INTRODUCTION

Phosphorus (P) is one of the major essential macronutrients for biological growth and
development of plants. Phosphorous in soil is mainly found as mineral phosphorous or
organic phosphorous which is however insoluble and unavailable to the plants.
Microorganisms, both bacteria and fungi play a central role in the natural phosphorus cycle
and convert insoluble forms of phosphorus to an accessible form which is an important trait
for the growth and survival of plants. Among the phosphate solubilizing microbes, strains
from the bacterial genera Pseudomonas, Bacillus and Rhizobium and fungi such
as Pencillium, Aspergillus, Fusarium, Helminthosparium, Alternaria, etc. are the most
powerful phosphate solubilizers. Phosphorous solubilization by microorganisms is a complex
phenomenon, which depends on many factors such as nutritional, physiological and growth
condition of the culture. The principal mechanism for mineral phosphate solubilization is the
production of organic acids where the enzyme phosphatases play a major role in the
mineralization of organic phosphorous in soil. In recent years several phosphatases encoding
genes have been cloned and characterized and a few genes involved in mineral phosphate
solubilization have been isolated. Therefore, genetic manipulation for improvement of
phosphate-solubilizing bacteria to improve plant growth may include cloning genes involved
in both mineral and organic phosphate solubilization, followed by their expression in
selected rhizobacterial strains is an interesting approach. . Soil fixed P can only be solubilized
by phosphate solubilizing microorganisms (PSMs).These bacteria released different types of
organic acids in the soil which make P soluble and available to plants. The potential of these
PSMs to solubilize P varies and mainly depends upon mechanism adopted for solubilization,
their molecular genetics as well as their ability to release P in soil. The PSMs, having all the
characteristics of phytohormone production, nitrogen fixation, as well as, heavy metal
decontamination and creating salt stress tolerance in plants, are quite rare for sustainable
agriculture. Application of this environment friendly approach for increasing crop
productivity as well as its impact on soil and plant health is discussed in this review which
will not only open new avenues of research but also provide fruitful information about
phosphate solubilizing microbes for sustainable agriculture development.

Phosphate solubilising bacteria:

A variety number of microbial species have the capacity to solubilise P. These include
bacteria, fungi, actinomycetes and algae and are collectively called as Phosphate Solubilising
Microorganisms . Bacteria are more effective in P solubilization than fungi . Phosphate
solubilizing bacteria belong to the PGPR . PSB are ubiquitous and varies in shape and
population in different soils. Their population in soil depends on chemical and physical
properties, organic matter and P content of the soil . PSB were found in majority of soils
(Chhonker and Taraedar ). Their population was generally low in arid and semi-arid regions,
possibly due to the low level of organic matter and high temperature regime (Gupta et al.,
1986). The PSB population was higher in soils under mild and moist climates than in dry
ones. Agricultural and rangeland soils are found with larger populations of PSB . Among the
whole microbial population in soil, phosphate solubilizing bacteria (PSB) constitute 1 to 50%,
while phosphorus solubilizing fungi (PSF) are only 0.1 to 0.5% in P solubilization potential .
Evidence for naturally occurring phosphate solubilizing microorganism dates back to 1903.
PSB possess phosphate solubilizing ability and they can convert the insoluble phosphatic
compounds into soluble forms in soil and make them available for plants to absorb (Khosro
and Yousef, 2012). These bacteria in the presence of labile carbon serve as a sink for P by
rapidly immobilizing it even in low P soils. Subsequently, PSB become a source of P to plants
upon its release from their cells . The solubilization of insoluble phosphates in the
rhizosphere is one of the most common mode of action of plant growth promoting bacteria
(PGPB) that enhance nutrient availability to plants . The PSB may release several organic
acids including oxalic, citric, butyric, malonic, lactic, succinic, malic, gluconic, acetic, glyconic,
fumaric, adipic, and 2-ketogluconic acid . Salstorm in 1903 first demonstrated the microbial
solubilization of inorganic phosphates by incubating TCP (Tricalcium phosphate) with
bacteria from milk and soil infusions as mentioned by Deepshika et al., (2014) and Gerretson
in 1948 demonstrated the microbial activity in the rhizosphere could dissolve the sparingly
soluble inorganic P and increase plant growth, mentioned the same (Deepshika et al., 2014).
Different bacterial genera and within genera different bacterial species have been reported
to have P solubilising capacity. Strains from bacterial genera Pseudomonas, Bacillus,
Rhizobium and Enterobacter, Bacillus megaterium, B. circulans, B. subtilis, B. polymyxa, B.
sircalmous, Pseudomonas striata, and Enterobacter are the most powerful P solubilizers.
Acetobacter sp. (Joseph and Jisha, 2009), Acetobacter diazotropicus , Agrobacterium sp. and
Alcaligenes sp., Corynebacterium sp. , Azotobacter chroococcum , Burkholderia sp.,
Gluconacetobacter sp., Enterobacter sp., Flavobacterium sp. Micrococcus sp. (Goldstein,
2001), Pseudomonas, Bacillus, Rhizobium, Micrococcus, Flavobacterium,
Int.J.Curr.Microbiol.App.Sci (2017) Achromobacter, Erwinia, Acinetobacter sp. and
Agrobacterium are among the frequently reported PSB. Among all PSB, rhizobia has dual
advantage; they can provide N, besides P solubilization and also improve legume growth
with other PGPR or mycorrhizal fungi. The only phosphate inoculum presently sold
commercially on a large scale, is JumpStart, developed in Western Canada with a strain of
Penicillium bilaii, and now sold by Novozyme (Jump Start, 2012).

