B IOD I V E R S I TA S ISSN: 1412-033X

Volume 25, Number 1, January 2024 E-ISSN: 2085-4722

Pages: 177-185 DOI: 10.13057/biodiv/d250120

Assessment of the dynamic growth and potassium solubilization

capability of three novel bacteria

IMAM HARTONO BANGUN1, BUNGA RAYA KETAREN2,, ASRITANARNI MUNAR1,

PERDINANTA SEMBIRING3, NURHAJIJAH1, ANDRI ABDI1, REYZA SUWANTO SITORUS4
1Program of Agrotechnology, Faculty of Agriculture, Universitas Muhammadiyah Sumatera Utara. Jl. Kapten Muchtar Basri 3, Medan 20238, North
Sumatra, Indonesia
2Program of Agricultural Technology, Faculty of Agriculture, Universitas Muhammadiyah Sumatera Utara. Jl. Kapten Muchtar Basri 3, Medan 20238,

North Sumatra, Indonesia. Tel.: +62-616-622400, email: [email protected]

3Research Center for Horticulture and Estate Crops, National Research and Innovation Agency. Jl. Raya Jakarta-Bogor Km. 46, Cibinong, Bogor 16915,

West Java, Indonesia

4Program of Agribusiness, Faculty of Agriculture, Universitas Muhammadiyah Sumatera Utara. Jl. Kapten Muchtar Basri No. 3, Medan 20238, North

Sumatra, Indonesia. Tel.: +62-61-6622400

Manuscript received: 24 August 2023. Revision accepted: 21 January 2024.

Abstract. Bangun IH, Ketaren BR, Munar A, Sembiring P, Nurhajijah, Abdi A, Sitorus RS. 2024. Assessment of the dynamic growth and
potassium solubilization capability of three novel bacteria. Biodiversitas 25: 177-185. Clay minerals are essential components that play
a crucial role in soil cation exchangeable capacity and optimal plant growth. These components, including potassium (K), have the
ability to bind with mineral crystals in the soil. Several studies have shown that K-solubilizing bacteria can facilitate the solubility of
potassium, leading to optimal availability. However, the use of bacteria as biofertilizers is still limited due to challenges related to
survival and growth patterns. To address these challenges, it is important to monitor the growth of the microbes and enhance their
selection process to achieve effective utilization. Therefore, this study aimed to determine the bacterial growth dynamics and potential
of Burkholderia paludis IHB_01, Burkholderia cepacia IHB_02, and Paraburkholderia phymatum IHB_03 in enhancing soil cation
availability on clay minerals. The results showed a common initial adaptation phase among the 3 bacterial strains, followed by distinct
exponential growth patterns. Burkholderia cepacia IHB_02 had the longest exponential growth phase, showing efficient resource
utilization and extended growth. The results of soil cation enhancement by these bacteria showed that there were no major changes in
measured parameters, such as total K, CEC, pH, and organic carbon. Furthermore, the variation in K exchange, organic carbon, and Na
exchange provided insights into their unique interactions in the soil. The non-significant impact on potassium-related parameters in this
study could be attributed to the presence of antagonistic cation interactions.

Keywords: Biofertilizer, Burkholderia cepacia, Burkholderia paludis, clay minerals, feldspar, Paraburkholderia phymatum, potassium

INTRODUCTION reservoir for K, allowing for a quantitative evaluation of

the nutrient (Barré et al. 2008). Previous studies have
Soil is an essential natural resource for human shown that K ions, which are initially considered non-
civilization, which supports plant growth, provides exchangeable when trapped in the interlayer sites of clay
nutrients, and purifies water (Adhikari and Hartemink minerals, can still be partly accessible and available to
2016). Furthermore, soil fertility is crucial for productive plants (Hinsinger et al. 1992; Falk and Krogstad 2005;
and sustainable agriculture, and it is influenced by various Officer et al. 2006).
factors, such as mineralogy and microorganisms (Bhunia et The limited presence of K often leads to the use of
al. 2021). Among these factors, potassium (K) has been inorganic fertilizers by farmers to improve yields. However,
reported to play an essential role in fertility and plant the persistent application of these fertilizers, such as K
growth, and is often required in larger quantities compared chloride or K sulfate (K2SO4) in agriculture can lead to a
to other nutrients, except nitrogen (N) (Wang et al. 2013). negative K balance, showing a deficiency in acidic soils
Despite the importance of K in the soil, a significant (Qaswar et al. 2020). To address this challenge, there has
challenge remains its availability, particularly due to been widespread development in the use of environmentally
fixation in the clay mineral. Several studies have reported friendly alternatives derived from sources, such as poultry,
various factors influencing the fixation of the nutrient, fisheries, and agricultural waste (Idris et al. 2023). The use
including clay type and quantity, wetting-drying and of fertilizers has also been reported to have a significant
freezing-thawing cycles, pH, K fertilizer application, effect on the soil's bacterial and fungal communities, with
moisture level, and the ratio of K to calcium (Ca) and organic types promoting greater bacterial diversity
magnesium (Mg) (Shakeri and Abtahi 2019). Compared to compared to the chemical variant (He et al. 2008). The
ammonium, its fixation often occurs when it binds to clay intensification of agriculture, comprising the application of
minerals and organic matter, reducing its availability for high-yielding crop varieties, fertilizers, irrigation, and
plants (Scherer et al. 2014). Clay minerals also serve as a pesticides, contributes to soil degradation (Maranguit et al.
178 B I OD I V E R S ITA S 25 (1): 177-185, January 2024

