IJERD – International Journal of Environmental and Rural Development (2021) 12-2

Research article
erd

Bamboo Charcoal as a Lactic Acid Bacteria Carrier for
Phosphate Removal
TAKAHIKO NAKAMURA
Faculty of Regional Environment Science, Tokyo University of Agriculture, Tokyo, Japan

NARONG TOUCH*
Faculty of Regional Environment Science, Tokyo University of Agriculture, Tokyo, Japan
Email: nt207118@nodai.ac.jp
Received 8 January 2021

Accepted 15 October 2021(*Corresponding Author)

Abstract A decline in the demand for using bamboo products has led to abandoned bamboo
forests in Japan. To reduce the area of abandoned bamboo forests, the use of bamboo as aconstruction material and bamboo charcoal as an absorbent have gained considerable
attention in recent years. In the literature, many studies have described the formation of
biofilms on biochar, leading bamboo charcoal to be considered as a microbial carrier. The
aim of this study was to examine the potential of bamboo charcoal as the microbial carrier oflactic acid bacteria (LAB) for phosphate removal. Bamboo charcoal was immersed in a LAB
solution for 24 h for LAB to adhere to the bamboo charcoal. Then, the bamboo charcoal wasplaced in a bamboo fermented solution. Two types of bamboo charcoal, i.e., without
pretreatment and dissolved in tap water, were used in the experiments. The experiments werealso conducted with and without aeration to determine the effects of oxygenation. The
bamboo charcoal without pretreatment displayed an increase in the phosphate concentration,
indicating that phosphate was released from the bamboo charcoal. LAB-attached bamboo
charcoal demonstrated a much smaller increase in phosphate concentration, suggesting
phosphate was consumed by LAB. Experiments with dissolved bamboo charcoal also
indicated a reduction in the phosphate concentration. The removal rate of phosphatedecreased with an increase in the solution pH, suggesting that alkaline conditions limited theactivity of the bacteria. Furthermore, the redox potential of the solution became negative inthe solution without aeration. Overall, the results demonstrated that bamboo charcoal could
be a LAB carrier; however, the bamboo charcoal must be dissolved prior to its use forphosphate removal. Furthermore, aeration and an acidic during phosphate removal are needed
to obtain a higher removal rate when using LAB.
Keywords bamboo charcoal, microbial carrier, lactic acid bacteria, phosphate removal

INTRODUCTION
Although bamboo has been used in many different fields in Japan, a decrease in the demand forbamboo products has led to abandoned bamboo forests. According to a report by the Forestry Agencyof Japan, the total bamboo forest area in 2007 was approximately 1600 km2, which was 10% greaterthan in 1981. In 2007, the bamboo forest area covered approximately 0.6% of the total forest area inJapan, but up to 66% of the bamboo forest area was left unused. To reduce the area of abandoned
bamboo forests, several countermeasures have been proposed to increase the utilization rate ofbamboo. Historically, bamboo was primarily used as construction material and for household
furniture. In recent years, bamboo has been used as a fertilizer in agriculture (Cui and Wu, 2010; Liuet al., 2014), and bamboo biochar has been used as an absorbent for contaminant removal (Liu et al.,2012; Mohamed et al., 2015).
In previous reports, a large number of negative charges was observed on the surface of biochar(Yigit and Mazlum, 2007). These negative charges generate electrostatic repulsion between thebiochar surface and anions, making it difficult to adsorb phosphate ions on the surface. Furthermore,some kinds of biochar can release phosphate in aqueous solutions (Yao et al., 2012). For example,Ⓒ ISERD