Diversity of Phosphate Solubilizing Microorganisms

Phosphate solubilizing microorganisms (PSMs) are group of beneficial microorganisms
capable of hydrolyzing organic and inorganic phosphorus compounds from insoluble
compounds. Among these PSMs, strains from bacterial genera (Bacillus, Pseudomonas,
and Rhizobium), fungal genera (Penicillium and Aspergillus), actinomycetes, and arbuscular
mycorrhizal (AM) are notable. Soil is a natural basal media for microbial growth. Mostly, one
gram of fertile soil contains 101 to 1010 bacteria, and their live weight may exceed 2,000 kg
ha−1. Among the whole microbial population in soil P, solubilizing bacteria comprise 1–50%
and P solubilizing fungi 0.1 to 0.5% of the total respective population. PSMs are ubiquitous,
and their figures differ from soil to soil. Most PSMs were isolated from the rhizosphere of
various plants, where they are known to be metabolically more active .Apart from those
species, symbiotic nitrogenous rhizobia and nematofungus Arthrobotrys oligospora have
also shown phosphate solubilizing activity.
Phosphate solubilization mechanism

Phosphate-solubilizing bacteria play a significant role in solubilization of organic and/or

inorganic phosphate. The solubilization of inorganic phosphorus is carried out as a
consequence of the action of low-molecular-weight organic acids (gluconic and citric acid)
which are synthesized by soil bacteria. Moreover, organic phosphorus mineralization occurs
through the synthesis of a variety of different phosphatases, enhancing phosphoric esters
hydrolysis . It is worth noting that the phosphate solubilization and mineralization ability can
coexist in the same bacterial strain.

Joe et al. (2016) stated the phosphate-solubilizing bacterial role in plant growth promotion
by isolating Bacillus sp. and Acinetobacter sp. from Phyllanthus amarus. Such bacteria have
salt-tolerant and phosphate-solubilizing properties and are also able to decrease fertilizer
consumption. Moreover, it is reported that the inoculation of bacterial strains increased the
phosphorus content, percentage of germination, phenolic content, antioxidative activity,
and plant biomass with respect to uninoculated control. Inagaki et al. (2015) used several
plant growth-promoting bacterial strains in acidic sandy soil, which resulted in a higher
concentration of phosphorus in the maize leaf tissue.

