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Sains Malaysiana 54(11)(2025):
2645-2659
http://doi.org/10.17576/jsm-2025-5411-06
Homofermentative Lactic Acid- and Cellulase-Secreting Oryctes rhinoceros Larval Gut Bacterial
Strains for Lactic Acid Production
(Penghasilan Homofermentatif Asid Laktik dan Selulase Strain Bakteria Usus Larva Oryctes rhinoceros untuk Pengeluaran Asid Laktik)
SIDEEQOT TOYIN
ABDULLAHI1, AHMAD FARIS MOHD ADNAN1,2,*, MOHAMMAD MONERUZZAMAN KHANDAKER3 &
MINATO WAKISAKA4
1Institute of Biological Sciences,
Faculty of Sciences, Universiti Malaya, 50603 Kuala
Lumpur, Malaysia
2Centre of Ionics, Faculty of Science, Universiti Malaya, 50603 Kuala Lumpur, Malaysia
3School of
Agriculture Science & Biotechnology, Faculty of Bioresources and Food
Industry, Universiti Sultan Zainal Abidin, Besut Campus, 22200 Besut, Terengganu, Malaysia
4Food Study
Centre, Fukuoka Women's University, 1-1-1 Kasumigaoka,
Fukuoka 813-8529, Japan
Received:
7 March 2024/Accepted: 19 September 2025
Abstract
Global demand
for lactic acid (LA) has increased significantly in recent years, thus,
prompting increased interest in utilising lignocellulosic waste biomass for its production. This study investigated the
relationship between cellulase activity and LA production by lactic acid
bacteria (LAB) isolated from the gut of rhinoceros beetle (Oryctes rhinoceros) larvae. Out of 11 LAB strains tested, three isolates namely
L5-5, L2a-1 and L3-2 were observed to exhibit good growth using glucose as
substrate and were able to obtain final LA concentrations of 9.04 g/L, 9.26 g/L
and 9.34 g/L, respectively. These strains were further screened for
cellulolytic activity at various temperatures and pH levels using a carboxymethyl
cellulose (CMC) assay and were identified as Enterococcus thailandicus through 16S rDNA sequencing. Optimal
enzyme activity at 45 °C for strain L5-5 was 0.42 U/mL, L2a-1 was 0.61 U/mL,
and L3-2 was 0.62 U/mL. Notably, the strong
cellulolytic activity was positively correlated with the elevated LA
production. These LAB strains could tolerate a broad temperature range of 30 to
50 °C, salt concentrations of 2.5 to 10 % (w/v), furfural concentration of up
to 1% (v/v), and pH levels between 4.5 and 9.0. The findings highlighted the
potential of LAB isolates from O. rhinoceros larval gut as viable candidates for LA production in industrial applications.
Keywords: Cellulase; Enterococcus thailandicus;
lactic acid bacteria; lactic acid fermentation; Oryctes rhinoceros larva
Abstrak
Permintaan global untuk asid laktik (LA) telah meningkat dengan ketara dalam beberapa tahun kebelakangan ini, lalu mendorong peningkatan minat terhadap penggunaan biojisim sisa lignoselulosa untuk pengeluarannya. Penyelidikan ini mengkaji hubungan antara aktiviti selulase dengan pengeluaran LA oleh bakteria asid laktik (LAB) yang dipencilkan daripada usus larva kumbang badak (Oryctes rhinoceros). Daripada 11 strain
LAB yang telah diuji, tiga pencilan iaitu L5-5, L2a-1 dan L3-2 telah menunjukkan tumbesaran yang baik menggunakan glukosa sebagai substrat dan mampu menghasilkan kepekatan akhir LA masing-masing sebanyak 9.04 g/L, 9.26 g/L dan 9.34 g/L. Kesemua strain tersebut selanjutnya disaring untuk aktiviti selulolitik merentasi pelbagai suhu dan tahap pH menggunakan asai karboksimetil selulosa (CMC) dan dikenal pasti sebagai Enterococcus thailandicus melalui penjujukan 16S rDNA. Aktiviti enzim optimum pada 45 °C bagi strain L5-5 adalah 0.42 U/mL, L2a-1 adalah 0.61 U/mL dan L3-2 adalah 0.62 U/mL. Dengan jelasnya, aktiviti selulolitik yang kuat dikorelasikan secara positif dengan pengeluaran LA yang tinggi. Kesemua strain LAB ini mampu bertoleransi dengan julat suhu yang luas iaitu 30 hingga 50 °C, kepekatan garam pada
2.5 hingga 10 % (b/v), kepekatan furfural sehingga 1% (v/v) dan tahap pH antara 4.5 kepada 9.0. Penemuan ini menyerlahkan potensi pencilan LAB daripada usus larva O.
