 g/L NaCl, 0.03 g/L Fe(EDTA)); glucose-MMS (10 g/L glucose, 1 g/L yeast extract, 0.5 g/L K2HPO4, 0.2 g/L MgSO4·7H2O, 0.1 g/L NaCl, 0.03 g/L Fe(EDTA)); and mannitol-MMS (10 g/L mannitol, 1 g/L yeast extract, 0.5 g/L K2HPO4, 0.2 g/L MgSO4·7H2O, 0.1 g/L NaCl, 0.03 g/L Fe(EDTA)). Ammonium sulphate was added when indicated. The media for 1 L reactors were autoclaved at 121 °C for 20 min. Sugars and Fe(EDTA) were autoclaved separately and then added to the media. All chemicals were of analytical grade (Sigma-Aldrich, St. Louis, MO, USA).
2.3. Fermentations in 1 L Bioreactor
Batch and fed-batch fermentations for biomass production were performed in Sartorius Biostat A bioreactors (Sartorius Stedim, Biostat B-Plus, Gottingen, Germany) equipped with a 1.3 L vessel and up to 1 L working volume. Complex nutrient broth (NB) and semi-defined media were used as indicated for each experiment. Liquid media and reactors were autoclaved at 121 °C for 20 min. One litre of the autoclaved medium was transferred to each reactor under aseptic conditions. Reactors were inoculated with cultures grown for no more than 16 h. Initial optical density at 600 nm (OD600) was adjusted to be 0.1–0.2 units. The reactors were run at 30 °C. When indicated, pH was maintained by the addition of acid (2 M HCl) and base (2 M NaOH). When indicated, dissolved oxygen was maintained at 50% through a cascade control (stirring speed, 200–500 rpm; air flow rate, 0.5–3.0 vvm). For fed-batch fermentation, the initial volume was 500 mL. Fresh media were supplied at a constant flow rate (1 mL/min) using an external Masterflex peristaltic pump.
2.4. Assays for Plant-Growth Promotion: Seed Germination and Vigour Index
Sorghum (Sorghum bicolor L.) and Maize (Zea mays L.) Seeds Tretament
Seeds were coated with their specified treatment in a Petri dish and then left to dry (sorghum, 10 μL per seed; maize, 20 μL per seed). A sheet of sterile filter paper was placed into a Petri dish and soaked with sterile water (sorghum, 2 mL; maize, 3 mL). Seeds were evenly spread onto the paper using sterile forceps (sorghum, 20 seeds; maize, 10 seeds). Another piece of filter paper was placed on top and soaked with 2 mL sterile water. This process was repeated for all of the required treatments and their replicates. Each treatment consisted of four replicate Petri dishes. The plates were closed, covered in plastic wrap, and then incubated at 30 °C. Before harvest, the number of seeds that had germinated in each plate was counted. After five days, roots and shoots were manually separated from the seed and dried in an oven at 60 °C for three days. The dry weights were then measured. The effects of the microbial inoculant on germination was measured using the vigour index:
2.5. Glasshouse Trials–Full Term Growth
Sterilised zeolite (1–5 mm in size) was used to immobilise SOS3 cells recovered from fermentations [
13]. Cells grown in either MMS or glucose-MMS medium were immobilised onto zeolite at a 1 to 3 ratio (mL per g) and dried at 30 °C for 16 h. The number of viable CFU per gram of zeolite was determined by resuspending 1 g of coated zeolite in 10 mL of sterile water. Serial dilutions were made, and 1 mL of each was plated in R2A agar plates. Plates were grown for 48 h at 30 °C, and the number of CFU was calculated.