Mechanism of P solubilization:-
PSB employ different mechanisms of P solubilization based on the type of insoluble P source.
Both inorganic and organic forms of insoluble P are present in the soil. Thus, solubilization
mechanisms can be put into two categories i.e. inorganic and organic P solubilization.
Inorganic P solubilization:-
Phosphate rock deposits are about 260 million tons in India and inorganic forms contribute
more than 50% insoluble P in the soil. The principal mechanism of inorganic phosphate
solubilization is the synthesis and secretion of low molecular weight organic acids. The
production of these organic acids results in the acidification of microbial cells and its
surroundings. Subsequently ionization of the acid takes place and either proton produced
becoming responsible for P release from mineral phosphate by proton substitution for Ca, Al
and Fe or carboxylic anions chelate cations and release phosphate anions. Further,
production of organic acids by PSB has also been well documented ( Park et al., 2009; Kumar
and Rai., 2015). Among the various organic acids produced, gluconic and keto-gluconic acids
are the principal components for P solubilization. Production of these acids was reported
from the strains of Pseudomonas , Enterobacter ( Kumar et al. 2015) and Burkholderia .
Other organic acids produced by PSB are citric, lactic, isovaleric, isobutyric, malonic, oxalic,
glycolic, tartaric, pyruvic and succinic. Experimental evidence in support of the role of
organic acids in mineral P solubilization was provided by Halder et al. (1990). Organic acids
isolated from the culture of Rhizobium leguminosarum solubilized nearly equal amount of P
as was solubilized by the culture. Furthermore, treatment of the culture filtrates from
several rhizobial strains with pepsin or acetone (protein precipitation) had not affected the
phosphate solubilizing activity of the filtrates, while addition of sodium hydroxide lost the P
solubilization activity. Based on such studies cloning and characterization of the genes
involved in organic acid production has been carried out (Rodríguez et al., 2006) and it was
concluded that genes directly or indirectly involved in organic acid synthesis or regulation of
the expression of organic acid synthesizing genes are responsible for mineral P solubilization
(Buch et al., 2010). Some researchers believe that the protons extruded to the outer surface
with the help of proton translocation ATPase play an important role in P mineralization as
well as there is an exchange of protons for cation uptake. Likewise, Lin et al. (2006) also
demonstrated in-vitro that the protons of the organic acids are involved in P solubilization.
However, effective buffer systems are present in the soil. Therefore, carboxylic anions
formed by dissociation of organic acids and other high molecular weight organic substances
may probably have the highest efficiency under soil conditions. The carboxyl and hydroxyl
groups of organic acids compete with cations (Ca, Al and Fe), thus make chelate complexes
with metal ions and convert the insoluble P into soluble (Kpomblekou-A and Tabatabai,
1994). The organic acids secreted in the medium can be measured by using high
performance liquid chromatography apparatus (Park et al., 2009; Kumar and Rai, 2015).
Additionally, inorganic P solubilization occurs as a consequence of nitrogen assimilation
(nitrate formation), carbon dioxide evolution and sulphur oxidation. These processes led to
the production of nitric, carbonic and sulphuric acid . Such acids were secreted by
Nitrosomonas and Thiobacillus for P mobilization (Sharma et al., 2013). However,
acidification could not be presumed the sole mechanism of inorganic P solubilization
because the extent of soluble P released and pH drop are not always correlated (Gulati et al.,
2008; Pei-Xiang et al., 2012). Trichoderma harzianum Rifai has not produced any known
organic acid but solubilized P by chelating and reducing molecules. Additionally,
siderophores and exopolysaccharides synthesized by PSB bring out locked P into soluble
form probably by charge related interactions ( Sharma et al., 2013). Although, their role and
mechanism need to be understand. Thus, the organic acids as well as chelating and reducing
molecules produced by PSB are responsible for inorganic P solubilization. As can be seen
from the above account, majority of the studies are relying on the organic acid concept of P
solubilization and these organic acids were utilized as an alternate source of energy by PSB
resulting in the increased biomass yield (Buch et al., 2010; Kumar and Rai, 2015).

