The economic growth of South Africa is primarily fuelled by land use activities, mining and non-renewable resource exploitation. However, the environmental impacts associated to these activities are extensive both in scale and severity. Natural resource utilization driven economic growth in South Africa urgently needs to become uncoupled from environmental degradation for sustainable development (Soderholm, 2020). Biotechnology offers a solution to overcoming the challenges associated with sustainable development in South Africa. However, biotechnology development and its integration into policy and management must be conceptualized and emerge upon the foundation of practical applicability in a developing-country context. Innovation and technology transfer offer such an avenue to allow the introduction, advent, and expansion of environmental biotechnology into South Africa.

 

The use of biofertilizers for sustainable agriculture represents one of the most advanced biotechnological tools in modern times. Across the world, at an annual increase of ~10%, there is an expanding market for microbial inoculants (Berg, 2009). When compared to chemical pesticides and fertilisers, microbial inoculants offer several advantages: They are safer with more targeted activity and reduced environmental damage. They are also effective in smaller quantities with the ability to multiply, and have decomposition procedures that are quicker (Berg, 2009).

 

The legacy of environmental pollution has followed in the wake of worldwide industrialisation and extensive agricultural and anthropogenic practices (Shinwari et al., 2015). Toxic compounds, metals and effluents have heavily contaminated soils, water and air leaving an environment that is unsuitable for sustaining life. Conventional methods for the remediation of contaminated soils and mine sites are high energy consuming, costly and in large unsustainable (Jeyasingh and Philip, 2005). In this regard, microbial based technologies, known as bioremediation, to remediate or assist remediation practices have gained considerable attention over the last few decades due to their superior performance, low cost, and environmentally friendly nature (Iqbal and Edyvean, 2004; Wu et al., 2006). Bioremediation is defined as the implementation of biological systems, almost universal microorganisms, to clean up or restore contaminated sites (Kumar et al., 2018).

 

The Pseudomonas genus is one of the best-studied and most important bacterial taxa in soil (Raaijmakers et al., 2002). The reasons for this are that the Pseudomonas species are isolated easily from the natural environment, effortless to cultivate and easy to manipulate genetically (Whipps, 2001; Raaijmakers et al., 2002). The members of the genus can colonise a wide range of niches as they demonstrate wide metabolic diversity (Palleroni and Cornelis, 2008). Other important characteristics of the genus Pseudomonas include rapid growth, production of metabolites (siderophores and growth promoters) and their ability to adapt to environmental stress and compete with other microorganisms (Goldberg, 2000; Kraemer, 2004; Hider and Kong, 2010; Cornelis, 2010; David et al., 2018). These features combine to make this microbial inoculant a strongly desired agent in applications such as bioremediation and biocontrol (Kloepper et al., 2004; Weller, 2007). Pseudomonas fluorescens is one species of this diverse group of bacteria that is particularly well known for its role in biocontrol, biodegradation, and bioremediation (Roca and Olsson, 2001; Palleroni, 2010).

 

The environmental biotechnological applicability of certain microbial species is generally performed by isolating soil microbes from the contaminated site and testing whether they possess the capacity to accomplish the desired function. This technique is however, limited by being site-specific and if the microbe presents as being applicable for bioremediation further time, money and workforce are required to developing the bacteria into a formulated inoculant that is safe for both the environment and human handling. This process is in all regard paradigmatic in the developing world where environmental conciseness and funds are not as broadly or extensively prioritized. Considering this, the use of commercially available, safe and relatively cheap PGPB biofertilizers exhibiting multiple plant beneficial properties, abiotic stress and metal tolerance is an encouraging, environmentally friendly and cost competitive soil bioremediating tool. The prospect of using commercially available biofertilizers for bioremediation technology and establishing whether the administration of PGPR biofertilizers, largely used to improve agricultural yields, for solving environmental problems is a concept that has in large been overlooked (Figure 1).

 

 

P. fluorescens containing biofertilizers produced specifically for their phosphate solubilising properties are a commercially available agricultural biotechnology (Goddard et al., 2001). Compared to fungi, the faster growth rate and ease for manipulation of bacteria make them convenient to culture. Thus, may serve potentially as more suitable effectors for the bioremediation of soil. Being commercially available and widely used, its performance for scale-up and field employment has already been confirmed and is a significant factor to why the commission of bioinoculants developed for biotechnological employment are commonly short-lived (Goddard et al., 2001). Numerous in-depth studies have exposed the bioremediation potentials of P. fluorescens (Juhasz and Naidu, 2000; Barathi and Vasudevan, 2001; Janek et al., 2010) and therefore this species was selected for this research. Thus, the microbial biofertilizer was investigated for its potential to be used beyond its current agricultural purpose in the realm of bioremediation and bioaugmentation-assisted rehabilitation of soils.

