Isolation and Identification of Phosphate Solubilizing and Nitrogen-Fixing Bacteria from Lake Ol’Bolossat Sediments, Kenya

Phosphate solubilizing and nitrogen-fixing bacteria have a vital role in improving soil fertility and reverting adversely affected soil properties. These bacteria could contribute towards sustainable agriculture with a focus on reducing excessive use of commercial fertilizers. This study aimed at investigating autochthonous populations of phosphate solubilizing and nitrogen-fixing bacteria from Lake Ol’Bolossat sediments. The total microbial counts ranged between 4.8 x 103 to 8.5 x 105 cfu/ml. A total of 50 bacteria were isolated, 34 were obtained from Pikovskaya’s agar medium while 16 were obtained from Norris Glucose Nitrogen free medium. Based on morphological and 16S rRNA gene analyses, the isolates were clustered under the genera Bacillus, Arthrobacter, Pseudomonas, Paenibacillus, Fictibacillus and Acinetobacter. Among potentially novel strains, four strains NFDA2, PKGBC1 (MT799539), PKGB5 and SCEC2 (MT799543) belonged to genus Bacillus, three strains NFGA1 (MT799529), NFGA4 and SCDB3 belonged to the genus Pseudomonas, two strains NFEB6 (MT799528) and NFDC5 belonged to the genus Paenibacillus, one strain PKHC3 (MT7995441) belonged to the genus Arthrobacter while one strain NFDC4, belonged to the genus Acinetobacter. Generally, the phosphate solubilizing bacteria were the most diverse and genera Bacillus, Fictibacillus and Pseudomonas were the most dominant, however, nitrogen-fixing bacteria were dominated by genera Arthrobacter and Pseudomonas.


Introduction
Rapid growth in industrialization along with the increasing population has led to the increase in the demand for crops (Chennappa et al., 2019) which further increases the pressure on the use of land, water and nutrients to increase crop yields. This is affecting food security, poverty eradication and agricultural sustainability of food systems since the world's population is projected to reach nearly 9.8 billion by 2050 (United Nations, 2017). Among the commonly practiced measure to overcome and increase productivity on existing agricultural lands, is the use of commercial fertilizer and pesticides. All growing plants need seventeen essential elements to grow to their full genetic potential. Out of these seventeen, fourteen are absorbed by plants through the soil, while the remaining three come from air and water (Njinga et al., 2013;Vatansever et al., 2017). Nitrogen, phosphorous, and potassium (NPK) are primary nutrient components in commercial fertilizers that play an important role in plant nutrition. Overreliance on these commercial nutrients, however, have a direct effect on soil microbiological aspects, environmental pollution, and health hazards on reaching the soil in significant quantities (Çakmakçi et al., 2006). These results to alterations in the soil microbial composition, soil fertility and crop productivity; altering soil nitrogen balance, interfering with ammonification, and hindering mycorrhizal symbiosis or nodulation in plants, as well as plant growth, soil structure, organic matter decomposition, and nutrients recycling (Chennappa et al., 2019). A recent study by Kaminsky et al. (2018) showed that excessive fertilization of inorganic phosphorous change microbiome composition thus affecting plant growth.
With the widespread public awareness, the negative effects of chemical fertilizers on soils, there has been increasing interest among scientists and engineers to develop environmentally friendly technologies to boost and sustain agricultural production systems. Therefore, innovative methods are needed to protect and enhance natural resources, increase agricultural productivity and sustainability. Several claims have been reported on the positive effect of organic-rich sediments bio-deposit on plant growth (Grantina-Ievina et al., 2014). The nutrients, microorganisms and enzymes contained in the bio-deposit assist in plant growth by affecting the activity of organic substrates. They can revive dead ground, thus reactivate soil functions, and give it highly fertile properties by forming humus. Further, sediment microorganisms have unique properties since they have to adapt to extreme environmental conditions (John and Salim, 2020).
Conversion of phosphate into readily available form for mineralization and solubilization processes is done by phosphate solubilizing microbes (Behera et al., 2017) making it available for plants uptake. Although soil possesses total phosphorous in organic and inorganic forms, essentially, they remain inactive and or bound to soil constituents making them unavailable for utilization by plants. Even with supplemented phosphorous, about 70-90% of chemical phosphorus fertilizers added to the soil become fixed by forming metal-cation precipitate complexes thus making them unavailable (Kalayu, 2019). Therefore, the insoluble forms of phosphate are converted into soluble forms via phosphate solubilizing microbes through the organic acid production, chelation and exchange reactions (Behera et al., 2017). The process is also achieved by the action of phosphatase enzymes (acid phosphatases) (Liu et al., 2015). The requirements of nitrogenous fertilizer can be reduced by converting inert nitrogen to ammonia through biological nitrogen fixation process using microbes like rhizobia as they form nodules in the roots of leguminous plants and fix atmospheric nitrogen (Singha et al., 2017). They are also known for different plant growth-promoting activities including indole acetic acid (IAA) production, siderophore and ammonia, solubilization of inorganic phosphate among others. Numerous genera such as Azotobacter, Bacillus, Klebsiella, Enterobacter, Arthrobacter, Burkholderia, Pseudomonas, Serratia among others have been studied and used as biofertilizers as reported by various authors and identified them as plant growth-promoting bacteria (PGPB) (Singh et al., 2019).
Recent studies have also been reported worldwide indicating the importance of phosphate solubilizing and nitrogen-fixing bacteria as biofertilizers, yet scanty information is available on the microorganisms from the Lake sediments in Kenya. Also, many microbes may be indigenous isolates well adapted to a specific ecological niche, local climatic environment conditions, as well as cropping systems (Panda et al., 2016). Undocumented information shows the use of the sediment from Lake Ol'Bolossat by farmers around the lake, which recorded an increase in crop yield. The study aimed at isolating, characterizing, and identifying indigenous nitrogen-fixing bacteria and phosphate solubilizing bacteria from Lake Ol'Bolossat sediments.