2017). To ensure environmental sustainability and improve growth and their mechanisms, as well as the potential to
the nutritional value of food, agriculture must adopt enhance the availability of soil cations in clay minerals.
innovative methods and advanced fertilizer programs. This Understanding the bacterial growth of these bacterial
includes the utilization of bacteria to solubilize minerals, strains and their potential in K solubilization can provide
thereby enhancing nutrient availability and promoting information on their efficacy as plant growth-promoting
optimal plant growth. A promising method to mitigate the agents. Therefore, this study aimed to investigate the
adverse impacts of climate change on agriculture comprises bacterial growth dynamics and potential of B. paludis
the use of biofertilizers. IHB_01, B. cepacia IHB_02, and P. phymatum IHB_03 in
Recent discoveries have identified 2 novel isolates, namely enhancing soil cation availability in clay minerals.
Burkholderia paludis IHB_01, and Paraburkholderia
phymatum IHB_03, as proficient in solubilizing K within
clay minerals. Furthermore, the well-established isolate, MATERIALS AND METHODS
Burkholderia cepacia IHB_02, has showed K
solubilization capabilities (Bangun et al. 2023). Study site
Burkholderia cepacia is recognized for diverse The study was conducted in the tissue culture laboratory of
mechanisms supporting plant growth, including K and the Faculty of Agriculture, Universitas Muhammadiyah
phosphorus solubilization (Zhang and Kong 2014; Zhao et Sumatera Utara, Medan, Indonesia. Furthermore, the soil
al. 2014; Ghosh et al. 2016), phytohormone production specimen was collected in Besitang, Langkat, North
(Compant et al. 2008), N fixation (Marciano Marra et al. Sumatra, Indonesia (latitude 98°14’65.21” N; longitude
2012), antifungal properties (Zhao et al. 2014), plant 4°04’86.84” E; elevation 17 meters). The study procedures
symbiosis (Tapia-García et al. 2020), and contribution to were carried out from January to May 2021.
organic matter decomposition (Elshafie and Camele 2021).
Previous studies also reported the ability of P. phymatum to Materials
form associations with the roots of leguminous plants and The materials used in the implementation of this study,
engage in N fixation (Vandamme et al. 2002; Moulin et al. included Aleksandrov medium (feldspar, CaPO4, glucose,
2014; Cai-xia et al. 2018). Based on results, limited yeast extract, MgSO4·7H2O, FeCl3, CaCO3, distilled water,
documentation exists concerning B. paludis, including its NaCl), nutrient broth (NB) medium (peptone, beef extract,
capacity to synthesize enzymes, such as phosphatase, NaCl, yeast extract), 0.85% NaCl, KCl, isolate of KSB (K
esterase, and esterase lipase, along with producing specific solubilizing bacteria) from a previous study (Bangun et al.
types of siderophore with antimicrobial properties, 2023) on Alexandrov media (Table 3), and soil samples as
including pyochelin (Ong et al. 2017). Investigation is materials for potency testing (Table 1). Furthermore, the
essential to prepare bacterial isolates for field application, equipment used was a spectrophotometer, analytical
comprising the crucial step of observing bacterial growth balance, digital pH meter, centrifuge, flame photometer,
curves. This process requires close observation of growth and rotary shaker. The procedures were divided into two
trends in these isolates over time, providing insights into stages, and the first comprised the examination of bacterial
their behavior and potential effectiveness in real-world growth curves. The second stage consisted of an
agriculture. This information has significantly contributed experimental study for potency testing in soil media.
to the commercial manufacturing of various microbial
products, including antibiotics, vitamins, amino acids, Research implementation
enzymes, yeast, vinegar, and biofertilizers (Zakaria et al. The growth curve analysis of KSB was conducted to
2019; Zahri et al. 2020). investigate the effects of factors, such as temperature, pH,
Environmental microbiologists currently face and media concentration on growth according to the
challenges centered on comprehending microbial growth in Monod methodology (Monod 1949). Furthermore, the
natural environments. This understanding is essential for process began by reproducing three KSB isolates in 250
predicting nutrient cycle rates, microbial responses to mL liquid NB medium, followed by culture for a day at 28
environmental disturbances, and microbial interactions and ± 2°C. To establish a standard curve, Total Plate Count
survival (Maier and Pepper 2015; Ibrahim et al. 2020). To (TPC) method was used, as shown in Table 2. This method
address these challenges, further information is needed on comprised creating serial dilutions with ratios of 1:1, 1:2,
bacterial growth, potential, and mechanisms to enhance the 1:4, 1:8, and 1:16. In this procedure, 4 mL of liquid NB
availability of soil cations within clay minerals. This study was added to each dilution tube, except for the 1:1 tube
provides insights into the developmental of bacterial (Figure 1).

Table 1. The initial soil characteristic for the pot experiment

Soil K Total CEC Organic K-exchange Ca- exchange Mg- exchange Na- exchange
pH
samples (%) (ppm) carbon (%) (ppm) (ppm) (ppm) (ppm)
A 0.08 11.42 4.72 2.44 0.13 5.73 0.64 0.04
B 0.05 11.27 4.92 1.41 0.20 2.83 0.54 0.02
Average 0.06 11.34 4.82 1.92 0.16 4.28 0.59 0.03
Note: A: depth 0-5 cm, B: depth 6-10 cm
 BANGUN et al. – Dynamic growth and potassium solubilization capability of three novel bacteria 179

Figure 1. Standard curve of KSB based on different serial dilution conditions. A. B. cepacia strain IHB_02, B. B. paludis strain
IHB_01, and C. P. phymatum strain IHB_03

Table 2. The standard density of KSB based on different serial dilution conditions

B. paludis strain IHB_01 P. phymatum strain IHB_03 B. cepacia strain IHB_02

Serial dilutions
Density* Log sel* Absorb Density* Log sel* Absorb Density* Log sel* Absorb
6 6 6
01:16 175 x 10 6.24 0.011 200 x 10 6.3 0.022 181 x 10 6.25 0.030
01:08 35 x 107 7.54 0.027 40 x 107 7.6 0.041 36 x 107 7.55 0.058
01:04 70 x 107 7.85 0.051 80 x 107 7.9 0.082 72 x 107 7.86 0.144
01:02 140 x 107 8.15 0.115 160 x 107 8.2 0.168 145 x 107 8.16 0.257
01:01 28 x 108 9.45 0.224 32 x 108 9.5 0.324 29 x 108 9.46 0.532
Note: *CFU mL-1 (CFU : colony forming unit)