Ninety pots (125 mm) were filled with University of California (UC) potting mix C (0.33 m
3 sand, 0.17 m
3 bale peat, 4.15 kg fertiliser mix) [
16]. The fertiliser mix (10 kg dolomite, 6 kg blood and bone, 6 kg superphosphate, 6 kg hydrated lime, 3 kg gypsum, 1.2 kg Micromax, 1 kg potassium nitrate, 0.5 kg potassium sulphate) was prepared separately and then added. All pots received an additional 1.8 g (per pot) of Osmocote fertiliser, a standard application as instructed by the company (Osmocote Exact 3M, Scotts. Evergreen Garden Care Australia, New South Wales, Australia). A small hole was pressed into the pot, and two maize seeds were added along with 1 g of zeolite with or without SOS3 that was previously cultured in MMS or Glucose-MMS. Each treatment consisted of 30 replicate pots. This hole was filled in, and then the pots were watered and left to grow in a glasshouse for 22 days. The pots were watered twice daily. The number of emerged seedlings for each pot was counted at various intervals, and pots with two emerged seedlings had one transplanted into a pot with no emerged seedlings of the same treatment group. Once there were no more empty pots for that treatment group, the additionally emerged seedlings were discarded. Before the harvest, the height measurements were taken from the top of the soil to the growth point of the newest leaf, and the number of leaves was counted. The pots were then upturned, and the soil shaken off the roots. The roots were then washed gently to ensure that as little of the root mass as possible was lost during cleaning.
2.6. Polyhydroxybutyrate (PHB) Extraction
PHB content in the cells may affect the shelf-life of inoculum or their in-field competitive efficacy. We measured PHB content by taking 1 mL of SOS3 culture. The sample was transferred into a clean Eppendorf tube and spun down for 3 min at 20,000 g. The supernatant was removed and the pellet frozen at −20 °C until processing. Pellets were thawed and washed twice with 1 mL of Milli-Q water. Then, 1 mL of 18 M H2SO4 was added to each tube before vortexing to dissolve the pellet. The cell suspension was transferred to a 10 mL glass vial and incubated in a 90 °C water bath for 1 h. Every 20 min, the vials were removed and vortexed to mix. The vials were cooled down, and 4 mL of 7 mM H
2SO
4 was added to each vial. The solution was then filtered using a 0.22 μm PES filter. Samples were diluted 1:10 with 7 mM H
2SO
4, and 50 μL of the diluted samples were mixed with 50 μL of 0.2 g/L adipic acid solution. The samples were sent to the Queensland node of Metabolomics Australia for crotonic acid analysis [
17].
2.7. Proteomics Analysis
Liquid culture samples of SOS3 were centrifuged for 3 min at 20,000 g. The supernatant was removed, and the pellet was stored at −20 °C for later processing. The frozen pellet was thawed and washed with 1 mL of PBS buffer. The pellet was then resuspended in 1 mL of lysis buffer (5% SDS, 100 mM TEAB buffer, 100 mM DTT; pH 7.55) and transferred to a 2 mL screwcap microcentrifuge tube prefilled with 0.1 mm glass beads (10% of the volume of the tube was filled with glass beads). Cells were disrupted using a Bead Ruptor 24 elite homogeniser (Omni International, Kennesaw, GA, USA) using the following cycle setting: speed, 6 m/s, 30 s on, 45 s off, 20 cycles. Samples were centrifuged at 20,000 g for 10 min, and the supernatant was recovered. Protein digestion was performed in S-trap mini-columns according to the manufacturer’s instruction using Promega trypsin. The recovered peptides were eluted from the column and the residual acetonitrile was removed by vacuum centrifugation (Eppendorf, Hamburg, Germany). Peptides were resuspended in 0.1% formic acid for LC-MS/MS analysis. Peptide identification was performed using an UltiMate 3000 RSLCnano LC-MS/MS system (ThermoFisher, Bremen, Germany). The LC () was equipped with a Waters Acquity 1.7 µm CSH C18 130Å, 100 mm × 300 µm column (Waters, MA, USA) operated at 40 °C with a 4–76% acetonitrile gradient in 0.1% formic acid for 60 min at a flow rate of 2.0 μL/min. Eluted peptides were directly analysed on a Q Exactive HF mass spectrometer equipped with a Nanoflex interface. Gas pressures were set according to the manufacturer recommendation, and the ion spray source was operated at 2100 V. Protein Discoverer 2.4.1.15 software (ThermoFisher) was used to identify the proteins. The mass tolerance values for precursor ions and fragment ions were set to 10 ppm and 0.05 Da, respectively. The genome annotation for SOS3 was taken from NCBI (GenBank assembly accession: GCA_001922345.1)
2.8. HPLC Analysis
Supernatant samples were retrieved from the freezer and left to thaw. A total of 400 μL of each sample was transferred into an HPLC vial. The Queensland node of Metabolomics Australia performed the sample analysis. Organic acids, carbohydrates, and alcohols were quantified by ion-exclusion chromatography using an Agilent 1200 HPLC system and an Agilent Hi-Plex H column (300 × 7.7 mm, PL1170-6830) with a guard column (SecurityGuard Carbo-H, Phenomenex PN: AJO-4490) as previously reported [
18].
