IJERD – International Journal of Environmental and Rural Development (2023) 14-1

Research article
erd

Research article
Evaluating Water Purification Capacity of Patinopecten
yessoensis Shells Inoculated with Effective Microorganisms

KOSEI MOROISHI
Graduate School of Agro-Environmental Science, Tokyo University of Agriculture, Japan

MACHITO MIHARA*Faculty of Regional Environment Science, Tokyo University of Agriculture, Japan
Email: m-mihara@nodai.ac.jp
Received 15 January 2023

Accepted 25 May 2023(*Corresponding Author)

Abstract In Japan, the installation of biotopes, defined as aquatic systems aiming to recreatea natural environment, has been popular since the 20th century. Due to the closed character ofmany biotopes and high nutrient inputs, one of the main issues related to water quality in abiotope is the occurrence of blue-green algae. In this environment, reduction and control ofthe blue-green algae cannot be done by chemicals. Water quality must be maintained by othernon-chemical means. Therefore, this study aims to investigate the effectiveness of
patinopecten yessoensis shells inoculated with effective microorganisms as a method fornutrient and water quality control in a biotope environment. In Japan, huge numbers of
patinopecten yessoensis shells are left out in the open because they cannot be properly handled.
At the same time, patinopecten yessoensis shells are porous and have been studied for water
purification. Accordingly, this research has focused on the water purification capacity ofpatinopecten yessoensis shells inoculated with effective microorganisms. In the study
experiment, patinopecten yessoensis shells inoculated with lactobacillus, bacillus subtilis var.
natto, and saccharomyces cerevisiae were used to purify water samples taken from a pond in
Yatoyama Park in Kanagawa Prefecture, Japan. To compare nitrogen removal capacity, theexperiments consisted of 8 treatments in total. In the first group of four treatments,
patinopecten yessoensis shells were burned at 600 degrees to increase pore size anddistribution, and were then inoculated with the aforementioned microorganisms. In the second
group of four treatments, patinopecten yessoensis shells were not burned, and were only
inoculated with the microorganisms. Uninoculated burned and unburned shells were used ascontrol treatments. The experimental results showed total nitrogen removal rates of 42% for
unburned and uninoculated shells, 0% for lactobacillus-inoculated unburned shells, 45% forbacillus subtilis var. natto-inoculated unburned shells, and 58% for saccharomyces cerevisiaeinoculated unburned shells. Regarding the burned shells, results showed 51% nitrogen
reduction at the uninoculated shells treatment. Lactobacillus-inoculated burned shells reduced
nitrogen by 47%, bacillus subtilis var. natto-inoculated burned shells reduced by 34% andsaccharomyces cerevisiae-inoculated burned shells reduced the nitrogen in the water sample
by 69%. Since this was a short-term experiment, a long-term experiment as well as fieldapplicable methods need to be discussed in the future.
Keywords water purification, microorganism, patinopecten yessoensis, lactobacillus,
bacillus subtilis var. natto, saccharomyces cerevisiae

INTRODUCTION
Biotope is a term used to describe a biological space; biotopes attracted attention in Germany in the
1970s when environmental problems arose. In Japan, the expansion of urban areas due to rapid
population growth since the period of high economic growth has led to a decrease in farmland andforest land. Accordingly, the habitat and growth environment for living organisms has decreased,and biodiversity has been damaged due to environmental changes, invasions, and pollution. For thesereasons, biotopes have begun to be created in many places since the end of the 20th century. However,Ⓒ ISERD106