rhinoceros sebagai calon yang berpotensi untuk pengeluaran LA dalam aplikasi industri.
Kata kunci: Bakteria asid laktik; Enterococcus thailandicus; fermentasi asid laktik; larva Oryctes rhinoceros; selulase
REFERENCES
Abdel-Rahman, M.A., Tashiro, Y., Zendo, T.,
Sakai, K. & Sonomoto, K. 2015. Enterococcus faecium QU 50: A novel thermophilic lactic acid bacterium for high-yield l-lactic acid
production from xylose. FEMS Microbiology Letters 362(2): 1-7.
Adnan, A.F.M. & Tan, I.K. 2007. Isolation
of lactic acid bacteria from Malaysian foods and assessment of the isolates for
industrial potential. Bioresource Technology 98(7): 1380-1385.
Baruah, J., Nath, B.K., Sharma, R., Kumar, S.,
Deka, R.C., Baruah, D.C. & Kalita, E. 2018. Recent trends in the
pretreatment of lignocellulosic biomass for value-added products. Frontiers
in Energy Research 6: 141.
Calumby, R.J., de Almeida, L.M., de Barros, Y.N., Segura, W.D., Barbosa, V.T.,
da Silva, A.T., Dornelas, C.B., Alvino, V. & Grillo, L.A. 2022.
Characterization of cultivable intestinal microbiota in Rhynchophorus palmarum Linnaeus (Coleoptera: Curculionidae)
and determination of its cellulolytic activity. Archives of Insect
Biochemistry and Physiology 110(2): 21881.
Chacón, M.G., Ibenegbu, C.
& Leak, D.J. 2021. Simultaneous saccharification and lactic acid
fermentation of the cellulosic fraction of municipal solid waste using Bacillus
smithii. Biotechnology Letters 43: 667-675.
Chandel, A.K., Garlapati, V.K., Singh, A.K.,
Antunes, F.A.F. & da Silva, S.S. 2018. The path forward for lignocellulose
biorefineries: Bottlenecks, solutions, and perspective on
commercialization. Bioresource Technology 264: 370-381.
Chantarasiri, A. 2015. Aquatic Bacillus cereus JD0404 isolated from the
muddy sediments of mangrove swamps in Thailand and characterization of its
cellulolytic activity. The Egyptian Journal of Aquatic Research 41(3):
257-264.
Chatgasem, C., Suwan, W., Attapong,
M., Siripornadulsil, W. & Siripornadulsil,
S. 2023. Single-step conversion of rice straw to lactic acid by thermotolerant
cellulolytic lactic acid bacteria. Biocatalysis and Agricultural
Biotechnology 47: 102546.
Chen, Z., Wang, Y., Cheng, H. and
Zhou, H., 2023. Integrated chemo‐and biocatalytic processes: a new
fashion toward renewable chemicals production from lignocellulosic biomass. Journal
of Chemical Technology & Biotechnology 98(2): 331-345.