Sample Site and Collection
Lake Ol'Bolossat is located in highlands of central Kenya in Nyandarua County ( Figure 1) with a catchment area of 340 km 2 , it covers the land area of 38 km 2 , it is the headwater for the 210, 0000 km 2 Ewaso Ngiro North Basin and it flows through Thompson falls. Sediments from the bottom of the lake were collected from four different locations in lake Ol'Bolossat site 1 (0 o 10'.12.6 S/36 o 26'.48E), site 2 (0 o 08'.24S/36 o 26'.1.8E), site 3 (0 o 4'.45.7S/36 o 24'.51E), and site 4 (0 o 6' 40.2S/36 o 25.51.6E). The sediments were collected in triplicates in a 7.62 cm diameter and 90 cm long aluminium core pipes using Specialty Device Inc (SDI) Sediment Sampler Vibecore and Accessories (Wylie Texas, USA). After collection, the core pipes were divided into three sections (0-30 cm, 30-60 cm and 60-90 cm) each representing a separate independent sample. The core pipes were then split open and the sediments were collected separately in sterile Ziplock plastic bags. They were labelled clearly indicating sample details and collection dates and transported at 4 o C to Jomo Kenyatta University of Agriculture and Technology (JKUAT) laboratories for bacterial isolation.

Isolation and Enumeration of Phosphate Solubilizing and Nitrogen-Fixing Bacteria
Ten grams of the sediments were suspended in 90 ml of sterile physiological saline (0.85% NaCl) to obtain 10 -1 dilution, followed by filtration through sterile 125 mm Whatman® qualitative filter paper, Grade 1 (Merck). One ml of the filtrate was transferred to 9 ml of sterile physiological saline to make 10 -2 dilution. Subsequently, other dilutions were serially made in the range of 10 -3 to 10 -8 . The inoculation mixture with serial dilution was then spread in triplicate on the plates containing Pikovskaya's (PKV) agar medium (Pikovskaya, 1948) (Ranganayaki and Mohan, 1981). Bacteria counts were determined by plating on both PKV and NGNFM while the total count was carried on Soybean-Casein Digest Agar (Casein Soyabean Digest Agar) (Himedia-MH290). Soybean-Casein Digest Agar medium consisted of tryptone (pancreatic digest of casein) 15.0 g, soya peptone (papaic digest of soyabean (soybean) 5.0 g, NaCl 5.0 g, and agar 15.0 g in 1 litre distilled water. The inoculated plates were incubated at 30 o C for up to 6 days, and the colony-forming units (CFU) were counted. To measure the survival efficiency the number of bacteria present per gram of original sample were enumerated using the following formula: Viable cell count CFU/g sample umber of colonies 25 300 CFU The volume of inoculum 0.1 ml X Dilution factor The colonies with clear zone on PKV agar were considered positive for phosphate solubilization hence were subcultured on the same media (PKV agar) three times to obtain pure cultures. Colonies that formed on NGNFM agar were considered nitrogen-fixing bacteria thus, were selected and sub-cultured three times by streak method to obtain pure cultures were on the same NGNFM agar media. The pure isolates were cryopreserved in PKV broth for PSB and NGNFM broth for NFB containing 20% glycerol for long term storage at -75 o C and for further analyses.