Table 3. The details of the experimental treatments

Accession numbers Bacterial isolates Codes Dosage KSB Bacterial density*

-1
- Control (aquadest) KSB0 10 mL pot -
OR342153 Burkholderia cepacia strain IHB_02 KSB1 10 mL pot-1 34 x 108
OR342197 Paraburkholderia phymatum strain IHB_03 KSB2 10 mL pot-1 35 x 108
OR342151 Burkholderia paludis strain IHB_01 KSB3 10 mL pot-1 62 x 108
Note: *CFU mL-1(CFU = colony forming unit). Note: To ensure uniform density

The selected KSB isolates were introduced into the Measurements of cell density were then conducted at
dilution tubes containing 8 mL of liquid NB. Subsequently, intervals of 30 minutes, facilitated by the
4 mL of the solution was transferred to 1:1 tube, and 4 mL spectrophotometer, extending until the culture reached a
was transferred to 1:2 tube, which initially held 4 mL of phase of stability or until a 24-hour period elapsed. The
liquid NB. These mixtures were thoroughly mixed, and the determination of cell count was accomplished by linking
process was continued by transferring 4 mL of the solution the logarithm of the cell count with the turbidity data
from 1:2 tube to 1:4 tube and homogenized it. A total of 4 derived from the 3 selected isolates, which were earlier
mL of the solution from the 1:4 tube was transferred to the prepared. After the calculation, the growth curve was set
1:8 tube and mixed until it achieved consistent uniformity. for analysis and assessment to determine whether
Approximately 4 mL of the solution from the 1:8 tube was noticeable alterations in growth rate occurred during the
transferred to the 1:16 tube and mixed until it became incubation period. The equation used to calculate the
homogeneous. The subsequent step comprised calibrating concentration of bacteria at a specified time is as follows
the spectrophotometer using liquid nutrient broth media, to Eq. 1 (Maier and Pepper 2015):
adjust the measured transmittance to show a value of 100% (Eq.1)
when using this specific NB medium. Within the
experiment geared towards determining the bacterial cell Where:
count using the turbidimetric method, a wavelength of 640 X0 : initial concentration of cells
nm was used. A total of 1 mL of the initial culture from X : concentration after time t
each of the 3 isolates was introduced into 250 mL of NB n : number of generations or cell division
medium and placed within an incubator for 24 hours. Initial In the exponential growth phase, when the initial cell
measurements were taken to ascertain the KSB cell count count and the count at a subsequent specific time were
through a spectrophotometer set at a wavelength of 640 provided, it was possible to determine the number of
nm. This initial measurement established the cell generations using the equation provided in Eq. 2 (Maier
concentration at the outset denoted as time t=0. and Pepper 2015):
180 B I OD I V E R S ITA S 25 (1): 177-185, January 2024

metabolites) reactions. Furthermore, these biosynthetic

(Eq. 2)
processes led to cell division, where bacterial development
could be deeply observed by monitoring the bacterial
Where: growth curve. In this study, the bacterial growth curves B.
n : number of generations or cell division cepacia IHB_02, P. phymatum IHB_03, and B. paludis
In : Natural logarithm (a logarithm using Euler's IHB_01 are presented in Figure 2. In a consistent and
number (e) as the base logarithm) nutrient-rich culture medium, a cell could divide within 10
X : concentration after time t minutes under optimal conditions. Therefore, observations
This evaluation could be performed using an on KSB (specific term or context needed for clarity) were
appropriate equation designed for growth rate assessment conducted at 30-minute intervals.
Eq. 3 (Maier and Pepper 2015): Bacterial proliferation was monitored by observing the
progressive increase in the number of bacterial cells over a
(Eq. 3) particular duration. Throughout the observation period, the
adaptation stage of B. cepacia strain IHB_02 spanned from
30 to 240 minutes. Furthermore, the initial population of
Where: bacterial cells introduced through inoculation was
μ : Growth rate approximately 0.0066 log cells (CFU mL-1), equivalent to
In : Natural logarithm (a logarithm using Euler's roughly 10 cells. The exponential phase was then observed
number (e) as the base logarithm) for 240 to 960 minutes.
n2 and n1: Number of cells at time t2 and t1 From the growth graph, the generation time for 8 hours
t2-t1 : Difference in time between the two was computed using Eq. 2, yielding a calculated result of n
measurements = Log 2.5890-Log 0.0066/0.693 = 4 generations. The
The soil application experiment began by cultivating number of generations within 8-hour timeframe was then
the 3 isolates in the Aleksandrov medium, followed by a determined as n = 4 generations. Based on this dataset, the
24-hour incubation period for each of them. Population cellular concentration (X) was determined at 8, 16, and 32
quantification was achieved through TPC method with the hours using Eq. 1, leading to the following: at 8 hours, X =
Aleksandrov medium. Subsequently, a total of 20 pots were 24 (10) = 16x10 cells; while at 16 hours with n = 8
prepared, each containing 500 grams of sieved soil (mesh generations, X = 28 (10) = 26x102 cells; at 32 hours with n
size: 40). Bacterial potency testing in soil was carried out = 16 generations, X = 216 (10) = 65x104 cells. The specific
with a non-factorial RCBD (randomized complete block rate of growth during the exponential phase (240-960
design), with 5 repetitions and 4 treatment levels. A total of minutes) was determined using Eq. 3: μ = (Log 4.23-Log
500 grams of soil per pot (Table 3) was used and incubated 0.0066)/(16 - 0) = 0.1755 cells/mL/hour.
at 28 ± 2°C for 30 days. After incubation, a comprehensive
total K measurement was conducted using the HNO3
method along with an Atomic Absorption
Spectrophotometer (AAS). Further analysis, such as
comprising CEC, exchangeable-K, exchangeable-Ca,
exchangeable-Mg, and exchangeable-Na, were performed
with the Ammonium acetate pH 7 method, while the
Walkey and Black method was used to determine the
organic carbon content.

Data analysis
The data obtained were subjected to statistical analysis
using ANOVA. When a significant impact was detected,
the subsequent examination comprised Duncan's Multiple
Range Test (DMRT) at a 5% significance level. The
analysis was carried out using Microsoft Excel 2016 and
GraphPad Prism (Version 9.5.1) software.