Dantur, K.I., Enrique, R., Welin, B. &
Castagnaro, A.P. 2015. Isolation of cellulolytic bacteria from the intestine of Diatraea saccharalis larvae and evaluation of their capacity to degrade sugarcane biomass. AMB
Express 5: 1-11.
da
Cunha, V.L., Leonarski, E., de Oliveira, J., Fireck, J.F., Rodrigues, M.X., da Silva, V.G., Ramos,
C.J.R. & dos Passos Francisco, C.T. 2024. Screening and characterization of Enterococcus durans isolates from raw organic
milk in Southern Brazil: Assessing technological potential. Food and
Humanity 2: 100276.
de Man, J.D., Rogosa,
D. & Sharpe, M.E. 1960. A medium for the cultivation of lactobacilli. Journal
of Applied Microbiology 23(1): 130-135.
de Oliveira, R.A., Komesu,
A., Rossell, C.E.V. & Maciel Filho, R. 2018. Challenges and opportunities
in lactic acid bioprocess design - From economic to production aspects. Biochemical
Engineering Journal 133: 219-239.
Dong, R., Zhang, J., Huan, H., Bai, C., Chen,
Z. & Liu, G. 2017. High salt tolerance of a Bradyrhizobium strain and its promotion of the growth of Stylosanthes guianensis. International Journal of
Molecular Sciences 18(8): 1625.
Du, Z., Yamasaki, S.,
Oya, T. & Cai, Y. 2023. Cellulase–lactic acid bacteria synergy action
regulates silage fermentation of woody plant. Biotechnology for
Biofuels and Bioproducts 16(1): 125.
Elzeini, H.M., Ali, A.R.A.A., Nasr, N.F., Elenany, Y.E. & Hassan, A.A.M. 2021. Isolation and
identification of lactic acid bacteria from the intestinal tracts of honeybees, Apis mellifera L., in Egypt. Journal of Apicultural Research 60(2):
349-357.
Gibson, T. & Abdel-Malek, Y. 1945. The
formation of carbon dioxide by lactic acid bacteria and Bacillus
licheniformis and a cultural method of detecting the process. Journal
of Dairy Research 14(1-2): 35-44.
Grewal, J., Sadaf, A., Yadav, N. & Khare,
S.K. 2020. Agroindustrial waste based biorefineries
for sustainable production of lactic acid. In Waste Biorefinery, edited
by Bhaskar, T., Pandey, A., Rene, E.R. & Tsang, D.C.W. Elsevier, Amsterdam.
pp.125-153.
Hankin, L. & Anagnostakis, S.L. 1977. Solid
media containing carboxymethylcellulose to detect Cx cellulase activity of micro-organisms. Microbiology 98(1):
109-115.
Harindintwali, J.D., Zhou, J. & Yu, X. 2020.
Lignocellulosic crop residue composting by cellulolytic nitrogen-fixing
bacteria: A novel tool for environmental sustainability. Science of the
Total Environment 715: 136912.
Herdian, H., Istiqomah, L.,
Damayanti, E., Suryani, A.E., Anggraeni,
A.S., Rosyada, N. & Susilowati,
A. 2018. Isolation of cellulolytic lactic-acid bacteria from Mentok (Anas moschata)
gastro-intestinal tract. Tropical Animal Science Journal 41(3):
200-206.
Huang, Y., Wang, Y., Shang, N. & Li, P.
2023. Microbial fermentation processes of lactic acid: Challenges, solutions,
and future prospects. Foods 12(12): 2311.
Jannah, A., Aulanniam,
A., Ardyati, T. & Suharjono,
S. 2018. Isolation, cellulase activity test and molecular identification of
selected cellulolytic bacteria indigenous rice bran. Indonesian Journal
of Chemistry 18(3): 514-521.
Jurášková, D., Ribeiro, S.C. & Silva, C.C. 2022.
Exopolysaccharides produced by lactic acid bacteria: From biosynthesis to
health-promoting properties. Foods 11(2): 156.
Kasana, R.C., Salwan, R.,
Dhar, H., Dutt, S. & Gulati, A. 2008. A rapid and easy method for the
detection of microbial cellulases on agar plates using Gram’s iodine. Current
Microbiology 57: 503-507.
Kaur, N., Panesar, P.S. & Ahluwalia, S.