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Jung et al. (2015) reported the release of phosphate from bamboo biochar. Therefore, when using
biochar for phosphate removal, it is necessary to modify the biochar surface with treatments using
acids, alkali, iron oxide, or magnesium ions (Stratful et al., 2001; Tansel et al., 2018). Biochar
impregnated with colloidal and nanosized metal oxyhydroxides can enhance the removal rate ofphosphate (Zhang and Gao 2013; Zhang et al., 2012). Dai et al. (2020) also reported phosphorus
removal in a solution using magnesium-modified biochar from bamboo. Li et al. (2020) reported theenhancement of phosphate adsorption using polyethyleneimine-modified biochar derived frombamboo biomass.
In addition to adsorption, the biological method is one of the various approaches for phosphateremoval. As biochar is a porous material, it can serve as a habitat for microorganisms (Egamberdieva
et al., 2018). Many studies have examined biofilm formation in biochar and its effect on soil
properties (Yang et al., 2019; Ajeng et al., 2020; Gorovtsov et al., 2020). According to the literature,
it is believed that organisms-attached biochar can increase the removal rate of phosphate in aqueous
solutions. However, to the best of our knowledge, there is a lack of information related to theemployment of organisms-attached bamboo charcoal for phosphate removal. In addition, as theefficacy of phosphate removal strongly depends on the properties of the biochar, a study of phosphateremoval capabilities is necessary before the widespread use of bamboo charcoal.
OBJECTIVE
This study examines the potential of bamboo charcoal as a microbial carrier for lactic acid bacteria(LAB) and its phosphate removal capabilities. It is believed that LAB-attached bamboo charcoal canincrease the removal rate of phosphate in aqueous solutions. Furthermore, this study focuses on
deciphering the effects of the pretreatment of bamboo charcoal for phosphate removal. As the releaseof phosphate from bamboo charcoal has been previously reported in the literature (Yao et al., 2012),
and bacteria need oxygen for their activities, the effects of oxygen supply (aeration) and solution pH
on the phosphate removal by bamboo charcoal are also examined.
METHODOLOGY
Material Used for the Experiments
Bamboo charcoal (Fig. 1a) and bamboo powder (Fig. 1b) are readily available products. The bamboo
charcoal was passed through 3.75 cm and 5.6 mm-mesh sieves. Only the residual on 5.6 mm-meshsieve was used in experiments, i.e., the diameter range of 0.56-3.75 cm. Two kinds of bamboo
charcoal were prepared, i.e., without pretreatment and dissolved in tap water (50 g of bamboo
charcoal in 500 mL of water). For the bamboo charcoal dissolved in tap water, the solution was
replaced until no further release of phosphate was observed. The bamboo powder was fermented for
four days, and then the supernatant (hereafter, called bamboo supernatant) was extracted and used in
the experiments.

(a) Bamboo charcoal(b) Bamboo powder

Fig. 1 Bamboo charcoal and bamboo powder used in the experimentsⒸ ISERD

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Experimental Procedures and Measurements
First, approximately 50 g of bamboo charcoal was placed in a LAB solution for 24 h to attach LAB
to the bamboo charcoal. Then, the bamboo charcoal was placed in the bamboo supernatant to assess
the phosphate removal. The LAB solution was produced by mixing 50 mL of LAB beverage with 1
L of deionized water. The experiments were conducted with and without aeration to examine theeffects of oxygen on LAB for phosphate removal. The experiments were conducted in a room withthe ambient temperature range of 25-30℃.
Temporal measurements of the solution pH, redox potential (ORP), and phosphateconcentration were conducted. The pH and ORP were directly measured by placing a pH/ORP meter(Horiba, D-73) into the solution. The solution in the bamboo charcoal layer was extracted using asyringe, and the phosphate concentration in the extracted solution was measured using a digital
Packtest (Kyoritsu, DPM2-PO4-D). A summary of these experimental procedures is shown below in
Fig. 2. A duplicate measurement was conducted, and the average value was used for discussion.
Phosphate concentration, pH and ORP were measured with relative errors of 0.1 mg/L, 0.1, and 1
mV, respectively.
Measurements
Phosphate

pH/ORP

Extracted solution

Fermentation (4 days)

Aeration
Supernatant
Phosphate: 2 mg/L

Tap water
Bamboo powder

Attachment (24h)Bamboocharcoal

LAB beverage(50 mL)