4. Discussion
The adoption of semi-defined MMS media to replace the complex NB medium greatly increases biomass production in SOS3 cultures (
Figure 3). NB batch cultures showed higher growth rates during the exponential phase than MMS cultures, however, these cultures have a reduced yield due to a much shorter growth phase. This higher growth rate is likely due to the increased presence of essential complex nutrients in NB (peptone and yeast extract) compared to MMS, which only has 1 g/L of yeast extract. In a complex medium, specific growth rates are almost always higher than a defined minimal medium because cells do not need to synthesise many essential compounds from base nutrients [
25]. Despite this, NB batch cultures quickly reached the stationary phase at 1.5 OD
600 compared to MMS batch cultures, where OD
600 reached around 5 units with an extended growth phase. NB cultures are likely being limited in growth, as they fully deplete their carbon source. The concentration of peptone and yeast extract makes nitrogen limitation unlikely. While supplementation of NB media with additional carbon sources could lead to improvements in biomass production as observed in MMS derived media, this strategy is not compatible with the ultimate goal, which is the use of chemically defined media [
5]. Recently, Lobo et al. (2019) [
6] summarised several studies on biomass production of bioinoculants. In most cases, media composition consists of complex mixtures, including corn flour (
Pseudomonas putida Rs-198) and dairy sludge (
Rhizobium trifolii). While these strategies aim to be sustainable by using relatively cheap material, still the final formulation can lead to inconsistent results [
5].
The apparent diauxic growth seen in SOS3 fermentations in MMS batch cultures suggested that one of the carbon sources is preferred by the bacterium (
Figure 3). It is not uncommon for rhizobacteria to exhibit this behaviour when grown in controlled conditions [
26] The carbon source analysis confirmed that glucose is consumed preferentially. Once glucose has been depleted, the culture exhibits a small diauxic shift as the bacterium seemingly adjusts its metabolism to primarily consume mannitol. However, the data suggest that mannitol is still being co-consumed during this first stage of growth. While mannitol and glucose have similar biomass yields, glucose is consumed much more rapidly than mannitol (even when mannitol was the sole carbon source). Despite these differences in substrate preference and yield per biomass during growth, all MMS variants showed similar yields in preliminary fermentations. This can be related to the fact that the cultures were nitrogen-limited.
It was clear that nitrogen supplementation in SOS3 culture promotes higher cell densities during the fed-batch stage, followed by a substantial reduction in the biomass concentration. Is has been reported that ammonia (NH3
+) uptake is coupled to carbon uptake [
27]. On the other hand, the non-supplemented cultures showed a rise and a relatively consistent stationary phase whilst consuming the remaining carbon sources. The supplemented cultures likely grew until they had depleted their carbon sources, and then the bacteria began to die off and eventually plateau. This was confirmed in
Figure 5, where it can be seen that total depletion of glucose and mannitol was achieved at roughly the same period as the cultures peaked in OD
600. Whilst this increase in yield was one of the key goals, it brings a new parameter to consider: harvest time. Whereas non-supplemented cultures remain relatively stable after the fed-batch stage, cultures supplemented with nitrogen must be harvested in a window of a few hours to collect the maximum amount of biomass. Late harvesting could lead to a weaker performance associated not only with the lower cell density but also with a reduced PHB content. Reduced PHB content in the cells may also have a detrimental effect on their potential as shelf-stable biofertilisers.
The PHB production profiles of SOS3 cultures seen in
Figure 5 are associated with the availability of nitrogen and carbon sources, a feature observed in several PGPR [
28,
29]. However, while most PHB producer accumulate the bioplastic when nitrogen is limiting [
30], SOS3 accumulates PHB when both carbon and nitrogen sources are available. PHB synthesis during the exponential growth phase can be associated with both nutrient limitation in the culture, such as phosphate and accumulation due to an excess of carbon (storage mechanism) [
30]. PHB accumulation is desirable in liquid formulations of biofetilisers where the PGPB does not produce spores. This can also help cells to remain viable after immobilisation into a solid matrix or onto seeds [
31].