RESULTS AND DISCUSSION

Growth curve of KSB2 Figure 2. Growth curves of Burkholderia cepacia strain IHB_02,
B. paludis strain IHB_01, and P. phymatum strain IHB_03 over
Metabolism is a complex process comprising several 16 hours
anabolic (the synthesis of cell components and metabolites)
and catabolic (the breakdown of cell components and
 BANGUN et al. – Dynamic growth and potassium solubilization capability of three novel bacteria 181

Burkholderia paludis strain IHB_01 showed that the KSB influence on soil fertility
bacterial adaptation phase occurred within 30 to 330- The results from the second phase of the experiment
minute timeframe. After this process, the initial population showed that 3 distinct KSB isolates, namely B. cepacia
of bacterial cells introduced through inoculation was IHB_02, P. phymatum IHB_03, and B. paludis IHB_01,
approximately 0.0066 log cells (CFU mL-1), which roughly into the soil for 30 days yielded outcomes with no
translated to 8 cells. The phase of exponential growth in significant impact on the quantified values of total K, CEC,
these bacteria took place between 360 and 960 minutes. pH levels, and organic carbon content. These outcomes
From the growth curve, generation time for an 8-hour underscored the absence of significant alterations from this
duration was computed using Eq. 2, yielding a result of n = specific application, as shown in Figure 3
Log 2.1010-Log 0.0066/0.693 = 4 generations. Based on The results showed significant differences among the
this dataset, the cell X was calculated at 8, 16, and 32 hours treatment groups regarding the total soil K content. The
using Eq. 1 in the following manner: at 8 hours, X = 24 (8) implementation of KSB1 led to a reduction of
= 128 cells; similarly, at 16 hours with n = 8 generations, X approximately 9.26% in the overall K levels compared to
= 28 (8) = 204x102 cells; and at 32 hours with n = 16 KSB0, while KSB2 and KSB3 exhibited decreases of 5.72
generations, X = 216 (8) = 52x104 cells. The specific growth and 10.00%, respectively. The assessment of CEC
rate during the exponential phase (360 - 960 minutes) was measurements also showed notable variations. KSB1,
determined using Equation 3: μ = (Log 6.58-Log KSB2, and KSB3 showed differences of approximately
0.0066)/(16-0) = 0.1875 cells/mL/hour. 5.85, -3.72, and +2.98%, respectively, relative to KSB0.
For P. phymatum strain IHB_03, exhibited bacterial These shifts in CEC could show alterations in the soil's
adaptation phase occurred within the time range of 30 to ability to retain and exchangeable ion levels. The
270 minutes. Furthermore, the initial population of examination of pH levels showed intriguing dynamics,
bacterial cells introduced through inoculation was where KSB1 and KSB2 caused reductions of -3.47 and -
approximately 0.0066 Log cells (CFU mL-1), which was 5.00%, respectively. The present results suggested the
approximately equivalent to 10 cells. The bacterial engagement of a K dissolution mechanism triggered by the
population then entered an exponential growth phase production of organic acids by these bacteria.
spanning from 300 to 930 minutes. From the growth curve, KSB3 displayed a distinctive trend, showing an
the generation time for 8 hours was calculated using Eq. 2, increase in pH levels by 2.17% compared to KSB0. This
yielded a result of n = Log 2.8084-Log 0.0066/0.693 = 4 divergence in alterations suggested varying metabolic
generations. The number of generations occurring within activities among the bacterial isolates. Regarding the
the 8 hours was determined as n = 4 generations. Based on organic carbon observation, a similar pattern to the pH
this data, the cell concentration (X) was calculated at 8, 16, trend was observed. Organic material content decreased,
and 32 hours using Eq. 1 as follows: at 8 hours, X = 24 (10) with KSB1 and KSB2 contributing to reductions of -6.17%
= 160 cells; while at 16 hours with n = 8 generations, X = and -7.10%, respectively. The results showed that KSB3
28 (10) = 25x103 cells; and at 32 hours with n = 16 showed a different effect, contributing to a 5.56% increase
generations, X = 216 (10) = 65x105 cells. The specific in organic carbon content compared to KSB0. These shifts
growth rate during the exponential phase (300-930 in organic carbon content reflected the interplay between
minutes) was calculated using Eq. 3: μ = (Log 4.6312-Log bacterial activities and the breakdown of organic matter.
0.0066)/(16-0) = 0.1780 cells/mL/hour. Furthermore, the illustration presented in Figure 4 provided
valuable insights into the cation exchangeable process
within clay minerals following a 30-day incubation period.

Figure 3. The effect of all KSB on the soil after 30 days of Figure 4. Cations exchangeable in clay minerals. (A) K-
incubation on clay mineral KSB 10 ml pot-1 (108 CFU mL-1). (A) exchangeable, (B) Ca-exchangeable, (C) Mg-exchangeable, and
K total, (B) CEC, (C) pH, and (D) organic carbon (D) Na-exchangeable after 30 days of incubation
182 B I OD I V E R S ITA S 25 (1): 177-185, January 2024

