IJERD – International Journal of Environmental and Rural Development (2013) 4-1

Research article
erd

Utilization of Inoculated Eri Cocoon for Water Pollutant
RemovalCARLOS ONAN MENDOZA TOVAR
Graduate School of Agriculture, Tokyo University of Agriculture, Tokyo, Japan
Email: onan14@yahoo.com

KUMIKO KAWABE
Graduate School of Agriculture, Tokyo University of Agriculture, Tokyo, Japan

HIROMU OKAZAWA
Faculty of Regional Environment Science, Tokyo University of Agriculture, Tokyo, Japan

MACHITO MIHARA*
Faculty of Regional Environment Science, Tokyo University of Agriculture, Tokyo, Japan /Institute of Environment Rehabilitation and Conservation, Tokyo, Japan
Email: m-mihara@nodai.ac.jp
Received 18 December 2012

Accepted 27 March 2013(*Corresponding Author)

Abstract The eri cocoon may have the potential to immobilize effective microorganisms
although it needs to be tested in order to be used as a method to remove pollutants fromwater. Lactobacillus acidophilus has proven to be capable of removing up to 60% of
Arsenic (III) from water within 3 hours. The main objective of this study was to quantify
the amount of glucose absorbed by inoculated eri cocoon as an indicator of the capacity forpollutant removal. Lactobacillus spp. was incubated at 37 oC for 48 hours. By microscopic
observation, structures were described. The rate of inoculation was calculated as colony
forming units (cfu) in the eri cocoon divided by cfu of the inoculation solution times one
hundred. An absorption trial was set up. The absorption rate was calculated as final
glucose concentration minus initial divided by time. The untreated (UT) eri cocoonpresented nano-tubes where bacteria can easily penetrate. By soaking eri cocoons in
distilled water (WS), cavern-like structures appeared within the silk fibers. When the ericocoon was autoclaved (AC), the cavern structures were more frequent. The twisted yarn(TY) showed almost no difference with the fibers in the UT eri cocoon. The sample counts
gave no statistical differences between UT and AC. Eri cocoon has the natural structures toaccommodate microorganisms within its fibers. In the inoculation rate there was nodifference between UT and AC treatments. The consumption of glucose showed nodifference between UT, AC and WS; but there was a significant difference between thefirst three and TY. TY had the lowest glucose consumption.
Keywords pollutants, eri cocoon, lactobacillus, glucose, absorption

INTRODUCTION
Sericulture is defined as the breeding and raising of silk worms for the production of silk. There aredifferent species of silk worm used for this purpose. This study will be focused on the eri silk worm
(Samia cynthia ricini). The eri culture, as is called the rearing of eri silk worm, takes place indifferent areas of Asia and India.
In the North-East region of India, eri farmers adopt various traditional indigenous practices forrearing (Sarmah et. al., 2010). Eri culture has different uses such as a poverty alleviation strategy(Ramalakshmi, 2009) and the use of the eri pupa as a food for human consumption (Sarmah, 2011).
But the main purpose of eri culture is for the production of silk.