IJERD – International Journal of Environmental and Rural Development (2023) 14-1

since biotopes are closed water systems with high nutrient inflow, the occurrence of blue-green algae
has become one of the problems. The use of chemicals is an effective means to suppress and control
the occurrence of blue-green algae. However, the use of chemicals in biotopes with biodiversity canlead to the destruction of biodiversity. Hence, it is necessary to maintain water quality by meansother than chemicals.
In Japan, about 300,000 to 600,000 tons of patinopecten yessoensis are caught annually, and it
is said that “about half of the catch is shells of patinopecten yessoensis” (Fishery Waste Disposal
Guidelines Revised, Ministry of the Environment, 2020). The patinopecten yessoensis shells would
normally be properly disposed of at a processing plant, but the huge number of shells is causing ashortage at the plant. As a result, many shells are left out in the open without being properly handled.
However, patinopecten yessoensis shells are porous and have been studied for water purification.
Accordingly, research attention has been focused on the water purification potential of patinopecten
yessoensis shells inoculated with effective microorganisms.
OBJECTIVE
The purpose of this study is to investigate the effectiveness of patinopecten yessoensis shells
inoculated with effective microorganisms as a method of nutrient removal and water quality
management in a biotope environment. There has been little discussion of water purification using
patinopecten yessoensis shells inoculated with effective microorganisms.
The objectives of this study were to investigate the water quality in the biotope, to evaluate theinoculation capacity of microorganisms in patinopecten yessoensis shells, and to evaluate andcompare the water purification capacity of patinopecten yessoensis shells inoculated with effective
microorganisms.
METHODOLOGY
Field Survey
This study was conducted at 3 biotopes in Yatoyama Park, Zama City, Kanagawa Prefecture, Japan(Site 1 - Site 3). Yatoyama Park has been developed as a natural ecology observation park in a placethat retains a satoyama atmosphere. It is a place being rich in biodiversity with fireflies in the summer
and waterflow in the fall and winter. However, according to the manager of Yatoyama Park,
eutrophication of the biotope and the occurrence of blue-green algae have been confirmed. There areconcerns about the impact on people who visit the park and the negative effects on the surrounding
environment. Water samples were collected approximately once a month from June 2021 to February2022, and total nitrogen (TN), total phosphorus (TP), potential hydrogen (pH), electric conductivity(EC), and suspended solid (SS) were measured. The water quality level in the biotope was determinedbased on the environmental standards listed by the Ministry of the Environment.
Water Purification Experiment Using Patinopecten yessoensis Shells Inoculated with
Effective Microorganisms
In the study experiment, discarded patinopecten yessoensis shells from Aomori Prefecture,
lactobacillus, bacillus subtilis var. natto, and saccharomyces cerevisiae were used. Thesemicroorganisms were used because they have been used in water purification and are relatively easy
to obtain.
Discarded patinopecten yessoensis shells were crushed with a hammer and then divided into
two groups, with one group being unburned (U) and the second group being burned at 600 degrees
to increase pore size and distribution (B). In addition, the burned shells were rinsed once in water toremove the calcium hydroxide generated after burning.

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The unburned and burned shells were inoculated with the above-mentioned microorganisms for
3 days. Uninoculated burned and unburned shells were used as control treatments, in total theexperiments consisted of 8 treatments.
After inoculation, the shells were rinsed under running water to wash away the easily detachedmicroorganisms. The inoculation capacity of patinopecten yessoensis shells was evaluated by
performing a colony count using a mixed culture method for lactobacillus and a plate dilution method
for bacillus subtilis var. natto, and saccharomyces cerevisiae.
In addition, to compare the nitrogen removal capacity, patinopecten yessoensis shells wereimmersed in 1 L of water from the biotope at Yatoyama Park for a water purification experiment for
3 days. The ultraviolet absorption spectrophotometric method was used to measure total nitrogen.
The details of the 8 treatments are as follows.
Table 1 Combination of shell processing and microorganisms
Unburned and uninoculated shells
Lactobacillus-inoculated unburnedshells
Bacillus subtilis var. natto-inoculated
unburned shells
Saccharomyces cerevisiae-inoculated
unburned shells

UCBurned and uninoculated shells

BCUL

Lactobacillus-inoculated burned shells

BLBacillus subtilis var. natto-inoculatedburned shells
Saccharomyces cerevisiae-inoculated
unburned shells

UBUS

BB
BS

RESULTS AND DISCUSSION
Water Quality In Biotope

Fig. 1 Changes in total nitrogen (TN) in the biotope (26 July 2021 to 4 February 2022)

Fig. 2 Changes in total phosphrus (TP) in the biotope (26 July 2021 to 4 February 2022)

Figure 1 indicates the change in total nitrogen. The average value of total nitrogen measured at eachwater sampling site was 1.84 mg L-1 at Site 1, 0.66 mg L-1 at Site 2, and 0.73 mg L-1 at Site 3. The
data tended to be higher in summer and fall, which seems to be due to precipitation, fallen leaves,Ⓒ ISERD108

IJERD – International Journal of Environmental and Rural Development (2023) 14-1and waterfowl. Figure 2 indicates the change in total phosphorus. The average total phosphorus
measurement data for each water sampling site was 0.29 mg L-1 for Site 1, 0.03 mg L-1 for Site 2 and0.07 mg L-1 for Site 3. As with the total nitrogen data, the data tended to be higher in the summer andfall, which seems to be due to precipitation, fallen leaves, and waterfowl. Also, the reason there is nodata for Site 1 on the 112th day is that the water source was buried by sediment, and water sampling
was impossible.
In addition, both total nitrogen and total phosphorus tended to be higher at Site 1. This is because
Site 1 is closer to the inflow point than other sampling sites, resulting in a greater inflow of sediment.
The biotope at Site 1 has been found to have poor water quality compared to the environmental
standards set by the Japanese Ministry of the Environment (Table 2). Therefore, there is a need forwater purification. The source of nitrogen inflows and their relationship to the surrounding
environment, such as waterfowl and vegetation, also need to be analyzed in detail.
Table 2 Environmental standards for total nitrogen (TN) and phosphorus (TP) in lakes
Item typeⅠⅡⅢⅣⅤStandard value
Total nitrogen
0.1 mg/L or less0.2 mg/L or less
0.4 mg/L or less
0.6 mg/L or less
1.0 mg/L or lessTotal phosphorus
0.005 mg/L or less
0.010 mg/L or less
0.030 mg/L or less0.050 mg/L or less
0.100 mg/L or lessNote: Ministry of the Environment, 2022