In this study, visual cues in the form of demarcations 16 hours, and 52x104 at 32 hours. Meanwhile, P.
were integrated to aid in distinguishing between values on phymatum IHB_03 had concentrations of 160 cells at 8
the lower and higher ends, and a central line was used to hours, 25x103 at 16 hours, and 65x105 at 32 hours. The
show the reference point of the mean. The data, comprising growth rate during the exponential phase was measured
the concentrations of each cation within different treatment using the specific growth rate. During the exponential
KSB provided intriguing insights. A closer analysis showed phase, the specific growth rate for B. cepacia IHB_02 was
significant noteworthy trends and variations among the 0.1755 cells/mL/hours, for B. paludis IHB_01 it was
treatment groups. In observing the Na exchangeable data, it 0.1875 cells/mL/hours, and for P. phymatum IHB_03 it
had a significant effect compared to after application to the was 0.1780 cells/mL/hours.
KSB treatment. KSB1, KSB2, and KSB3 treatments In the second phase, experimental results indicate that
showed elevated Na concentrations compared to the KSB0 the application of KSB did not lead to substantial variations
group. Among these treatments, KSB2 stood out with the in measured parameters such as total K, CEC, pH, and
most significant surge in Na content, recording a organic carbon content. Although there were differences in
concentration increase of approximately 318%. KSB3 the total soil K content, these differences were not
followed closely with an increase of approximately 393%, statistically significant among the three novel bacterial
and KSB1 exhibited a substantial increase of 406% strains. Additionally, differences are observed in CEC
compared to KSB0. observations among treatment groups, though not
Based on the results, K exchangeable alterations statistically significant. This may indicate changes in the
showed no significant impact. The KSB1 treatment caused soil's capacity to retain and exchange ions. Based on soil
a reduction in soil K content of approximately 5.56% pH observations, KSB3 treatment shows an increase in pH
compared to the KSB0 group. Furthermore, both KSB2 and compared to the control and KSB1 and KSB2 treatments.
KSB3 showed decreases in K exchangeable, showing Observations related to organic carbon content also exhibit
reductions of approximately 5.72% and 10.00% a similar pattern to the pH trend. Data related to Na
respectively. This effectively underscored and strengthened exchange show a significant impact, with all three
these treatments' influence on K levels. The data related to treatments (KSB1, KSB2, KSB3) able to increase Na
the exchangeable Ca did not show an easily identifiable concentrations compared to the control. Meanwhile, data
pattern. Although there were slight fluctuations in Ca on K exchange show a different pattern, where KSB2
concentrations among the different treatment groups, these shows higher exchangeable K concentrations than KSB1
variations were not significant to definitively establish a and KSB3. Regarding cation exchange, Ca does not show a
clear impact of the treatments on the exchangeability of Ca clear pattern in all treatments and does not have sufficient
within clay minerals. The exchangeable Mg did not show significance to determine treatment effects conclusively.
significant disparities among the treatment groups. The Mg Meanwhile, exchangeable Mg concentrations remain fairly
concentrations remained relatively consistent across all consistent in all treatments, without noticeable trends or
treatments, without any pronounced trends or significant significant differences after a 30-day incubation period.
differences occurring after the 30-day incubation period. Based on the results, it was clear that all three bacteria
undergo an adaptation process to the new environmental
Discussion conditions. This indicates that the bacteria adapt to the
Research on bacterial growth through growth curves environmental conditions before entering the exponential
serves as a foundation for understanding the complex growth phase. Furthermore, B. cepacia IHB_02 exhibits the
metabolic processes in bacterial cell development. Three longest exponential growth phase, followed by P.
novel discovered potassium-dissolving bacteria, namely B. phymatum IHB_03 and B. paludis IHB_01. Bacteria B.
cepacia IHB_02, P. phymatum IHB_03, and B. paludis cepacia IHB_02 may be more efficient in utilizing
IHB_01, were the focal points of growth analysis. resources and experiencing a broader period of growth. The
Spectroscopy methods were employed to calculate bacterial exponential growth phase emphasizes rapid bacterial
growth by referring to the standard curves of each division and an exponential increase in the number of cells
bacterium. during this phase. During the exponential phase, bacteria
In the bacterial growth graph, it was observed that all divide rapidly, and the number of cells increases
three strains undergo an initial adaptation phase from exponentially over time (Sood et al. 2011). Disparities in
around minute 30 to 240. The exponential phase of the cell concentrations at specific time intervals also indicate
three bacteria differs, lasting for 12 hours (minutes 240 to fluctuations in the length or intensity of the exponential
960) for B. cepacia IHB_02, 10 hours (minutes 360 to 960) growth phase (Schuurmans et al. 2017). It was observed
for B. paludis IHB_01, and 10.5 hours (minutes 300 to that bacteria with higher concentrations may undergo a
930) for P. phymatum IHB_03. The number of generations more intense exponential growth phase, a similar
for all three bacteria tends to be similar, occurring over 8 phenomenon also observed by Bertrand (2019), who
hours with a total of 4 generations. The concentration of suggesting that strains with higher concentrations may
cells for the three bacteria varies at each observation point experience a longer or more intense exponential growth
(8, 16, and 32 hours). Burkholderia cepacia IHB_02 had a phase, indicating a higher ability to divide rapidly. From
concentration of 16x10 cells at 8 hours, 26x102 at 16 hours, the data on concentrations at specific time intervals, each
and 65x104 at 32 hours. In the case of B. paludis IHB_01, strain exhibits different growth rates. Burkholderia cepacia
the cell concentration was 128 cells at 8 hours, 204 x 102 at IHB_02 shows a lower growth rate compared to B. paludis
 BANGUN et al. – Dynamic growth and potassium solubilization capability of three novel bacteria 183