Organic P solubilization:-
Solubilization of organic P is also known as mineralization of organic P, which occurs on the
expense of plant, animal and microbial residues. Mineralization of organic P is carried out
with the help of different enzymes. In this process mainly three categories of enzymes are
involved i.e. phosphatases, phytases and phosphonatases. Phosphatases hydrolyze organic
phospho-ester and phosphoanyhydride bonds, and classified as acid (pH < 6) or alkaline (pH
> 7) phosphatase depending on their pH optima (Behera et al., 2013). Their dominance is
determined by soil pH, in acidic soils acid phosphatases play major role while in neutral to
alkaline soils alkaline phosphates are prevalent (Rodríguez and Fraga, 1999; Sharma et al.,
2013). Several genes encoding for acid and alkaline phosphatases with broad substrate
specificity have been cloned and characterized (Rosollini et al; Li et al., 2007; Nilgiriwala et
al., 2008). Interest in these enzymes has increased from last decade due to their suggested
biotechnological potential (Rodríguez et al., 2006). A newer approach is the integration of
best screened gene into the plant growth promoting bacterial chromosome to obtain a
promising PSB strain without the risk of horizontal gene transfer (Behera et al., 2013).
Phytate is the major source of inositol phosphate and accounts more than 50% organic P
form in the soil. It is synthesized by microorganisms, plant seeds and pollen grains . Phytases
release utilizable P from inositol phosphate component of phytate. Initially phytases were
used to improve animal nutrition; however, the current approach may be the use of phytase
secreting PSB to improve the plant growth and development (Richardson and Simpson,
2011). The above approach is based on the concept that Arabidopsis plants genetically
engineered with phytase gene from Aspergillus niger were competent to acquire P from
phytate. The growth and P content of the plant was comparable to those plants supplied
with soluble P. Phosphonatases and C-P lyases release P by hydrolyzing C-P bond of
oraganophosphonates . However, they would not be major contributors in soil solution P
due to scarce availability of their substrates.
Effect Of Some PSM on Crops
PSM TEST CROS RESULT
Aspergillus niger Wheat Improved growth
Serratia sp Wheat Improved growth
Aspergillus awamoriS29 Mung bean Increased plant growth, total
P content, and plant biomass
Burkholderia gladioli Sweetleaf Increased plant growth
Pseudomonas aeruginosa Chinese cabbage Increased total weight and
total length
P. putida Moss Increased growth
Azotobacter chroococcum, Moringaoleifera Increased shoot and root
Saccharomyces cerevisiae, and lengths, increased shoot and
Bacillus megaterium root dry weights, increased
vitamin C and protein content
g/g dry weight leaves
Burkholderia gladioli Oil palm Increased growth and
phosphate uptake
Aspergillus niger Penicillium Chinese cabbage Increased growth
aculeatum
Bacillus sp. and Pseudomonas sp Sesame Increased seed yield

Bacillus thuringiensis Rice Increased shoot length

Pseudomonas striata and Soybean-wheat Better root property and
Glomus fasciculatum increased grain yield
Burkholderia cepacia Maize Improved plant growth
Azotobacter chroococcum and Wheat Enhanced productivity of
Bacillus subtilis wheat
P. favisporus TG1R2 Soybeans Increased dry biomass
Rhizobium tropici CIAT899 Beans Enhanced increased; nodule
number, nodule mass, shoot
dry weight, and root growth

PSM Enhance Soil P Cycle through Organic P Mineralization

Soil Po, which derives mainly from biomolecules, including nucleotides, phosphides, co-
enzymes, phosphoproteins, sugar phosphates, and phosphonates, plays an essential role in
soil P cycling . Po substances (e.g., orthophosphate esters, phosphonates, and
polyphosphates) are mostly short-lived compounds, and may comprise as much as 65% of
Pt in most soils . Based on its sources, soil Po can be considered to exist in a rapidly cycling
pool (fast Po) and a slowly cycling pool (slow Po) . The fast pool consists of the constant
Po from soil solution immobilized in microbial biomass and resupplies the slow pool
following cell death. Soil soluble orthophosphate ions can be immobilized in microbial cells
to improve biomass growth. It is found that most of the P mineralized from organic P by
PSM is incorporated into the bacterial cells as cellular P . Concurrently, these soil microbes
can rapidly release Po to the slow pool following cell lysis, cell death, and soil fauna
predation . Plant detritus, dead animals and microbes, and non-living Po fertilizers (e.g., dry
straw and animal manure) are the most common slow Po sources that can directly replenish
soil orthophosphate contents through geochemical or biological decomposition, beneficial
for plant-available P supplies and soil quality improvement . Hence, manipulation of the
orthophosphate release from soil Po sources is an important soil P cycle, which has the
potential to increase the availability of soil Po for plant uptake and reduce reliance on the P
fertilizer inputs. Soil microbes, especially PSM, can enhance soil Po cycle through
Po mineralization and decomposition. By analyzing soil P pools and oxygen isotope ratios in
P (δ18OP), Bi et al. have uncovered that soil microbes could activate the soil P cycle by
promoting extracellular hydrolysis of Po compounds and facilitating the turnover of
bioavailable P pools (H2O-Pi, NaHCO3-Pi, and NaOH-Pi). These biogeochemical processes
are mainly moderated by the activities of phosphatase enzymes in PSM and soils .