 

The aim of this work is to determine whether this biofertilizer shows morphological, behavioral, and metabolic phenotypes pertaining to rhizosphere colonization ability and survival. The objectives of this study were to use a culture-based strategy to examine colony morphology expressions, abiotic stress tolerance and heavy metal tolerance of P. fluorescens. PGPR P. fluorescens was cultured on a variety of variable culture media and colony growth times and the observed phenotypic expressions (colony morphology, alterations in colony traits, growth rate, fluorescent pigments and colouration) were examined and compared. Abiotic stress and heavy metal tolerance was also studied using standard culturing techniques and determined by adjusting the individual environmental parameters for each and inspecting the effects on growth. Identifying the impacts of some variables on the microbiological effects and phenotypic expressions of P. fluorescens provides inferences on their potential environmental adaptability, virulence and resistance following introduction into the soil environment (Sousa et al., 2013). As this microbe is already used in agriculture for its plant growth promoting abilities, these properties were not studied as the goal of this study was to determine if the use of this agricultural bioproduct in environmental biotechnological applications is a feasible recourse which mainly relying on its ability to survive, replicate, tolerate and perform the desired biotechnological appointment.

Microorganism, maintenance and storage

 

The bacterial strain of P. fluorescens used in this study was donated as a commercially sold inoculant labelled Rizofos maize from Microbial Biological Fertilizers International (MBFi), Delmas, South Africa. Rizofos maize is a liquid inoculant containing the phosphorus solubilizing bacteria (Pseudomonas fluorescens) (1 × 10⁹ cfu/mL in a sterile liquid broth culture). This strain is however also capable of producing siderophores under iron limited conditions a trait desired for this experiment. This biofertilizer is a special liquid formulation that contains not only the desired micro-organism and their nutrients, but also special cell protectants or substances that encourage longer shelf life and tolerance to stressful condition. The P. fluorescens was maintained at 4°C on nutrient agar (Merck, Darmstadt, Germany) plates. For long time storage, stock cultures were made from liquid cultures (1200 µl culture, 300 µl 80% glycerol) and frozen at -20°C.

 

Several growth-based assays were used to phenotypically characterise the P. fluorescens biofertilizer. These assays are important to increase understanding of the ecology of this significant rhizosphere related microorganisms and have major implications for its application as a bioinoculant for environmental restoration and remediation biotechnologies.

 

Media composition and preparation of experimental plates

 

P. fluorescens was cultured in four different media, Pseudomonas agar, Kings B agar, LB agar and nutrient agar to investigate the effects of the composition of different media and growth time on (i) morphological features (that is, colony growth, colour and edge), (ii) rate of bacterial growth and (iii) physiological features (that is, colour changes and fluorescens). To assess the impact of solid media composition on colony morphology, growth and fluorescens, P. fluorescens was streaked on different solid media (Table 1).

 

 

The media were prepared, autoclaved at 121°C for 20 min, allowed to cool and poured into Petri dishes under sterile conditions in triplicate. As the amount of solid medium in the Petri plates has been observed to impact colony morphogenesis (Sousa et al., 2013), the height of the solid media in each plate was standardised to 0.5 cm thick or ~15 mL of medium per 9 cm diameter plates with an area of 7226.4 mm².

 

Detection of motility

 

Motility can be divided into (i) swimming and (ii) swarming forms (Robertson et al., 2013). A positive result for swimming motility is shown by a ring of colony expansion after 24 to 48 h of growth. Swarming motility is observed if the edge of isolated colonies demonstrates irregular extensions and/or projections after 24 to 48 h of growth. Colony morphogenesis and motility were monitored visually over the incubation period.

 

Detection of fluorescence

 

After 24, 48 and 72 h of incubation, the plates were examined for fluorescence under an ultraviolet light at 360 nm.