Similar fed-batch and media supplementation strategies have been implemented in the cultivation of bioinoculants such as
Azospirillum sp., with growth-promoting properties associated with the production of growth regulators [
5,
32]. Trujillo et al. (2013) [
30] implemented a control strategy where pH, dissolved oxygen, stirring speed, and aeration were kept constant while varying the carbon to nitrogen ratio of the feed. Their process reached up to 3 OD
600 units, which is ten times lower than the highest OD
600 reached by the SOS3 cultures tested in this work. While SOS3 reached higher biomass production, it should be noted that SOS3 does not produce spores, as
Azospirillum sp. does. The higher cells densities reached in SOS3 are a desirable characteristic for biofertiliser formulation in non-sporulating organisms [
33].
Generation of by-products such as organic acids and alcohols seemed to vary with AS supplementation (
Supplementary Materials File 2). In particular, non-supplemented cultures produced significant amounts of formate with a small amount of acetate production. On the other hand, the supplemented cultures show no formate production but significant acetate production. It might be possible that the addition of AS triggered multiple metabolic functions other than improving growth and promoting PHB accumulation.
Implementing multi-omics approaches has been instrumental in deciphering the mechanisms that lead to growth promotion [
34]. While omics’ implementation has focused primarily on the plant-bacterium interaction to build accurate and reliable data [
34], there is little information regarding quality controls for bioinoculant formulation. We sought to identify potential biomarkers for the pleiotropic growth-promoting effects observed during trials by identifying key expressed proteins using proteomics. The results obtained provide an initial snapshot of the potential biomarkers that can can be used as a quality parameter during the formulation of inoculants. Still, further work is required to demonstrate to what extent these biomarkers contribute to growth promotion as long as they are present at the time of harvesting.
Overall, SOS3 exhibits four main pathways during fed-batch cultivation that can arguably be associated with an immediate response in plant growth post-inoculation. While phosphate solubilisation, synthesis of ACC deaminase, synthesis of zeatin, and synthesis of IAA are active in harvested cells, targeted experiments to assess the translation of such effects into plant growth are needed to confirm the robustness of the biofertiliser [
35,
36]. Only zeatin biosynthesis seems to be incomplete as IspD was not found in any sample. While
nif genes are present in the SOS3 genome and the proteins were present in the analysed samples, no nitrogenase production was detected in SOS3 during cultivation in bioreactors. Proteomic analysis of SOS3 in the rhizosphere of growing plants may hold evidence for the production and activity of nitrogenases. Still, trial experiments show the positive effect that SOS3 has regarding germination, emergence, and vigour.
High-cell density cultures maintained the growth-promoting effects previously observed. In both sorghum and maize coated with culture supernatant, an increased germination rate is likely associated with metabolites or peptides secreted by SOS3. Although this is an exciting result, the germination experiments are not quality substitutes for real field conditions. Many plant growth-promoting mechanisms involve longer-term interactions, and so only the production of phytohormones would be expected to play a role in germination improvements over only a few days. The identification of the molecules (small molecules, peptides, glycoproteins) in the supernatant that trigger growth-promotion in plants can provide further information to build the next generation of fertiliser.
Using a solid matrix as a delivery method allowed us to test the benefits of SOS3 in model crops. The most valuable experiment for assessing the effects of SOS3 on plant growth was the maize glasshouse trial. Significant increases in all metrics of plant growth were observed in SOS3 treatments. This shows the potential for SOS3 use in farming applications as a drop-in biofertiliser (with zeolite as the carrier).
Preliminary data on the effectiveness of SOS3 cultures grown on MMS medium suggest that cultures harvested during the late stationary phase have a detrimental effect on the germination, vigour index, and dry mass of sorghum seeds. This observation becomes relevant as it influences the shelf life of a liquid formulation. Interestingly, the inhibition of maize seed germination by treatment with SOS3 grown in MMS compared to G-MMS was not seen in the glasshouse trial, where MMS and G-MMS yields were similar. This further highlights the importance of the glasshouse trial, as the germination experiments alone suggest that MMS is not a suitable growth medium for maize treatments. Whether this was due to the use of zeolite as a carrier instead of direct seed coating or some impacts from actually being planted in the soil is not yet known. By planting maize seeds coated with MMS culture and comparing them with maize seeds treated with MMS culture zeolite, this cause should be identified.