PSMs isolated from bulk soils and rhizospheres have been shown to hydrolyze Po by the
release of phosphatases . Phosphatases are enzymes responsible for Po decomposition and
mineralization by catalyzing the hydrolysis of both esters and anhydrides of phosphoric acid,
and they are usually classified as phosphomonoesterases, phosphodiesterases, and enzymes
acting on phosphoryl–containing anhydrides or on P–N bonds . These enzymes originate
mainly from soil microorganisms and plant cells, and the enzyme activities are always higher
in rhizosphere than in bulk soil . The Po hydrolysis activities of extracellular phosphatase
enzymes are affected by soil properties, microbial interactions, plant cover, and
environmental inhibitors and activators . Some key environmental traits associated with
phosphatase enzyme activities can be genetically manipulated by the regulation of P-
cycling-related genes in PSMs and other microorganisms under P deficiency conditions .
Although soil PSM is involved in Po mineralization and P cycle at various scales, the
dominant enzymes and the functional genes are always similar. It has been established that
the dynamics of microbial genes and expression of phosphatase enzymes are the key factors
governing mineralization of Po into bioavailable orthophosphates by PSMs .

The non-specific acid phosphatases (phosphohydrolases, or NSAPs) released by PSMs

perform dephosphorylation of phosphodiester or phosphoanhydride bonds in Po matters,
and they play a major role in Po mineralization in most soils . These NSAPs may be either
acid or alkaline phosphomonoesterases , and several NSAP genes have been isolated and
characterized in PSMs . For example, olpA gene of Chryseobacterium
meningosepticum encodes and expresses a broad–spectrum of phosphatases that efficiently
hydrolyze monophosphates and sugar phosphates . PhoN and PhoK from indigenous soil
PSMs could express periplasmic acid phosphatase and extracellular alkaline phosphatase, in
genetically modified Deinococcus radiodurans and Escherichia coli, respectively, to enhance
the biomineralization of toxic ions in polluted soil . Cyanobacterial Microcystis
aeruginosa harbors genes encoding extracellular alkaline phosphatase to utilize a variety of
inorganic or organic insoluble P . Phytase enzyme, encoded by appA or phyA genes, is
another important Po mineralization enzyme responsible for P release from phytate in soil .
Previous studies have focused on applications of phytase in the Po mineralization as phytate
is the major component of Po forms in soil . Approximately 30–48% of culturable soil
microorganisms were reported to utilize phytate by producing phytase enzyme . Yeasts
(including Pichia acacia and Candida argentea) were also demonstrated to produce phytase
and utilize phytic acid as their sole P source , while a great diversity of phytase exists in the
vast majority of unculturable soil microorganisms, which have been rarely studied. Using
metagenomics, Farias et al. constructed environmental genomic libraries to determine the
complete sequencing of the clone phytase gene from unculturable soil microorganisms in
red rice crop residues and castor bean cake. The newly isolated phytase enzyme showed
high hydrolase activity at neutral pH under β-propeller structure. Therefore, it is crucial to
develop and utilize more advanced approaches to support the roles of PSM–derived
enzymes in releasing free orthophosphate from organic P forms in the soil P cycle .

Potential uses of PSMs in agriculture and environmental engineering

There is great potential and expectation for the future of PSMs in agriculture and
environmental engineering. As the world’s population continues to grow, the demand for
food and other resources will increase, putting pressure on the world’s finite resources,
including phosphates. PSMs have the potential to help address this challenge by improving
soil fertility and reducing the need for chemical fertilizers (Sarmah and Sarma, 2022; Tian et
al., 2022).

PSMs are an emerging biotechnology tools in agriculture and environmental engineering,

but the application is still in its early stages of development and not yet for
commercialization. While there have been several studies demonstrating the potential of
PSMs for enhancing plant growth, improving soil fertility, and detoxifying contaminated
environments, much of the research is still in the laboratory or experimental stage (Chen et
al., 2020; 2019b; Lai et al., 2022a).
For Agriculture

PSMs can regulate plant metabolism by providing an increased availability of soluble

phosphorus. This increased availability of phosphorus can lead to changes in plant growth
and metabolism , such as an increased rate of photosynthesis, improved root development,
and an enhanced ability to defend against stressors such as drought or disease (Billah et al.,
2019; Kour et al., 2020). PSMs can enhance plant endogenous gene expression and help
plants cope with abiotic stress by providing an increased availability of soluble phosphorus.
This increased availability of phosphorus can activate signaling pathways and trigger the
expression of stress-responsive genes in plants, which can enhance their ability to tolerate
and resist environmental stressors such as drought, salinity, heavy metal toxicity, and
temperature extremes. For example, research has shown that PSMs can increase the
expression of genes involved in the regulation of water uptake, antioxidant defense, and
heat shock response, which can help plants better cope with abiotic stress (Paul et al.,
2017). In addition, PSMs can also stimulate the production of phytohormones such as
auxins, cytokinin, gibberellins et al., which can enhance plant growth and stress tolerance .