Morphological Characterization of Phosphate Solubilizing and Nitrogen-Fixing Bacteria
Preliminary characterization was performed using morphological and cultural characteristics as described by Benson, (2002). Morphological characterization of the isolates was under the dissecting and compound microscopes to observe cell shape, size and arrangement characteristics after classical Gram-staining and catalase tests were performed as described by Cappuccino and Sherman, (2014) the Gram-reaction was confirmed by 3% (w/v) KOH test (Gregersen, 1978).

Extraction of Genomic DNA, PCR Amplification and Sequencing of 16S rRNA Gene
Total genomic DNA was extracted from overnight cell cultures grown on PKV broth for PSB and NGNFM broth at 30 o C using QIAamp DNA Mini Kit (Qiagen, Germany) according to manufacturer's instructions. Polymerase chain reaction (PCR) was performed in Primus 96 advanced thermal cycler (PEQLAB, Erlangen, Germany). The 16S rRNA gene amplification was performed using the universal primers 27F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492R (5′-GGT TAC CTT GTT ACG ACT T-3′). PCR was performed in a 50 µl mixture containing 25 µl 3X Taq PCR master mix (Qiagen, Germany), 2.5 µl of each primer, 10 µl of DNA template (50 ng) and 10 µl RNase free water. The reaction mixtures were subjected to the following PCR conditions: initial denaturation at 95 °C for 5 min, followed by 32 cycles consisting of 1 minute denaturation at 95 °C, 1 minute annealing at 55 °C, 2 minutes extension at 72 °C, and a final extension step of 10 minutes at 72 °C. The amplified PCR products were resolved in 1.2% agarose gel stained with ethidium bromide (1 μg/ml) and visualized using a Biotec-Fischer Felix 6050 gel documentation system (ProfiLab24, Germany). PCR products were purified using the QIAquick PCR purification Kit (Qiagen, Germany) according to the manufacturer's instructions. The purified amplicons were Sanger sequenced at Human Genomics Macrogen Europe (Macrogen Europe B.V, Amsterdam, Netherlands).

Phylogenetic Analysis
The 16S rRNA gene sequences of the bacterial isolates were viewed for quality checks and edited using ChromasPro 2.1.8 software package (http://technelysium.com.au/wp/). They were then compared with available standard sequences of bacteria lineages in the public nucleotide sequence databases in the National Center for Biotechnology Information (NCBI) using nucleotide blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to find closely related bacterial 16S rRNA gene sequences. The 16S rRNA gene sequences of the isolates and those of the unknown closely related bacteria strains were aligned using Clustal W software, phylogenetic trees were constructed using Maximum Likelihood method based on the Tamura-Nei model (Tamura and Nei, 1993) with MEGA (Molecular Evolutionary Genetics analysis) 7.0 software package (Kumar et al., 2016). The trees topologies were evaluated using the bootstrap resampling method (Felsenstein, 1985) based on 1000 replicates.