2022. Production of organic acids from agro-industrial waste and their
industrial utilization. In Valorization of Agro-Industrial Byproducts, edited
by Anal, A.K. & Panesar, P.A. Boca Raton: CRC Press. pp. 227-264.
Kim, J., Kim, Y.M., Lebaka,
V.R. & Wee, Y.J. 2022. Lactic acid for green chemical industry: Recent
advances in and future prospects for production technology, recovery, and
applications. Fermentation 8(11): 609.
Malacara-Becerra, A., Melchor-Martínez, E.M.,
Sosa-Hernández, J.E., Riquelme-Jiménez, L.M., Mansouri, S.S., Iqbal, H.M. &
Parra-Saldívar, R. 2022. Bioconversion of corn crop residues: Lactic acid
production through simultaneous saccharification and fermentation. Sustainability 14(19):
11799.
Marheni, M., Martono, E. & Sijabat,
O.S. 2021. Exploration of symbiotic bacteria of Oryctes rhinoceros (Coleoptera: Scarabaeidae)
larvae from oil palm empty fruit bunches. AGRIVITA Journal of
Agricultural Science 43(1): 190-197.
Miller, G.L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry 31(3):
426-428.
Mohamad Zabidi, N.A., Foo, H.L., Loh, T.C.,
Mohamad, R. & Abdul Rahim, R. 2020. Enhancement of versatile extracellular
cellulolytic and hemicellulolytic enzyme productions by Lactobacillus
plantarum RI 11 isolated from Malaysian food using renewable natural
polymers. Molecules 25(11): 2607.
Motta, E.V. & Moran, N.A. 2024. The
honeybee microbiota and its impact on health and disease. Nature
Reviews Microbiology 22(3): 122-137.
Ngouénam, J.R., Kenfack,
C.H.M., Kouam, E.M.F., Kaktcham,
P.M., Maharjan, R. & Ngoufack, F.Z. 2021. Lactic
acid production ability of Lactobacillus sp. from four tropical fruits
using their by-products as carbon source. Heliyon 7(5): e07079.
Ojo, A.O. & de Smidt, O. 2023. Lactic acid:
A comprehensive review of production to purification. Processes 11(3):
688.
Peng, L., Wang, L., Che, C., Yang, G., Yu, B.
& Ma, Y. 2013. Bacillus sp. strain P38: An efficient producer of
L-lactate from cellulosic hydrolysate, with high tolerance for
2-furfural. Bioresource Technology 149: 169-176.
Pitiwittayakul,
N., Bureenok, S. & Schonewille,
J.T. 2021. Selective thermotolerant lactic acid bacteria isolated from
fermented juice of epiphytic lactic acid bacteria and their effects on
fermentation quality of stylo silages. Frontiers
in Microbiology 12: 673946.
Pleissner, D., Demichelis, F., Mariano, S., Fiore, S.,
Gutiérrez, I.M.N., Schneider, R. & Venus, J. 2017. Direct production of
lactic acid based on simultaneous saccharification and fermentation of mixed
restaurant food waste. Journal of Cleaner Production 143:
615-623.
Rawoof, S.A.A., Kumar, P.S., Vo, D.V.N., Devaraj, K.,
Mani, Y., Devaraj, T. & Subramanian, S. 2021. Production of optically pure
lactic acid by microbial fermentation: A review. Environmental
Chemistry Letters 19: 39-556.
Shao, Y., Arias-Cordero, E., Guo, H., Bartram, S. &
Boland, W. 2014. In vivo Pyro-SIP assessing active gut microbiota of the
cotton leafworm, Spodoptera littoralis. PLoS ONE 9(1): e85948.
Sharpe, M. 1976. Identification of lactic acid bacteria. In Identification
Methods for Microbiology, edited by Skinner, F.A. & Lovelock,
D.W. London: Academic Press. pp. 233-259.
Shelomi, M. & Chen, M.J. 2020. Culturing-enriched
metabarcoding analysis of the Oryctes rhinoceros gut microbiome. Insects 11: 782.