IHB_01 and P. phymatum IHB_03 during the observed organic matter decomposition process (Gougoulias et al.
time intervals. This variation indicates that each strain 2014; Chen et al. 2023).
experiences a different growth rate during the exponential The ability of all treatments to increase Na exchange
phase (Berney et al. 2006). This could imply differences in can be influenced because, in general, KSB inoculation can
resource utilization efficiency and reproductive capabilities lead to the release of K from the soil ion exchange layers,
among the bacteria. potentially increasing Na concentrations in the soil solution
Furthermore, this study also revealed that each (Wakeel 2013). Additionally, clay soil, known for its high
bacterium has its specific growth rate. The specific growth ion exchange capacity, may experience Na release due to
rate of B. paludis IHB_01 was higher compared to B. KSB activity, resulting in an increase in Na concentration
cepacia IHB_02 and P. phymatum IHB_03. Variations in in the soil solution (Endo et al. 2002). The impact of KSB
specific growth rates indicate differences in genetic in clay soil affects other microbial communities and
regulation and metabolic capacity among the bacteria influences soil chemical conditions and ion balance,
(Klumpp et al. 2009). Additionally, some studies suggest including Na. The decrease in Ca exchange after the
that the culture environment can also influence these application of KSB1, KSB2, and KSB3 may occur for
parameters (Ihssen and Egli 2004). These differing growth several reasons. Firstly, the reduction in soil K levels due to
rates demonstrate the bacteria's potential for adaptation to KSB1 treatment makes K less available for ion exchange.
their environment (Gonzalez and Aranda 2023). Strains Secondly, although KSB2 and KSB3 treatments show a
with higher specific growth rate values have a better and decrease in K, the impact on the sufficiency of K exchange
faster ability to adapt to environmental changes (Chacón et may be due to changes in how K binds to the soil structure
al. 2020). Differences in temperature, nutrition, and other or interacts with other ions. Thirdly, the observed decrease
environmental conditions can lead to varying growth rates in K exchange after the application of KSB2 and KSB3
among the three bacteria. Understanding these parameters may also be related to the interaction between KSB
has implications for various applications. Strains with bacteria and other microbes in the soil, affecting ion
higher growth rates are often more valuable for industrial exchange properties. The lack of a significant influence on
production or biotechnological applications. Data from total K, as well as exchangeable K, Mg, and Ca, was due to
growth curves, generation time, cell concentration, and antagonistic interactions among cations in the soil.
specific growth rate provide insights into how bacterial According to Tan et al. (1991), antagonistic relationships
metabolism and growth occur in specific culture conditions between K, Ca, and Mg ions occur at soil adsorption sites
(Rolfe et al. 2012). Expanding this understanding beyond due to their similar physical and chemical properties. An
laboratory conditions is crucial for comprehending growth excess of one element can interfere with the absorption of
in natural settings or aquatic ecosystems, where challenges other elements, leading to competition for soil adsorption
arise due to the complex nature of the extreme environment sites. Ratios such as K/Ca, K/(Ca+Mg), and K/(Ca+Mg)
(Maier and Pepper 2015). provide information about soil acidity levels. Fageria
The utilization of three bacteria in the soil did not result (2002) suggested that despite having antagonistic
in distinguishable variations, primarily because the K properties, the ideal percentage of cation exchange
solubilization process could not occur due to the lack of saturation is 65% Ca, 10% Mg, and 5% K, or a Ca:K ratio
supportive environmental conditions, as indicated by the of 13:1 and a Mg:K ratio of 2:1. This also explains the
total soil K content after the application of all bacteria. A antagonism between K, Ca, and Mg. A deficiency in Mg or
key indicator in the K solubilization process, as found in an excess of K can often lead to enzyme unit dissociation,
some studies, involves a decrease in pH, signaling that halting protein formation.
bacteria trigger K solubilization mechanisms by producing In conclusion, the results of the assessment of the
organic acids, which is also observed in some bacteria in growth of three novel potassium-solubilizing bacteria
this study (Meena et al. 2015; Olaniyan et al. 2022). reveal that, despite experiencing similar initial adaptation
However, the differences in pH in each treatment indicate phases, B. cepacia IHB_02 exhibited a longer exponential
varying metabolic activities among bacterial isolates. growth phase, indicating efficiency in resource utilization
Consistent with previous research, KSB3 can solubilize K and an extended growth period. This suggests potential
and increase pH through mechanisms that are not yet fully added value in terms of reproduction and resource
understood (Bangun et al. 2023). The differences in organic utilization. The distinct metabolic specificities among the
carbon content in the inoculation of KSB1, KSB2, and three bacteria are reflected in their respective specific
KSB3 are attributed to disparities in the metabolic growth rates. Burkholderia paludis IHB_01 displays a
capabilities of these three strains. KSB1 and KSB2 exhibit higher specific growth rate, indicating differences in
additional activities related to the decomposition of organic genetic regulation and metabolic capacity. The three KSB
matter, contributing to the decrease in organic carbon. A novels do not significantly impact certain parameters, such
study suggests that bacteria belonging to the as total K, CEC, pH, and organic carbon content, but
Paraburkholderia and B. cepacia groups can degrade variations are observed in specific ion exchanges.
phenolic acid compounds, thus triggering the Burkholderia cepacia IHB_02 and P. phymatum IHB_03
decomposition process (Wilhelm et al. 2021). lead to a decrease in pH and organic carbon content, while
Modifications in organic carbon content reflect the B. paludis IHB_01 shows an increase in pH and organic
complex interactions between bacterial activities and the carbon content. This indicates the complex role of KSB in
influencing soil chemical properties. The weaknesses of
184 B I OD I V E R S ITA S 25 (1): 177-185, January 2024