Statistical Microbial Analysis
The microbial enumeration results were expressed as the mean and standard deviation of triplicate experiments of the counts and sampling sites using two-way ANOVA (GraphPad Prism version 8.4.2 software, GraphPad LLC, San Diego, California, USA), at a significance level of p<0.05, multiple range tests were performed using Tukey's test.

Isolation of Phosphate Solubilizing and Nitrogen-Fixing Bacteria from the Sediment
A total of 50 bacteria were isolated from lake Ol'Bolossat sediment out of which 34 were isolated on PKV agar medium (Table 2) and had the potential for phosphate solubilizers due to clear zone around the colonies. Similarly, 16 were isolated on Norris Glucose Nitrogen free medium (Table 1)

Enumeration of Phosphate Solubilizing, Nitrogen-Fixing and Total Microbial Counts
The microbial counts varied significantly between the sampling site and depth ( Figure 3). In general, the highest microbial counts were recorded at 0-30 cm depth for all sampling points, the results showed microbial counts decreased with increase in depth, notably at the depth of 0-30 cm, 30-60 cm and 60-90 cm, for example, phosphate solubilizing bacteria counts ranged between 2.7 x 10 3 to 7.4 x 10 5 cfu/ml, nitrogen-fixing bacteria counts were 1.9 x 10 3 to 4.6 x 10 5 cfu/ml while total microbial counts ranged between 4.8 x 10 3 to 8.5 x 10 5 cfu/ml ( Figure 3). The analysis of variance (ANOVA) showed a statistically significant difference between microbial counts and sampling points (p< 0.05).

Colony Morphologies and Identities of Phosphate Solubilizing and Nitrogen-Fixing Bacterial Strains
Morphological characterization of phosphate solubilizing and nitrogen-fixing bacteria was based on classical macroscopic techniques of colour, form, shape, and elevation of pure colonies. Most colonies were able to grow within 2-6 days of incubation at 30 o C. Bacterial species were further examined for their Gram's reaction, shape and catalase activity. Characteristically, all the isolates were catalase-positive, 86% were Gram-positive, while 14% were Gram-negative and all were rod-shaped. Nitrogen-fixing bacteria strains are shown in Table 1 while phosphate solubilizing bacteria strains are shown in Table 2.