For environmental engineering

PSMs can play a role in remediation of heavy metal pollution in soil by indirectly reducing
the bioavailability of heavy metals in the soil, as showed in Figure. When PSMs solubilize
phosphates, they create a competition for metal ions, which can reduce the uptake of heavy
metals by plants and lower their concentration in the soil alone or combining with other
species or materials (Lai et al., 2022b; Feng et al., 2022; Chen et al., 2023). In addition, some
PSMs have been shown to produce organic acids, such as citric acid, that can chelate heavy
metals and make them less available for uptake by plants (Direct assistance).

Another way PSMs can help remediate heavy metal pollution in soil is by promoting the
growth of plants that can tolerate high levels of heavy metals. This is known as
phytoremediation, and it involves using plants to remove heavy metals from the soil by
absorbing and accumulating them in their tissues (Indirect facilitation). PSMs can help
improve the growth and health of these phytoremediation plants by providing them with an
available source of phosphates, which are essential for plant growth and development.

As all the function and potential use of PMS, it worth to note that here is still a need for
further research to fully understand the mechanisms by which PSMs interact with plants
and the environment; and to develop cost-effective and scalable methods for their
application in agriculture and environmental engineering. Additionally, there are challenges
associated with the large-scale production, formulation, and storage of PSMs, which must
be overcome in order to make their application more practical and widespread.

The potential uses of PSMs in agriculture and environmental engineering were summarized
in Table.

Agriculture Environmental Engineering

Improve soil fertility Improve water quality

Increase nutrient uptake Reduce Eutrophication

Boost plant immunity Enhance Bioremediation of contaminated

soil

Reduce the need for chemical fertilizers Improve soil quality in mine reclamation

Improve crop yields Help plants establish on degraded land

Help plant tolerate stress Enhance microbial diversity in ecosystems

Increase soil organic matter Mitigate the impact of agricultural runoff

Improve soil structure Improve soil carbon sequestration

Reduce soil erosion Increase sustainability of land use practices

Acid phosphatase

The results showed that the effect of year, types of biochar and PSM, as well as the co-
treatment of them, was significant on the acid phosphatase enzyme activity. Results showed
that the acid phosphatase enzyme in 2018 (186.21 µg PNPg−1h−1) was higher than those in
2017 (162.25 µg PNPg−1h−1). As mentioned, temperature and rainfall in the second year of
our experiment were increased. Temperature has a profound effect and controls acid
phosphatase, changing enzyme kinetics and stability, substrate affinity and enzyme
production because it can influence the size and activity of microbial biomass (Kumari et
al. Citation2017).The compare means (Figure ) showed that the maximum activity of acid
phosphatase was obtained in soils inoculated with P. fluorescence. In addition, this
superiority was more pronounced when cow manure biochar was used. Nitrogen can
increase the activity of acid phosphatase (Wang, Wang, and Liu Citation2008). In a study by
Jiao et al. (Citation2011), N and P concentrations in soil were correlated with phosphatase
activity. In our study cow manure biochar has the highest nitrogen as compared to other
biochars and therefore in this treatment, the highest acid phosphatase was observed.
Phosphatase activity in the soil has a microbial origin (Li et al. Citation2017). Bacteria such
as Pseudomonas can cause acid phosphatase expression (Luo, Meng, and Gu Citation2017).
Figure. Effects of co-treatment of biochar application and PSM on acid phosphatase (a),
alkaline phosphatase (b) and grain P (c). The PSM with the same letter within each biochar
types are not significantly different according to fisher’s protected LSD test at the 5% level
of probability. Error bars represent standard errors of the means (n = 3). As observed in
(Figure), in using cow manure biochar, the activity of acid phosphatase increased
significantly in the soils inoculated with mycorrhiza species and B. lentus as compared to the
non-inoculated. These three inoculum treatments did not differ significantly. When using
wheat straw biochar, the highest activity of acid phosphatase was obtained in inoculum
with P. fluorescence, which was significantly different from other inoculum and inoculation
treatments. Safflower inoculation with B. lentus was in the next rank. It increased the
activity of acid phosphatase significantly compared to the plants inoculated with mycorrhiza
and non-inoculated plants. The inoculation of safflower with mycorrhiza in the presences of
straw biochar did not affect the activity of acid phosphatase, as it was in the same statistical
group as the non-inoculated plants in terms of the activity of this enzyme. When using wood
biochar, inoculation with B. lentus and P.
fluorescence significantly increased acid phosphatase activity compared to other inoculum
and inoculation treatments. Inoculation with G. etunicatum increased enzyme activity, but
no significant difference was observed with the control. The lowest levels of acid
phosphatase activity were obtained in safflower inoculated with G. mosseae, which were in
the same statistical group as the control. When no biochar was used, the highest enzyme
activity was obtained in the inoculum with bacteria. However, they did not differ
significantly with mycorrhizal-treated safflower. In other words, all the inoculum treatments
with microorganisms were in the same statistical group: they all significantly increased acid
phosphatase activity compared to the non-inoculated conditions. The type of
microorganisms used in the inoculation of safflower was more effective on the activity of
acid phosphatase as compared to the type of used biochar. In fact, bacteria, fungi, and
plants are the main sources of activity of acid phosphatase in the soil (Narendrula-kotha and
Nkongolo Citation2017).
 Alkaline phosphatase
The activity of alkaline phosphatase enzyme was significantly affected by the year, type of
biochar and PSM. Furthermore, the interaction of these two factors significantly affected
the enzyme activity at the level of 5% . Results shows that the alkaline phosphatase enzyme
in 2018 (2752 µg PNPg−1h−1) were higher than last year (2562 µg PNPg−1h−1). The main
reason could be the increase in precipitation and temperature in 2018. The inoculation of
safflower by microorganisms significantly increased alkaline phosphatase activity compared
to non-inoculated conditions (Figure ). Soil enzymes are mainly produced by soil
microorganisms, and enzyme activity is closely related to the number, diversity and
microbial biomass of the soil (Lehmann et al. Citation2011). When using different biochar
types, the highest activity of alkaline phosphatase was attributed to the use of cow manure
biochar followed by wood biochar. There was no significant difference between the use of
wheat straw biochar and biochar-free treatments.