this study were that it did not observe different nutritional belt of Birbhum District of West Bengal, India. Microbiol Res 183:
80-91. DOI: 10.1016/j.micres.2015.11.011.
and environmental conditions. Therefore, further Gonzalez JM, Aranda B. 2023. Microbial growth under limiting
investigations should be needed on the effect of variations conditions-future perspectives. Microorganisms 11 (7): 1641. DOI:
in nutrition, temperature, pH, and other environmental 10.3390/microorganisms11071641.
factors on bacterial growth. Gougoulias C, Clark JM, Shaw LJ. 2014. The role of soil microbes in the
global carbon cycle: Tracking the below-ground microbial processing
of plant-derived carbon for manipulating carbon dynamics in
agricultural systems. J Sci Food Agric 94 (12): 2362-2371. DOI:
ACKNOWLEDGEMENTS 10.1002/jsfa.6577.
He J-Z, Zheng Y, Chen C-R, He Y-Q, Zhang L-M. 2008. Microbial
composition and diversity of an upland red soil under long-term
The authors are grateful to the Rector of the Universitas fertilization treatments as revealed by culture-dependent and culture-
Muhammadiyah Sumatera Utara, Medan, Indonesia for independent approaches. J Soils Sediments 8: 349-358. DOI:
generously providing the laboratory facilities that have 10.1007/s11368-008-0025-1.
been instrumental in supporting the study process. Hinsinger P, Jaillard B, Dufey JE. 1992. Rapid weathering of a
trioctahedral mica by the roots of ryegrass. Soil Sci Soc Am J 56 (3):
977-982. DOI: 10.2136/sssaj1992.03615995005600030049x.
Ibrahim S, Abdul Khalil K, Zahri KN, Gomez-Fuentes C, Convey P,
REFERENCES Zulkharnain A, Sabri S, Alias SA, González-Rocha G, Ahmad SA.
2020. Biosurfactant production and growth kinetics studies of the
waste canola oil-degrading bacterium Rhodococcus erythropolis
Adhikari K, Hartemink AE. 2016. Linking soils to ecosystem services - A
AQ5-07 from Antarctica. Molecules 25 (17): 3878. DOI:
global review. Geoderma 262: 101-111. DOI: 10.3390/molecules25173878.
10.1016/j.geoderma.2015.08.009. Idris M, Bangun IH, Ani N, Hutagaol D, Siddik F. 2023. The effect of fish
Bangun IH, Hanum H, Sabrina T. 2023. Isolation and molecular
waste and duck manure on the growth and yield of pak choi. J Water
characterization of potassium-solubilizing bacteria from limestone Land Dev 59 (X-XII):100-107. DOI: 10.24425/jwld.2023.147234.
mountain of Bahorok, Langkat District, Indonesia. Biodiversitas 24 Ihssen J, Egli T. 2004. Specific growth rate and not cell density controls
(7): 4175-4184. DOI: 10.13057/biodiv/d240757.
the general stress response in Escherichia coli. Microbiology 150 (6):
Barré P, Velde B, Fontaine C, Catel N, Abbadie L. 2008. Which 2: 1 clay 1637-1648. DOI: 10.1099/mic.0.26849-0.
minerals are involved in the soil potassium reservoir? insights from Klumpp S, Zhang Z, Hwa T. 2009. Growth rate-dependent global effects
potassium addition or removal experiments on three temperate
on gene expression in bacteria. Cell 139 (7): 1366-1375. DOI:
grassland soil clay assemblages. Geoderma 146 (1-2): 216-223. DOI: 10.1016/j.cell.2009.12.001.
10.1016/j.geoderma.2008.05.022. Maier RM, Pepper IL. 2015. Bacterial growth. E Environmental
Berney M, Weilenmann H-U, Ihssen J, Bassin C, Egli T. 2006. Specific
Microbiology (Third edition), Elsevier, Academic Press. DOI:
growth rate determines the sensitivity of Escherichia coli to thermal, 10.1016/B978-0-12-394626-3.00003-X.
UVA, and solar disinfection. Appl Environ Microbiol 72 (4): 2586- Maranguit D, Guillaume T, Kuzyakov Y. 2017. Land-use change affects
2593. DOI: 10.1128/AEM.72.4.2586-2593.2006.
phosphorus fractions in highly weathered tropical soils. Catena 149:
Bertrand RL. 2019. Lag phase is a dynamic, organized, adaptive, and 385-393. DOI: 10.1016/j.catena.2016.10.010.
evolvable period that prepares bacteria for cell division. J Bacteriol Marciano Marra L, Fonsêca Sousa Soares CR, de Oliveira SM, Avelar
201 (7): e00697-18. DOI: 10.1128/jb.00697-18.
Ferreira PA, Lima Soares B, de Fráguas Carvalho R, de Lima JM, de
Bhunia S, Bhowmik A, Mallick R, Mukherjee J. 2021. Agronomic Souza Moreira FM. 2012. Biological nitrogen fixation and phosphate
efficiency of animal-derived organic fertilizers and their effects on solubilization by bacteria isolated from tropical soils. Plant Soil 357:
biology and fertility of soil: A review. Agronomy 11 (5): 823. DOI:
289-307. DOI: 10.1007/s11104-012-1157-z.
10.3390/agronomy11050823. Meena VS, Maurya BR, Verma JP, Aeron A, Kumar A, Kim K, Bajpai
Cai-xia L, Jing-jing Z, Ru-zhen J. 2018. Studies on the growth VK. 2015. Potassium Solubilizing Rhizobacteria (KSR): Isolation,
characteristics of nitrogen-fixing bacterium in soil of Cunninghamia
identification, and K-release dynamics from waste mica. Ecol Eng 81:
lanceolata forest. 林业科学研究 31 (4): 98-103. 340-347. DOI: 10.1016/j.ecoleng.2015.04.065.
Chacón JM, Shaw AK, Harcombe WR. 2020. Increasing growth rate Monod J. 1949. The growth of bacterial cultures. Annu Rev Microbiol 3:
slows adaptation when genotypes compete for diffusing resources. 371-394. DOI: 10.1146/annurev.mi.03.100149.002103.
PLoS Comput Biol 16 (1): e1007585. DOI: Moulin L, Klonowska A, Caroline B, Booth K, Vriezen JA, Melkonian R,
10.1371/journal.pcbi.1007585. James EK, Young JP, Bena G, Hauser L, Land M. 2014. Complete
Chen W, Hu H, Heal K, Sohi S, Tigabu M, Qiu W, Zhou C. 2023. Linking genome sequence of Burkholderia phymatum STM815 T, a broad
microbial decomposition to dissolved organic matter composition in host range and efficient nitrogen-fixing symbiont of Mimosa species.
the revegetation of the red soil erosion area. Forests 14 (2): 270. DOI: Stand Genomic Sci 9: 763-774. DOI: 10.4056/sigs.4861021.
10.3390/f14020270. Officer SJ, Tillman RW, Palmer AS, Whitton JS. 2006. Variability of clay
Compant S, Nowak J, Coenye T, Clement C, Ait Barka E. 2008. Diversity mineralogy in two New Zealand steep-land topsoils under pasture.
and occurrence of Burkholderia spp. in the natural environment. Geoderma 132 (3-4): 427-440. DOI: 10.1016/j.geoderma.2004.11.027.
FEMS Microbiol Rev 32 (4): 607-626. DOI: 10.1111/j.1574- Olaniyan FT, Alori ET, Adekiya AO, Ayorinde BB, Daramola FY,
6976.2008.00113.x. Osemwegie OO, Babalola OO. 2022. The use of soil microbial
Elshafie HS, Camele I. 2021. An overview of metabolic activity, potassium solubilizers in potassium nutrient availability in soil and its
beneficial and pathogenic aspects of Burkholderia Spp. Metabolites dynamics. Ann Microbiol 72: 45. DOI: 10.1186/s13213-022-01701-8.
11 (5): 321. DOI: 10.3390/metabo11050321. Ong KS, Cheow YL, Lee SM. 2017. The role of reactive oxygen species
Endo T, Yamamoto S, Honna T, Eneji AE. 2002. Sodium-calcium in the antimicrobial activity of pyochelin. J Adv Res 8 (4): 393-398.
exchange selectivity as influenced by clay minerals and composition. DOI: 10.1016/j.jare.2017.05.007.
Soil Sci 167 (2): 117-125. DOI: 10.1097/00010694-200202000-00004. Qaswar M, Jing H, Ahmed W, Dongchu L, Shujun L, Lu Z, Cai A,
Fageria NK. 2002. Dry matter yield of common bean, lowland rice, corn, Lisheng L, Yongmei X, Jusheng G, Huimin Z. 2020. Yield
soybean, and wheat at different basic cation saturation ratios in acid sustainability, soil organic carbon sequestration and nutrients balance
soil. Commun Soil Sci Plant Anal 33 (3-4): 519-531. DOI: under long-term combined application of manure and inorganic
10.1081/CSS-120002761. fertilizers in acidic paddy soil. Soil Tillage Res 198: 104569. DOI:
Falk Ogaard A, Krogstad T. 2005. Release of interlayer potassium in 10.1016/j.still.2019.104569.
Norwegian grassland soils. J Plant Nutr Soil Sci 168 (1): 80-88. DOI: Rolfe MD, Rice CJ, Lucchini S, Pin C, Thompson A, Cameron AD,
10.1002/jpln.200421454. Alston M, Stringer MF, Betts RP, Baranyi J, Peck MW. 2012. Lag
Ghosh R, Barman S, Mukherjee R, Mandal NC. 2016. Role of phosphate phase is a distinct growth phase that prepares bacteria for exponential
solubilizing Burkholderia spp. for successful colonization and growth growth and involves transient metal accumulation. J Bacteriol 194
promotion of Lycopodium cernuum L. (Lycopodiaceae) in lateritic (3): 686-701. DOI: 10.1128/jb.06112-11.
 BANGUN et al. – Dynamic growth and potassium solubilization capability of three novel bacteria 185