Discussion
Lake Ol'Bolossat was selected for the isolation of phosphate solubilizing bacteria (PSB) and nitrogen-fixing bacteria (NFB) because of its greater potential as an alternative source of cheap biofertilizer. Currently, undocumented data indicates that local people living around this lake, are utilizing the sediment in agricultural production with positive results. Phosphate solubilizing bacteria (PSB) are usually screened by a plate assay method on Pikovskaya's medium. The medium allows the bacteria to grow and form clear zones around the colonies as a result of the conversion of tricalcium phosphate in the medium from insoluble to soluble forms (Pikovskaya, 1948). The formation of clear zone around the bacterial colonies ( Figure 2) could be attributed to the production of phosphatase enzymes by phosphate solubilizing bacteria (Halder and Chakrabartty, 1993;. The PSB and NFB counts were in line with Stankevica et al. (2015) who showed that freshwater sediments are highly populated with microorganisms ranging between 5.2 x 10 3 to 6.9 x 10 6 cfu/g of dry matter. PSB are commonly found in most soils, although their count varies depending upon the conditions of soil and climate with a high concentration in the rhizosphere in comparison with non-rhizosphere soils (Rafi et al., 2019). The quantity of living cells is among the quality parameters within the biofertilizer regulation in international standards for many countries (Malusá & Vassilev, 2014). The standard has different groups of microorganisms (rhizobia, for fast or slow-growing species; N-fixing bacteria; phosphorous solubilizing bacteria (PSB), classified based on the ability to act on organic or inorganic phosphate. However, the quantity of living cells varies from >0.5 x 10 9 cfu/ml to >0.1 x 10 9 cfu/ml and >1.5 x 10 9 cfu/g to >0.2 x 10 9 cfu/g, for liquid and solid products, respectively subject to the kind of bacteria used in the production of the biofertilizer (Malusá & Vassilev, 2014). The difference in the cfu in comparison to the obtained results could be attributed to the fact that microbes may be local isolates that are native to the location, sediment type and climatic conditions (Panda et al., 2016).
Based on morphological characteristics and 16S rRNA sequencing, PSB were grouped under the genus Bacillus, Arthrobacter and Pseudomonas ( Figure 5). From table 2 the most dominant phosphate solubilizing bacteria were Gram-positive and catalase-positive belonging to the genus Bacillus. Bacillus and Pseudomonas are common genera for solubilization of phosphate since they can convert the insoluble form of phosphate into soluble one (Rafi et al., 2019). Eleven isolates were affiliated with Bacillus megaterium, which have been described by Burgos et al. (2015) and Akinrinlola et al. (2018) as Gram-positive, endospore-forming, rod-shaped plant growth-promoting bacteria. This bacterium has been previously reported to increase the grain yield of rice by approximately 103%-256% in an autoclaved soil either as a single culture or in combination (Khan et al., 2003). According to El-Komy, (2005), wheat inoculated with B. megaterium exhibited higher shoot dry weight, total nitrogen yield and phosphate contents. Studies by Nascimento et al. (2020) and Wu et al. (2019) show B. megaterium possess genetic elements that play a role in xenobiotic degradation, stress resistance, pathogen antagonistic activities, and other soil rhizosphere colonization traits. Five isolates were closely related to B. simplex, a bacterium species reported by Schwartz et al. (2013) to modify the architecture of the roots by enhancing the formation of more lateral roots in pea plants. The bacterium species also enabled the development of larger and highly clustered nodules when pea roots were co-inoculated with either B. simplex or Rhizobium leguminosarum. According to El-Komy, (2005), B. simplex has the abilities to solubilize phosphate and enhancing iron availability to the plants.
From the results, five isolates were affiliated with genus Fictibacillus with four associated with Fictibacillus rigui (formerly known as Bacillus rigui) while one was associated with F. enclensis (Table 2 and Figure 4). Just like other members of the genus they are Gram-positive, aerobic and endospore-forming commonly isolated from varied habitats like freshwater wetlands, marine sediments, hot springs and industrial wastes (Baik et al., 2010;Glaeser et al., 2013). Members of genus Fictibacillus are known to produce siderophores, indole acetic acid as well as participate in phosphate solubilization and nitrogen-fixation (Battini et al., 2016). Studies by Ansari et al. (2019), have shown that B. pumilus can produce strong biofilm with enhanced indole acetic acid, exopolysaccharides, deaminase and phosphate solubilization activities. Bacillus zhangzhouensis was phylogenetically clustered together with B. pumilus ( Figure 5), this bacterium is mainly isolated from aquaculture water and sea sediments . B. zhangzhouensis has been reported as a good plant growth-promoting bacteria through phosphate solubilization (Emami et al., 2019). One isolate was identified and clustered together with B. thuringiensis ( Figure 5). According to Wang et al. (2014), B. thuringiensis is an excellent phosphate solubilizing bacteria since when inoculated with the soil, B. thuringiensis had a positive effect on the plant growth characteristics of peanuts (Arachis hypogeae) as well as seed weight and crude protein content.