Stephen, J.M. & Saleh, A.M. 2023.
Homofermentative Lactobacilli isolated from organic sources exhibit
potential ability of lactic acid production. Frontiers in Microbiology 14:
1297036.
Sun, Y., Liu, H., Yang, Y., Zhou, X. & Xiu,
Z. 2021. High-efficient l-lactic acid production from inedible starchy biomass
by one-step open fermentation using thermotolerant Lactobacillus rhamnosus DUT1908. Bioprocess and Biosystems
Engineering 44(9): 1935-1941.
Suwannaphan, S. 2021. Isolation, identification and
potential probiotic characterization of lactic acid bacteria from Thai
traditional fermented food. AIMS Microbiology 7(4): 431.
Teather, R.M. & Wood, P.J. 1982. Use of
Congo red-polysaccharide interactions in enumeration and characterization of
cellulolytic bacteria from the bovine rumen. Applied and Environmental
Microbiology 43(4): 777-780.
Trinh, H.N.P., Long, B.H.D., Thanh, N.N.,
Phong, H.X. & Dung, N.T.P. 2018. Characterization of newly isolated
thermotolerant lactic acid bacteria and lactic acid production at high
temperature. International Food Research Journal 25(2):
523-526.
Ubando, A.T., Felix, C.B. & Chen, W.H.
2020. Biorefineries in circular bioeconomy: A comprehensive review. Bioresource
Technology 299: 122585.
Upfold, J., Rejasse,
A., Nielsen-Leroux, C., Jensen, A.B. & Sanchis-Borja, V. 2023. The
immunostimulatory role of an Enterococcus-dominated gut microbiota in
host protection against bacterial and fungal pathogens in Galleria mellonella larvae. Frontiers in Insect Science 3:
1260333.
Wang, B., Rutherfurd-Markwick, K., Zhang, X.X.
& Mutukumira, A.N. 2022. Isolation and characterisation of dominant acetic acid bacteria and yeast
isolated from Kombucha samples at point of sale in New Zealand. Current
Research in Food Science 5: 835-844.
Xavier, J.R., Nallamuthu, I., Murugan, M.P.
& Chauhan, O.P. 2024. Optimisation of lactic acid
production using cost effective agro residue for food
applications. Sustainable Food Technology 2(3): 741-749.
Yankov, D. 2022. Fermentative lactic acid production
from lignocellulosic feedstocks: From source to purified product. Frontiers
in Chemistry 10: 823005.
Yang, H., He, M. & Wu, C. 2021. Cross
protection of lactic acid bacteria during environmental stresses: Stress
responses and underlying mechanisms. LWT 144: 111203.
Yang, Y., Wang, Y., Lu, X., Zheng, X., Yan, D.,
Xin, J., El-Sayed, I.E.T., Kang, Y. & Yang, J. 2022. Highly efficient enzymolysis and fermentation of corn stalk into L-lactic
acid by enzyme-bacteria friendly ionic liquid pretreatment. Green Chemical
Engineering 3(4): 321-327.
Zhang, X., Zhang, F. & Lu, X. 2022.
Diversity and functional roles of the gut microbiota in Lepidopteran insects. Microorganisms 10(6): 1234.
Zhang, Y.P., Hong, J. & Ye, X. 2009. Cellulase assays. Methods
in Molecular Biology 581: 213-231.
Zhang, Y., Ding, Z., Hossain, M.S., Maurya, R.,
Yang, Y., Singh, V., Kumar, D., Salama, E.S., Sun, X., Sindhu, R. & Binod,
P. 2023. Recent advances in lignocellulosic and algal biomass pretreatment and
its biorefinery approaches for biochemicals and bioenergy conversion. Bioresource
Technology 367: 128281.
Zhang, Z., Yang, D., Liu, L., Chang, Z. &
Peng, N. 2022. Effective gossypol removal from cottonseed meal through
optimized solid-state fermentation by Bacillus coagulans. Microbial
Cell Factories 21(1): 252.
*Corresponding author; email: ahmad_farisz@um.edu.my
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