Cow manure biochar has the highest pH among biochar types used in our experiments
(Table 3). Consistent with our results, increased alkaline phosphatase activity in biochar-
added soils is attributed to an increase in soil pH (Marzooqi and Yousef Citation2017). If the
initial soil pH is high, it may decrease the effect of biochar. However, biochar might increase
soil pH far above the optimal level for alkaline phosphatase activity. This suggests that,
when pH increases, the positive effects of cow manure biochar on alkaline phosphatase
activity are more likely to happen in acid than in neutral soils.

In the conditions of using cow manure biochar, the highest enzyme activity was obtained
for P. fluorescence inoculum, which had a difference with other inoculum and inoculation
treatments. The alkaline phosphatase solubilizes phosphate compounds (Yu, Xu, and Tian-
hui Citation2014). Phosphatase activity in soil has a microbial origin (Zimmerman, Gao, and
Ahn Citation2011). The main source of alkaline phosphatase activity is bacteria, while the
acid phosphatase activity is related to bacteria, fungi, and plants (Lammirato, Miltner, and
Kaestner Citation2011). In all bacteria, including all species of Pseudomonas, this
hydrolyzate enzyme catalyzes various types of phosphorus-monomers and trans-
phosphorylation reactions (reaction involving the transfer of phosphoryl group to alcohol in
the presence of phosphate) (Behera et al. Citation2017). The plants inoculated by B.
lentus and G. mosseae were placed in a statistical group in the next rank. The plants
inoculated with G. etunicatum fungi also increased the alkaline phosphatase activity
compared to the control. The lowest enzyme activity was obtained in non-inoculation
treatments. In the conditions of wheat straw biochar application, plant inoculation by
microorganisms significantly increased the activity of alkaline phosphatase compared to that
in non-inoculated plants. The plants inoculated with different microorganisms did not differ
significantly in terms of the activity of this enzyme as all were in the same statistical group.
When using biochar, all treatments increased alkaline phosphatase activity, but only
inoculation with P. fluorescence increased the activity significantly compared to the control.
Significant differences were not observed between inoculum treatments by different
microorganisms. Discussing the superiority of plant inoculation with P. fluorescence, it can
be stated that the Pseudomonas are the most important catalyst microorganisms
solubilizing soil phosphates (Behera et al. Citation2017).
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