An isolate PKGBC1 (MT799539) clustered together with B. mangrovi, was isolated from sediments of the mas.ccsenet.org Modern Applied Science Vol. 14, No. 10; 2020 mangrove forest in India (Gupta et al., 2017). Although it belongs to PGPB there is limited information on the exact role it plays in plant growth promotion. Members of the genus Pseudomonas, are usually Gram-negative, rod-shaped bacteria commonly found in soils, sediments, plant rhizosphere and freshwater as saprophytes (Bossis et al., 2000). Pseudomonas putida (Table 2), is an excellent plant growth-enhancing bacteria through the production of indole acetic acid (Bharucha et al., 2013). The study by Tiwari et al. (2016), has demonstrated the ability of the P. putida to ameliorate drought resistance stress through the production of abscisic acids (ABA) in chicken pea. P. mendocina has been reported as important nitrogen-fixing bacteria for improving soil nitrogen content (Sharma and Singh, 2020). Pseudomonas fluorescens was reported to produce, indole acetic acid, ABA and the gibberellins with potential as biocontrol, enhancement in the length of stem and roots, the germination rate of various plants (Salomon et al., 2014).
Various species of Arthrobacter are found in varied environments like freshwater, soil, plant rhizosphere and marine habitats and are implicated in the promotion of plant growth. Ten isolates phylogenetically clustered with A. oryzae (Figure 4), were isolated. Recent study indicate that this bacterium is responsible for the degradation of heavy metals from contaminated lake sediments (Cho et al., 2019). A. koreensis has shown to benefit plants withstand desiccation due production of xeroprotectants (Manzanera et al., 2015), while Arthrobacter pokkalii promotes plant growth under high salt concentrations in agricultural soils (Krishnan et al., 2016) thus protecting them from abiotic stress while improving plant health, nutrition and yield. Evidence shows some species of Arthrobacter can degrade various xenobiotic compounds like 4-chlorophenol and 4-nitrophenol (Sahoo et al., 2011;Arora and Jain, 2013).
Phylogenetic analysis showed two isolates formed a cluster with Paenibacillus sp (Figure 4). Members of this group are designated as nitrogen-fixing bacteria and are widely distributed in nature such as plant rhizosphere, soil, animal and humans (Grady et al., 2016). Members of genus Paenibacillus are usually Gram-positive, rod-shaped, facultatively anaerobic, endospore-forming bacteria . According to Liu et al. (2019), most Paenibacillus species possess nifH genes for encoding Fe protein with nitrogenase activities important in fixation of atmospheric nitrogen, production of siderophore and indole-acetic acid. There is also strong evidence that Paenibacillus polymyxa produces various antimicrobial and insecticidal compounds against different pathogenic fungi, bacteria and nematodes in cucumber, wheat and tomatoes Grady et al., 2016).
Bacillus subtilis is an important model organism commonly used in agriculture as plant growth-promoting rhizobium (PGPR), its crucial in solubilizing soil phosphates, increase nitrogen fixation, facilitates iron absorption by plants via siderophores production and promotes plant growth by suppressing fungal pathogens (Adam et al., 2014;Hashem et al., 2019). Protection of plants by this bacterium is thought to involve biofilms formation on the plant roots (Chen et al., 2012). According to Bais et al. (2004), B. subtilis is an excellent biocontrol agent against pathogenic Pseudomonas syringae in tomatoes. A study on kiwi fruit showed that the bacteria improves the root growth mass through the production of indole-acetic acid (Erturk et al., 2010). Another important PGPR isolated was clustered together with the genus Acinetobacter ( Figure 4). Members of this species are associated with plant growth promotion via mineral solubilization, siderophore production, inhibition of phytopathogenic fungi such as Fusarium oxysporum. They have also been reported to enhance plant growth characteristics in pearl millet (Rokhbakhsh-Zamin et al., 2011). Additionally, studies by Wafula et al. (2012) isolated Acinetobacter spp from tea soils in Kenya which showed promise as potential biofertilizers and indicators of soil health.

Conclusion
In total 50 bacteria were isolated and identified from the sediments of Lake Ol'Bolossat. Based on 16S rRNA gene analyses, the identified bacterial strains belonged to the genera Bacillus, Arthrobacter, Pseudomonas, Paenibacillus, Fictibacillus and Acinetobacter. The study has demonstrated that Lake Ol'Bolossat harbors diverse bacteria species with potential for plant growth promotion. The results highlighted the significance of the isolated strains in phosphate solubilization and nitrogen-fixation and could be used as potential biofertilizers.
Owing to complex factors of lake Ol'Bolossat sediments, production of a clear zone on a PKV solid agar medium alone cannot be used as a definitive proof for phosphate solubilization or growth on NGNFM as nitrogen-fixing properties. Therefore, further studies are needed to assess the effects of the isolates on the plant growth promotion characteristics under field and greenhouse conditions. acknowledge Prof. Elijah Ateka for providing the Molecular and Biotechnology laboratory and R. Rotich from Institute of Biotechnology JKUAT for technical support.