 

Abiotic stress tolerance

 

pH tolerance tests

 

To study the effect of temperature on growth of P. fluorescens, a loopful of 24-h old culture growth in nutrient broth were streaked onto nutrient agar plates with different pH values (4, 5, 6, 7, 8, 9 and 10). The final pH of the medium was adjusted using 1 mol/L HCl or 1 mol/L NaOH. The plates were incubated at 25°C for 48 h and visually inspected for the absence or presence of growth, which were then documented as intolerant (I) and tolerant (T), respectively.

 

Temperature tolerance tests

 

To study the effect of temperature on growth of P. fluorescens, a loopful of 24-h old culture growth in nutrient broth were streaked onto nutrient agar plates and incubated at different temperatures of 5, 10, 15, 20, 25, 30, 35 and 40°C for 48 to 96 h (Bruno et al., 2020). The plates were visually inspected for the absence or presence of growth which was then documented as intolerant (I) and tolerant (T), respectively.

 

Salinity tolerance tests

 

To study the effect of temperature on growth of P. fluorescens, a loopful of 24-h old culture growth in nutrient broth were streaked onto nutrient agar plates amended with different concentrations of NaCl (0.1, 1, 2, 3, 4, 5, 6 and 7%). The plates were incubated at 25°C for 48 h and visually inspected for the absence or presence of growth which was then documented as intolerant (I) and tolerant (T), respectively.

 

Heavy metal (HM) tolerance

 

The heavy metals used in the tolerance tests were iron, manganese, cobalt, zinc, copper, lead and chromium from FeSO₄.6H₂O, MnSO₄.4H₂O, CoCl₂.6H₂O, ZnCl₂, CuO₄S.5H₂O, Pb(NO₃)₂ and CrO₃, respectively. A 1 mol/L stock solution for each metal salt was made from which the appropriate volumes were taken to get the desired concentration of heavy metals in each of the 25 mL agar plates using C₁V₁ = C₂V₂ (Table 2).

 

 

The heavy metal (HM) tolerance of the PGPR P. fluorescens was tested using the agar dilution method (Lee et al., 2009). The P. fluorescens was grown in a nutrient broth for 24 h and then, using an inoculating loop, streaked onto nutrient agar plates amended individually with increasing concentrations (10, 50, 100, 200 and 300 ppm) of different heavy metals. Unamended nutrient agar plates were used as controls to examine tolerance. The plates were incubated at 25°C for 48 h and visually inspected for the absence or presence of growth which was then documented as intolerant (I) and tolerant (T), respectively.

 

 

 

In context of developing countries such as South Africa environment-based biotechnologies being economically and technically feasible for economic growth and environmental preservation are urgently demanded. Despite this necessity, the environmental biotechnology market for industrial sector application in South Africa is absent. Developing-country limitations to national environmental biotechnological capabilities must be acknowledged and the development of novel and leading environmentally sound technology undertaken in accordance.

 

For decades the introduction of desired bacteria and fungi into the soil has occurred for agricultural purposes (Chaudhary et al., 2020). These inoculations have been done to stimulate plant growth, supply nutrients, improve the structure of soil and control plant pathogen activity (Chaudhary et al., 2020). In more recent years, there have been other objectives for the introduction of microbial organisms into the soil (Rizvi et al., 2022). These include the bioaccumulation of inorganic compounds, microbial leaching and the bioremediation of pollutants in the soil (Nadeem et al., 2016).

 

Successful soil augmentation requires the selection of a microbial inoculant that is easily cultured; fast growing; occur and survive in a wide range of environmental conditions and can withstand high concentrations of contaminants (Mrozik and Piotrowska-Seget, 2010). The survival of strains introduced into the soil is one of the major difficulties associated with bioaugmentation procedures (Thompson et al., 2005). The effectiveness of bioaugmentation is influenced by both biotic and abiotic factors and the effect the have on the survival of the introduced microorganisms (Bento et al., 2005). Moreover, the efficiency of bioaugmentation is also determined by the soil type; nutrient content and aeration (Bento et al., 2005). Being an in-situ treatment, bioaugmentation, provides a safe and economic alternative to commonly used physicochemical strategies (Adams et al., 2015).

 

Insight into microorganisms displaying multifaceted beneficial traits such as plant growth promotion, rhizosphere competence, abiotic stress tolerance and/or heavy metal stress tolerance is of great importance in the realm of environmental biotechnology. Such microorganisms present novel agents in regulated and legally obligated ecological restoration efforts. The prospect of identifying and make use of already available and commercially sold bioproducts for environmental applications other than in agriculture is significant, especially in developing countries where research and funds restrict remediation practices. To the best of our knowledge this study is the first of its kind with regards to exploring the bioremediation potential of agronomic products sold in South Africa.