Scherer HW, Feils E, Beuters P. 2014. Ammonium fixation and release by Wakeel A. 2013. Potassium-sodium interactions in soil and plant under
clay minerals as influenced by potassium. Plant Soil Environ 60 (7): saline-sodic conditions. J Plant Nutr Soil Sci 176 (3): 344-354. DOI:
325-331. DOI: 10.17221/202/2014-PSE. 10.1002/jpln.201200417.
Schuurmans RM, Matthijs JCP, Hellingwerf KJ. 2017. Transition from Wang M, Zheng Q, Shen Q, Guo S. 2013. The critical role of potassium in
exponential to linear photoautotrophic growth changes the physiology plant stress response. Intl J Mol Sci 14 (4): 7370-7390. DOI:
of Synechocystis sp. PCC 6803. Photosynth Res 132: 69-82. DOI: 10.3390/ijms14047370.
10.1007/s11120-016-0329-8. Wilhelm RC, DeRito CM, Shapleigh JP, Madsen EL, Buckley DH. 2021.
Shakeri S, Abtahi A. 2019. Potassium fixation capacity of some highly Phenolic acid-degrading Paraburkholderia prime decomposition in
calcareous soils as a function of clay minerals and alternately wetting- forest soil. ISME Commun 1 (1): 4. DOI: 10.1038/s43705-021-
drying. Arch Agron Soil Sci 66 (4): 445-457. DOI: 00009-z.
10.1080/03650340.2019.1619176. Zahri KN, Zulkharnain A, Ibrahim S, Gomez-Fuentes C, Sabri S, Calisto-
Sood S, Singhal R, Bhat S, Kumar A. 2011. Inoculum preparation. In: Ulloa N, Ahmad SA. 2020. Kinetic analysis on the effects of lead
Comprehensive Biotechnology (Second Edition). Academic Press. (Pb) and silver (Ag) on waste canola oil (WCO) biodegradation by
Burlington. DOI: 10.1016/B978-0-08-088504-9.00090-8. selected Antarctic microbial consortium. Malays J Biochem Mol Biol
Tan Y, Bond WJ, Rovira AD, Brisbane PG, Griffin DM. 1991. Movement 23 (1): 20-23.
through soil of a biological control agent, Pseudomonas fluorescens. Zakaria NN, Roslee AF, Zulkharnain A, Gomez-Fuentes C, Abdulrasheed
Soil Biol Biochem 23 (9): 821-825. DOI: 10.1016/0038- M, Sabri S, Calisto-Ulloa N, Ahmad SA. 2019. Bacterial growth and
0717(91)90092-X. diesel biodegradation in the presence of As, Cu and Pb by Antarctic
Tapia-García EY, Hernández-Trejo V, Guevara-Luna J, Rojas-Rojas FU, marine bacteria. Malays J Biochem Mol Biol 22 (3): 8-15.
Arroyo-Herrera I, Meza-Radilla G, Vásquez-Murrieta MS, Estrada-de Zhang C, Kong F. 2014. Isolation and identification of potassium-
Los Santos P. 2020. Plant growth-promoting bacteria isolated from solubilizing bacteria from tobacco rhizospheric soil and their effect on
wild legume nodules and nodules of Phaseolus vulgaris L. trap plants tobacco plants. Appl Soil Ecol 82: 18-25. DOI:
in central and southern Mexico. Microbiol Res 239: 126522. DOI: 10.1016/j.apsoil.2014.05.002.
10.1016/j.micres.2020.126522. Zhao K, Penttinen P, Zhang X, Ao X, Liu M, Yu X, Chen Q. 2014. Maize
Vandamme P, Goris J, Chen W-M, de Vos P, Willems A. 2002. rhizosphere in Sichuan, China, hosts plant growth promoting
Burkholderia tuberum sp. nov. and Burkholderia phymatum sp. nov., Burkholderia cepacia with phosphate solubilizing and antifungal
nodulate the roots of tropical legumes. Syst Appl Microbiol 25 (4): abilities. Microbiol Res 169 (1): 76-82. DOI:
507-512. DOI: 10.1078/07232020260517634. 10.1016/j.micres.2013.07.003.