Bamboo charcoal(50 g)➢ No pretreatment

Deionized water(1 L)➢ Dissolved(50 g)
Diameter of 40 mm
Height of 185 mm

Fig. 2 Summary of experimental procedures
RESULTS AND DISCUSSION
Temporal Changes in Phosphate Concentrations (Bamboo Charcoal Without Pretreatment)
Figure 3 shows the temporal changes of phosphate concentration when using bamboo charcoal (nopretreatment) without aeration. The phosphate concentration in the bamboo supernatant wasapproximately 2 mg/L, and increased to 37 mg/L at day 20 after the experiment started (Fig. 3a).
This increase indicates a release of phosphate from the bamboo charcoal, which is in good agreementwith the findings by Jung et al. (2015).
When LAB-attached bamboo charcoal was used, the increase in the phosphate concentration
was smaller compared to the bamboo charcoal without LAB. This indicates phosphate removal by
LAB. Even though the LAB is attached to the bamboo charcoal, they effectively remove phosphatefrom the aqueous solution. These results suggest that LAB-attached bamboo charcoal can be anabsorbent for phosphate removal.
The removal rate of phosphate by LAB was 40% at day 2, 50% at day 6, and 56% at day 20,
see Fig. 3b. These removal rates are on the same scale as conventional biological methods, whichremove 20-40% of phosphorus (Ruzhitskaya and Gogina, 2017). Furthermore, it can be inferred from
Fig. 3b that the removal capacity rapidly decreased, as it was 40% at day 2, but only increased byⒸ ISERD

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40
Without LAB
LAB attached

35

Removal rate by LAB (%)

Phosphate concentration (mg/L)

10% from day 2 to day 6 and by 6% from day 6 to day 20. It is likely that the metabolic activity or
the bacteria growth was limited due to the lack of oxygen. Without aeration, the ORP decreased
significantly at day 20 when using LAB-attached bamboo charcoal to -300 mV vs. Ag/AgCl (nooxygen in water), while the ORP was maintained at 190 mV when using bamboo without LAB.
Moreover, the solution pH was 7 when using LAB-attached bamboo charcoal but climbed to 9 whenusing bamboo charcoal, indicating that the dissolution of bamboo charcoal increases the solution pH.
In summary, these results indicated that pretreatment and aeration are necessary when using LABattached bamboo charcoal for phosphate removal.

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Operation time (day)Operation time (day)(a) Phosphate concentration(b) Removal rate of phosphate by LAB

Fig. 3 Changes over time of phosphate concentration and removal rate of phosphate by LAB
using bamboo charcoal (no pretreatment) without aeration
Temporal Changes in Phosphate Concentrations (Dissolved Bamboo Charcoal)
Based on the findings shown in Fig. 3, pretreatment of the bamboo charcoal and aeration are bothneeded for phosphate removal. Figure 4 shows the temporal changes of phosphate concentration
when using dissolved bamboo charcoal with aeration. The phosphate concentration of the bamboo
supernatant was approximately 2 mg/L and slowly increased to 12 mg/L on day 13 after theexperiment began (Fig. 4). In comparison with Fig. 3a, the release of phosphate from the bamboo
charcoal was strongly suppressed due to the dissolution of bamboo charcoal before its use in theexperiments. This indicates that bamboo charcoal without pretreatment is not an effective absorbent
for phosphate removal.

Fig. 4 Temporal changes of phosphate concentration using dissolved bamboo charcoal
with aeration
When using LAB-attached bamboo charcoal, a decrease in the phosphate concentration wasobserved, indicating phosphate removal by LAB. Up to 95% of phosphate was removed during thefirst 3 days due to a low initial concentration of phosphate. According to Fig. 3a, LAB removed 5
mg/L of phosphate on day 2 and 11.5 mg/L on day 6. Interestingly, according to Fig. 4, LAB couldⒸ ISERD