Microorganism Inoculation Capacity of Patinopecten yessoensis Shells
Figure 3 shows the number of microorganisms inoculated on patinopecten yessoensis shells. The
number of microorganisms inoculated with lactobacillus on unburned shells was 2.8.E+05 cfu g-1,that with bacillus subtilis var. natto on unburned shells was 6.0.E+05 cfu g-1, and that withSaccharmyces cerevisiae on unburned shells was 2.3.E+07 cfu g-1. Also, the number ofmicroorganisms inoculated into the burned shells with lactobacillus was 1.9.E+07 cfu g-1, that into
the burned shells with bacillus subtilis var. natto was 2.6.E+05 cfu g-1, and that into the burned shells
with Saccharmyces cerevisiae was 1.6.E+07 cfu g-1.

Fig. 3 Amount of microorganisms inoculated in each shell
The amount of inoculated lactobacillus was increased by burning the shells at 600 degrees. The
amount of bacillus subtilis var. natto and Saccharmyces cerevisiae inoculated was not significantly
changed by burned shells. The change in the amount of inoculum seems to be caused by changes in
the pore size, specific surface area, and organic matter content of the patinopecten yessoensis shells.
A more detailed analysis of pore size, distribution, and specific surface area will be necessary todetermine the relation between the pore and the number of microorganisms inoculated.
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Nitrogen Removal Capacity of Patinopecten yessoensis Shells Inoculated with
Effective Microorganisms
The experimental result according to the Fig. 4 showed total nitrogen removal rates of 42% for
unburned and uninoculated shells, 0% for lactobacillus-inoculated unburned shells, 45% for bacillus
subtilis var. natto-inoculated unburned shells, 58% for saccharomyces cerevisiae-inoculated
unburned shells. Regarding the burned shells, results showed 51% nitrogen reduction at theuninoculated shells treatment. Lactobacillus-inoculated burned shells reduced nitrogen by 47%,
Bacillus subtilis var. natto-inoculated burned shells reduced by 34% and saccharomyces cerevisiaeinoculated burned shells reduced the nitrogen in the water sample by 69%. The highest total nitrogen
removal rate was indicated by the treatment of burned shells inoculated with saccharomyces
cerevisiae. Watabe et al. (2014) mentioned that saccharomyces cerevisiae is ableto incorporate
nitrogen into the fungus itself. Hence, its removal capacity seems to be significantly different fromthat of other microorganisms. The reason that the inoculation of burned shells with lactobacillus did
not remove total nitrogen seems to be that the rinsing process after the inoculation was not performedproperly.

Fig. 4 Changes in total nitrogen (TN) in water purification experiments
CONCLUSION
In this study, the water quality in the biotope was determined, and at the same time, themicroorganism inoculation capacity of patinopecten yessoensis shells and the water purification
ability of patinopecten yessoensis shells inoculated with effective microorganisms were evaluated.
The water quality of the biotope at the subject site was found to be inadequate and in need ofwater purification. It is necessary to conduct a more detailed survey because of the influence of thesurrounding environment, including fallen leaves, precipitation, waterfowl, and vegetation.
The microorganism inoculation capacity of patinopecten yessoensis shells was evaluated to acertain capacity for all microorganisms. Among them, Saccharmyces cerevisiae was found to have ahigh inoculation capacity. It is assumed that the size of the Saccharmyces cerevisiae was suitablewith the internal pore size and specific surface area of the patinopecten yessoensis shells. In the future,
it is necessary to study the relationship between microorganism inoculation capacity and pore size,and specific surface area.
The water purification ability of patinopecten yessoensis shells inoculated with effective
microorganisms was evaluated for total nitrogen removal except for lactobacillus-inoculated
unburned shells. The highest removal capacity was observed in burned shells inoculated with
Saccharmyces cerevisiae. This was a short-term and small-scale experiment. Future experiments
should be conducted to consider long-term and field applicability for the reuse of patinopecten
yessoensis shells.

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ACKNOWLEDGEMENTS
In preparing this paper, I would like to express my sincere gratitude to Associate Professor Toru
Nakajima of the Department of Production and Environmental Engineering, Faculty of Regional
Environmental Sciences, Tokyo University of Agriculture, for his enthusiastic guidance onexperimental methods, paper structure, and presentation, despite his busy schedules amidst theconfusion of the social situation. I would also like to thank the graduate students at the samelaboratory for their detailed guidance on experimental methods and analysis of the data obtained.
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