Ⓒ ISERD

164

IJERD – International Journal of Environmental and Rural Development (2013) 4-1

Some studies on the structure of the silk fiber have been conducted. However, these studies
have focused on the protein structure rather than the physical structure (Asakura and Nakazawa,2004). In another study the eri cocoon was analyzed in an aqueous solution and dry-frizzed toobtain a fibroin powder to be used for its thermal properties and protein structure study (Yaowalaket. al., 2009). Asakura and Nakazawa (2004) concluded that the silk fiber has a spiral protein
structure focusing in poly-Alanine chain and Glycerine areas with a Carbon and Nitrogen terminals.
Structural studies of the filaments have been done by Akai and Nagashima (2001, 2002) on
different silk moths. Porous and compact filament formations are caused or regulated by continued
lysosome releases which are affected by the progress of silk gland degradation (Akai andNagashima, 2001). These porous structures and shell holes, as described by Akai and Nagashima(2001, 2002), can supposedly be present in other Saturniids as well and used as a niche for
effective microorganisms.
The use of different methods for water pollutant removal has been considered one of theimportant actions taken to protect the environment. There have been some experiments toimmobilize microorganisms and used for bioremediation (Krumme et. al., 1994; Arango, 2004;
Hosseini et. al., 2007; Singh and Sarma, 2010). Most of the substrates used to immobilize effective
microorganisms are non-biological porous materials. The eri cocoon has the potential toimmobilize effective microorganisms and from there be used as a method to remove pollutantsfrom water. A study with Lactobacillus acidophilus has proven that this bacteria is capable ofremoving, at a concentration of 2 mg dry wt/ml biomass, up to 60% of Arsenic (III) from a 1000
ppb water solution at pH 7 within 3 hours (Singh and Sarma, 2010). In another experiment,Lactobacillus spp. isolated from shrimp farm water samples was capable of simultaneous removalof pathogenic bacteria and nitrogen (Ma et al., 2009).
OBJECTIVE
The main objective of this study was to quantify the amount of glucose absorbed by inoculated eri
cocoon as an indicator of the capacity for pollutant removal. Secondary objectives were: todescribe the structures that eri cocoon present in order to be a niche for microorganisms; to observe
the effects of different treatments on the physical structures of the cocoon fiber; and to determine
the ability of eri cocoon to allocate Lactobacillus spp. within its structure.
METHODOLOGY
The experiment was done in a two stages stile. Stage one the description and inoculation, and stage
two the glucose consumption. When analyzing the inoculation the treatments was cocoon
treatments only and when analyzing the glucose consumption the statistical differences werecalculated based on cocoon treatments and glucose concentration levels.
Physical description: Microscopic images were taken from different treatments of eri cocoon.
From the images, physical description and differences were observed to determine the space thatmicroorganisms could inhabit. The treatments were untreated cocoons (UT); the pupa was removed
and only the silk cocoon was utilized. 1 week soaked in water (WS); the cocoon was soaked in
distilled water for seven days without stirring at 25 oC. Autoclaved (AC); the eri cocoon wereimmersed in destilled water and autoclaved at a temperature of 110 oC and at a pressure of 152 kPa.
Twisted yarn (TY); an already processed silk yarn was used.
Inoculation: Lactobacillus spp. was incubated and inoculated to calculate the ability of thetreatments to allocate bacteria by the cocoon. The inoculation solution was added directly to thetreatments by 6 ml per sample in order to cover half of the sample and allowing the other half toabsorb by capillarity. The inoculated eri cocoon was incubated for 72 hours at 37 oC. Five countrepetitions were made to get an average of colony forming units (cfu) per milligram of eri cocoon
in order to observe the differences between treatments.
Glucose consumption: Two levels of glucose solutions were used: a high concentration of 15 g ofglucose dissolved in 100 ml of distilled water; and 5 g of glucose dissolved in 100 ml of distilledⒸ ISERD

165

IJERD – International Journal of Environmental and Rural Development (2013) 4-1water, which equals 15% glucose solution and 5% glucose solution respectively. The inoculated eri
cocoon treatments were placed in 60 ml of glucose solution and sampled at 3, 24, 48, 72, 120, 192,288, 360, 408, 480 hours. The four treatments had four repetitions and two repetitions of control.
The control treatment was distilled water instead of the glucose solution. The glucose consumptionwas recorded and analyzed by the SPSS 15 for windows system in a two factorial analysis. The
treatments resulted in UT15%, UT5%, WS15%, WS5%, AC15%, AC5%, TY15%, and TY5%.
RESULTS AND DISCUSSION
Physical description
The UT presented entrances and nanotubes where bacteria can easily penetrate (Fig. 1a). Afterusing a filter to observe the spaces within fibers the nanotubes were easily observed (Fig. 2). After
treating the cocoon by soaking it for seven days cavern like structures began to appear within thesilk fibers (Fig. 1b). Although no entrances where observed, these cavern-like structures present
more suitable and larger spaces than those on the normal nanotubes present on the UT. When theeri cocoon was autoclaved the cavern-like structures were more frequent and entrances to themwere observed. These cavern-like structures presented more space within the fiber and also werelonger that those present on the WS treatment (Fig. 1c). The twisted yarn showed almost nodifference to the fibers in the untreated, the only difference was the order that they aligned witheach other making a lower exposure area (Fig. 1d).
Inoculation
In Fig. 3, the inoculation data are shown. The inoculation solution showed an average of 1.15 x1016 colony forming units (cfu) per milligram of cocoon. UT and AC were not statistically different
and had the higher inoculation count in cfu per milligram of cocoon. WS and TY had the lowest
inoculation and were statistically different from AC and UT.

abcdFig. 1 Fig. 1 Microscopic cross-sectional views of eri-cocoon(a untreated, b 1week soaked in water, c autoclaved, d twisted yarn)Ⓒ ISERD

166

IJERD – International Journal of Environmental and Rural Development (2013) 4-1

Fig. 2 Negative light filter fiber picture
2000000a**

CCFU/mg
FU /m g

1500000a**

1000000

500000b**b**

0Untreated(cfu/mg)

Water Soaked(cfu/mg)

Autoclaved(cfu/mg)

Twisted Yarn
(cfu/mg)

Fig. 3 Average colony forming units per milligram of eri cocoon
The alignment in TY and the treatment that it received beforehand may be the cause of such low
cfu/mg count. It is also possible that if the TY was soaked in water before the inoculation this might
have given a similar result to WS. WS and TY were not statisticalyl different; nevertheless, they bothhave the capacity to allocate a certain amount of Lactobacillus spp. within their structures.
Glucose consumption

CConsumedo n s um e d G
l u c o s e( (g/ml)
g /m l )GlucoseThe glucose consumption was taken and corrected against the control samples. There was astatistical difference in glucose consumption between UT15%, AC15%, WS15% and TY15%,UT5%, AC5%, WS5%, TY5%; TY15% was statistically different from UT5%, AC5%, WS5%,TY5%. There was no statistical difference between UT15%, AC15%, and WS15%. Within theUT5%, AC5%, WS5%, TY5% there was no statistical difference. Fig.4 shows the net consumptionof glucose per treatment over time.