 

Phenotypic or phase variation is an important characteristic demonstrated in rhizosphere competent bacteria as it allows the achievement of population diversification within a species crucial in niche adaptation and enhance bacterial fitness when subjected to certain unfavourable conditions (Van den Broek et al., 2005). Colony morphotyping has been shown to be a definitive technique for assessing phenotypic variation which is an important measure colonization, survival and adaptive competence in the soil system (Sousa et al., 2013). The colony morphology method was applied in this study as means of rapidly predicting the competence of a selected strain and therefore its enduring performance following introduction into the field.

 

During colonisation of the different media, P. fluorescens underwent phenotypic variation which resulted in the emergence of colonies having different morphologies. The appearance of colony morphology variation of the P. fluorescens on and between each of the different media, representing structured environments, demonstrates the capability for both stress tolerance and niche specialisation. Additionally, the diffuse morphology observed for some colonies indicates that motility on the agar substrates may have occurred. This is not an unusual observation as P. fluorescens possess flagella required for swarming and swimming (Santoyo et al., 2021). Mobility has several advantages related to rhizosphere competence as it allows the microbe to colonise a greater area of the rhizosphere and populate potentially less microbially dense parts under competitive stress (Sánchez-Contreras et al., 2002). Colony colour and pigmentation variation was also observed for P. fluorescens on the different agar substrates. Under UV light the P. fluorescens demonstrated a blue pigmentation and fluorescence on the Pseudomonas agar, LB and nutrient agar and a vibrant green on Kings B. Pigmentation and fluorescence appears to be linked to the nutritional composition of the growth media. Pigment production is an important parameter as it is correlated to stress tolerance and survival under different environmental conditions (Ahmad et al., 2016).

 

Environmental stress is a major challenge for the sustainability of rehabilitation and therefore ecosystem recovery of mine sites, soils and rock wastes (Goswami and Deka, 2020; Khan et al., 2020). For a microbial inoculant to be considered effective in stress environments it is necessary that they display the ability to survive in adverse conditions. The results of this study suggest that the commercially available PGPR P. fluorescens demonstrates such a capacity for survival and persistence in unfavourable abiotic environments further supporting it suitability in applications of bioremediation.

 

The global repositioning to a greener economy and the subsequent biotechnological potentials of heavy metal tolerant microbes and their cellular products provided major momentum into their research. In general, microorganisms have many beneficial applications in the environment and are in large used for agricultural purposes such as promoting crop yields and/or increasing soil fertility (Nazli et al., 2020). However, biotechnological processes in large require that for microbial species to play a significant role, especially in matters regarding environmental and ecosystem recovery and/or heath, they need to demonstrate heavy metal tolerance (Nazli et al., 2020). Our study shows that PGPR P. fluorescens is of 100% tolerance to all heavy metals tested except for chromium. However, the extent of growth was affected by increasing heavy metal concentrations for all metals tested implying increased sensitivity to higher metal concentrations. Apart from chromium the heavy metal tolerance of the PGPR P. fluorescens strain indicates that they could survive and have bioremediation potential for HM-polluted environments. Tolerance to heavy metals by certain PGPR microorganisms indicates their suitability as bioinoculants to be applied to contaminated environments in terms of their probable survival. Based on the demonstrated heavy metal tolerance PGPR P. fluorescens strain should be further investigated for the possibility of being used for soil bioremediation purposes in heavy metal contaminated soils.

The success of using microbial agents as bioinoculants for soil bioremediation technologies will depend on our ability to manage the rhizosphere to enhance survival and competitiveness of these beneficial microorganisms. The use of commercially available PGPR P. fluorescens as a bioinoculant for the bioaugmentation and bioremediation of contaminated soils relies on its ability to survive, replicate and perform the desired biotechnological appointment. Rhizosphere competence, abiotic stress and heavy metal tolerance to certain common environmental parameters associated with mine lands and/or contaminated soils enables the survivability of the microbial inoculant to be gauged. This study showed that common, widely applied and available agronomic biofertilizers, P. fluorescens, exhibits an exceptional capacity to withstand hostile environmental conditions which in addition to its numerous plant growth promoting traits, make it a desirable microorganism using in environmental biotechnologies applied to ecosystem restoration.

The authors have not declared any conflict of interests.