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not completely remove the 2.8 mg/L of phosphate produced on day 7; however, it was able to remove
8.3 mg/L of phosphate on day 13. As bacteria are involved in this reaction, it is believed from theseresults that pH and dissolved oxygen (DO) concentration may also affect phosphate removal.
Effects of DO Concentration and pH on Phosphate Removal
By providing aeration, the ORP was maintained at an oxidizing potential of 275 mV when using
bamboo charcoal without LAB, and the ORP was 245 mV with LAB-attached charcoal on day 13.
The slightly lower ORP was observed for LAB-attached charcoal, indicating a small oxygen
consumption by LAB. In other words, DO was enough for bacteria respiration when aeration wasprovided, which was considered to be less effect on the bacteria’s activity. In addition, it is known
that LAB is more active in acidic conditions; however, the dissolution of bamboo charcoal increases
the solution pH, potentially influencing the bacteria’s activity.
Figure 5 depicts the temporal changes in the solution pH when using dissolved bamboo charcoal
with aeration. The dissolution of the bamboo charcoal increased the solution pH with and without
LAB; however, the pH was lower with LAB-attached bamboo charcoal (Fig. 5a). This was attributed
to the activities of LAB which released lactic acid. The data from Fig. 5a indicate that the solution
pH was 6.3 at day 1, which induced the high phosphate removal rate of 95% on day 3. The solution
pH increased from 6.3 on day 1 to 7.9 on day 3, and then decreased to 6.9 on day 7. Although thesolution pH decreased from day 3 to 7, the pH value of 7.9 influenced the removal rate from day 3
to 7, i.e., a decrease in the removal rate to 55% was observed on day 7.
Similarly, a pH value of 6.9 on day 7 led to an increase in the removal rate to 68% on day 13. It
can be understood from these results that the removal rate is dependent on the solution pH. By
plotting the removal rate versus the solution pH (Fig. 5b), it can be observed that the removal rate
and pH were strongly correlated (R=-0.947), as expected. These results indicate that the solution pH
should be maintained in an acidic state (lower than 6.5) to achieve a higher removal rate.

(a) Temporal changes in pH and removal rate(b) Relationship between pH and removal rate

Fig. 5 Relationship between pH and removal rate
CONCLUSION
In this study, the potential of bamboo charcoal as a microbial carrier for LAB was evaluated, and its
ability to remove phosphate was investigated. The effects of oxygen supply and solution pH on thephosphate removal by bamboo charcoal were also examined. Without aeration, a negative redox
potential (ORP) was observed, indicating a large oxygen consumption by LAB. Thus, aeration isneeded when using LAB. Even with dissolved bamboo charcoal, the release of phosphate wasobserved, indicating that bamboo charcoal alone is not an effective absorbent for phosphate removal.
When LAB-attached bamboo charcoal was used, a decrease in the phosphate concentration wasobserved. This suggests that LAB-attached bamboo charcoal is an effective adsorbent for phosphateremoval. With aeration, the ORP was maintained in an oxidizing state, which had no impact onⒸ ISERD