-100

5UT 15%4UT 5%
WS 15%

3WS 5%
AC 15%

2AC 5%TY 15%

1TY 5%

00100

200

300

400

500

Time(hours)(hours)
TimeFig. 4 Net glucose consumption by Lactobacillus spp. inoculated eri cocoonⒸ ISERD

167

IJERD – International Journal of Environmental and Rural Development (2013) 4-1

CONCLUSIONS AND RECOMMENDATIONS
Eri cocoon has the natural structures to accommodate microorganisms within its fibers. Applicationof treatments such as soaking the eri cocoon in distilled water, or using an autoclave can improve
the structures. Creation of yarn appeared to reduce the ability of eri cocoon fiber to accommodate
bacteria. Eri cocoon inoculated with Lactobacillus spp. is able to absorb up to 29% of glucose outof a glucose solution at 15g/100ml or 15% glucose solution within 450 hours; and up to 26%
within the first 3 hours.
Comparing physical structures, as the amount of space observable by microscopic imaging,and inoculation rate, the AC treatment presents the best structures and the highest rate of colony
formation. Regarding inoculation rate there was no statistical difference between UT and AC which
infers that the cheapest and best option for inoculation is the UT.
When comparing glucose consumption, the treatments AC15%, UT15%, and WS15% present
the highest consumption. Nevertheless, using the inoculation rates as a reference point, the mosteffective treatments are AC15% and UT15%. Even though AC presents all the advantages for a
filtering system, the use of UT is recommended to make a nitrogen absorption trial replacing
glucose in the solution. The combination of UT and TY may give a better result when used together
to make a net for water pollutant removal.
REFERENCES
Akai, H. and Nagashima, T. 2001. Structural characteristics of cocoon filaments from rothschildia silkmoth.
International Journal of Wild Silkmoth & Silk, 6, 25-32.
Akai, H. and Nagashima, T. 2002. Structural characteristics of cocoon filaments of the African silkmothArgema mimosa. International Journal of Wild Silkmoth & Silk, 7, 47-52.
Arango, A. 2004. La biofiltración, una alternativa para la potabilización del agua. Revista Lasallista deInvestigación, 7.
Asakura, T. and Nakazawa, Y. 2004. Structure and structural changes of the silk fibroin from Samia cynthia
ricini using nuclear magnetic resonance spectroscopy. Macromolecular Bioscience, 4, 175-185.
Hosseini, M., Mahmoudi, M. and Mohammadi, M. 2007. Removal of benzene emission by a bio filter withimmobilized bacteria. American-Eurasian Journal of Agriculture and Environmental Sciences, 2(6),756-760.
Krumme, M.L., Smith, R.L., Egestorff, J., Thiem, S., Tiedje, J., Timmis, K. and Dwyer, D. 1994. Behavior ofpollutan-degrading microorganisms in aquifers: predictions for genetically engineered organisms.
Journal of Environmental Science and Technology, 28, 1134-1138.
Ma, C. W., Cho, Y. S. and Oh, K.H. 2009. Removal of pathogenic bacteria and nitrogens by Lactobacillus
spp. jk-8 and jk-11. Aquaculture Journal, 287, 266-270.
Ramalakshmi, C.S. 2009. Potentiality of eri culture for poverty alleviation in dry land areas of andhra
pradesh, An economic analysis. Department of Sericulture, Jubilee Hills, Hyderabad, India. 11.
Sarmah, M.C., Rahman, S. and Barah, A. 2010, Traditional practices and terminologies in muga and eri
culture. Indian Journal of Traditional Knowledge, 9, 448-452.
Sarmah, M.C. 2011. Eri pupa: a delectable dish in north east india. Central Muga Eri Research And Training
Institute, Current Science, 100, 3.
Singh, A.L. and Sarma, P.N. 2010. Removal of arsenic (III) from waste water using Lactobacillus
acidophilus. Bioremediation Journal, 14 (2), 92-97.
Yaowalak, S., Wilaiwan, S. and Prasong, S., 2009. Preparation and characterization of eri (Philosamia ricini)silk fibroin powder. Journal of Applied Sciences, 9(16), 2992-2995.

Ⓒ ISERD168