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phosphate removal. However, the removal rate was found to depend on pH, where the removal rate
decreased with an increase in the solution pH. Therefore, the phosphate removal should be conductedunder acidic conditions (pH lower than 6.5) to obtain a higher removal rate.
REFERENCES
Ajeng, A.A., Abdullah, R., Ling, T.C., Ismail, S., Lau, B.F., Ong, H.C., Chew, K.W., Show, P.L. and Chang,
J.S. 2020. Bioformulation of biochar as a potential inoculant carrier for sustainable agriculture. Environ.
Technol. Innov., 20, Retrieved from https://doi.org/10.1016/j.eti.2020.101168
Cui, Y. and Wu, L.R. 2010. Present situation and prospect of bamboo vinegar study in China. J. Bamboo Res.,
29 (1), 11-16.
Dai, J., Zheng, X., Wang, H., Zhang, H., Zhang, L., Lin, T., Qin, R. and Qiu, M. 2020. The removal ofphosphorus in solution by the magnesium modified biochar from bamboo. Nat. Environ. Pollut. Technol.,
19 (1), 389-393.
Egamberdieva, D., Hua, M., Reckling, M., Wirth, S. and Bellingrath-Kimura, S.D. 2018. Potential effects ofbiochar based microbial inoculants in agriculture. Environ. Sustain., 1 (1), 19-24.
Gorovtsov, A.V., Minkina, T.M., Mandzhieva, S.S., Perelomov, L.V., Soja, G., Zamulina, I.V., Rajput, V.D.,
Sushkova, S.N., Mohan, D. and Yao, J. 2020. The mechanisms of biochar interactions withmicroorganisms in soil. Environ. Geochem. Health, 42, 2495-2518.
Huo, Y., Liu, Z., Xuan, H., Lu, C., Yu, L., Bao, W. and Zhao, G. 2016. Effects of bamboo vinegar powder on
growth performance and mRNA expression levels of interleukin-10, interleukin-22, and interleukin-25 in
immune organs of weaned piglets. Anim. Nutr., 2, 111-118.
Jessada, R. 2014. Effects of dietary bamboo charcoal powder including bamboo vinegar liquid supplementation
on growth performance, fecal microflora population and intestinal morphology in Betong chickens. J. Poult.
Sci., 51 (2), 165-171.
Jung, K.W., Hwang, M.J., Ahn, K.H. and Ok, Y.S. 2015. Kinetic study on phosphate removal from aqueous
solution by biochar derived from peanut shell as renewable adsorptive media. Int. J. Environ. Sci. Technol.,12, 3363-3372.
Li, T., Tong, Z., Gao, B., Li, Y.C., Smyth, A. and Bayabil, H.K. 2020. Polyethyleneimine-modified biochar
for enhanced phosphate adsorption. Environ. Sci. Pollut. Res., 27, 7420-7429.
Liu, Y., Gan, L., Chen, Z., Megharaj, M. and Naidu, R. 2012. Removal of nitrate using Paracoccus sp. YF1
immobilized on bamboo carbon. J. Hazard. Mater., 229, 419- 425.
Liu, Z.X., Cui, Y., Huo, Y.J., Yu, L.H., Bao, W.B. and Xu, Z.H. 2014. The comparison of the physicochemical
property of two different types of bamboo vinegar powder as feed additive. J. Bamboo Res., 33 (3), 27-31.
Mohamed, I., Zhang, G., Li, Z., Liu, Y., Chen, F. and Dai, K. 2015. Ecological restoration of an acidic Cd
contaminated soil using bamboo biochar application. Ecol. Eng., 84, 67-76.
Ruzhitskaya, O. and Gogina, E. 2017. Methods for removing of phosphates from wastewater. MATEC Web
of Conferences, 106, pp. 7, Retrieved from https://doi.org/10.1051/matecconf/20171060SPbWOSCE20167006
Stratful, I., Scrimshaw, M.D. and Lester, J.N. 2001. Conditions influencing the precipitation of magnesium
ammonium phosphate. Water Res., 35, 4191-4199.
Tansel, B., Lunn, G. and Monje, O. 2018. Struvite formation and decomposition characteristics for ammonia
and phosphorous recovery: A review of magnesium-ammonia-phosphate interaction. Chemosphere, 194,504-512.
Yang, F., Zhou, Y., Liu, W., Tang, W., Meng, J., Chen, W. and Li, X. 2019. Strain-specific effects of biochar
and its water-soluble compounds on bacterial growth. Appl. Sci., 9, 3209, pp. 11.
Yao, Y., Gao, B., Zhang, M., Inyang, M. and Zimmerman, A.R. 2012. Effect of biochar amendment on sorption
and leaching of nitrate, ammonium, and phosphate in a sandy soil. Chemosphere, 89, 1467-1471.
Yigit, N.O. and Mazlum, S. 2007. Phosphate recovery potential from wastewater by chemical precipitation atbatch conditions. Environ. Technol. Lett., 28, 83-93.
Zhang, M. and Gao, B. 2013. Removal of arsenic, methylene blue, and phosphate by biochar/AlOOH
nanocomposite. Chem. Eng. J., 226, 286-292.
Zhang, M., Gao, B., Yao, Y., Xue, Y.W. and Inyang, M. 2012. Synthesis of porous MgO-biochar
nanocomposites for removal of phosphate and nitrate from aqueous solutions. Chem. Eng. J., 210, 26-32.

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