CISAK 2015 – Paper Number (Will be replaced with paper registrationor alphabetical number if the manuscript is accepted)

Manufacture of Fermented Feed Using

Aspergillus terreus as Lovastatin Producer to
Reduce Methane Production Towards
Environmentally Friendly Farms
Hendra Nur Cahyo1, Resti1 and Saepul1
1
Department of Animal Science, Universitas Gadjah Mada
Jl. Fauna 3, Bulaksumur, Depok, Sleman, Yogyakarta, Indonesia
1
hendra.nur.c@mail.ugm.ac.id
1
restiugm@gmail.com
1
saepul170294@gmail.com

Abstract. Ruminants produce methane emissions (CH4) which has contributed to the accumulation of greenhouse gases in the
atmosphere that have an impact on global warming. Methane produced from enteric fermentation and animal feces. Digestion in the
rumen process involves several components of microorganisms such as fungi, bacteria, and protozoa. Rumen microbial role in the
production of methane that methanogenic bacteria and protozoa which mostly live in symbiosis. Protozoa is an agent that serves as a
unifier between methanogenic with her in a symbiotic ciliate, causing the formation of methane. With the decline in the numbe r of
protozoa was possible mechanism symbiosis between ciliate protozoa and methanogenic disrupted resulting in methane formation is
not sufficient. Bacteria and protozoa body structure has the form of cell membrane phospholipids composed of charged phosphat e
group and attached to the two-chain fatty acids are associated with the presence of cholesterol. The most important stage in the
formation of cholesterol is the formation of mevalonate from HMG-CoA which occurs in the first stage. This process involves the
enzyme HMG-CoA reductase. HMG-CoA reductase is the target of HMG-CoA reductase inhibitors (statins) to inhibit cholesterol
synthesis. Statins inhibit the synthesis of mevalonate are spesific. Writing this idea aims to assess the effect of the addition of
inhibitors lovastatin from Aspergillus terreus to decrease methane production in cattle towards environmental friendly farm. Based on
the ability of lovastatin to inhibit the synthesis of cholesterol which is a component of the cell membranes of bacteria and protozoa, it
can be concluded that the compound lovastatin may be inhibiting agents ruminant methane production. Fungi from the
Deuteromycetes genus (Aspergillus sp. and Penicillium sp.) have been reported to potentially produces lovastatin, a secondary
metabolites as an anti-hypercholesterolemia agent (Chang and Miles, 1973). Feed fermentation using Aspergillus terreus can reduce
methane production.

Keywords: Aspergillus terreus global warming, lovastatin, methane, protozoa.

A. INTRODUCTION
Solar energy that reaches to the earth will returned to the
atmosphere by way of emitted in the form of heat. Most of the
heat that passes will be absorbed by greenhouse gases so that the
earth becomes warmer [26]. Greenhouse gases are naturally
present in the earth and very important role in regulating the
temperature on the earth, but if there are in excess the amount
greenhouse gas would lead to global warming. Carbondioxyde is
a gas as the greatest proportion of greenhouse gases while
methane contributes 18% of total greenhouse gas [17].
Methane has a higher potential to affect global warming
compared with other gases with a standard value of 296 GWP
(Global Warming Potential). The concentration of greenhouse
gases in the atmosphere for hundreds of years is relatively
constant, but now is increasing due to the burned of coal,
transport, industry, waste, burning biomass, and agricultural
activities on the farm. Otherwise, the livestock sector contributes
9% CO2 and 37% CH4 (especially ruminants) to anthropogenic
greenhouse gas [26].
The ranch has a major contribution to the emission of
methane from enteric fermentation and animal feces. When the
organic matter content in the feces was under anaerobic
conditions, the population of methanogenic bacteria would
growth and produce large amounts of CH4. Total emissions from
enteric fermentation process varies. Based on current conditions,
global warming are our responsibility, not only the livestock
sector but also the world. Climate change will impact on all
aspects of life [1]. Many research has been carried out to reduce
methane production from livestock sector by decreasing
microbial fermentation activity in the rumen which is a methane-
producing agent. One way to reduce methane production is by
adding an agent which causes defaunation of protozoa.
CISAK 2015 – Paper Number (Will be replaced with paper registrationor alphabetical number if the manuscript is accepted)

B. DISSCUSSION inhibit the growth of rumen bacteria specific ferment cellulose,

I. DIGESTION SYSTEM OF RUMINANTS starch, and other polysaccharides into acetic acid, propionic, and
Ruminant digestive process performes occur in three butyric.
stages, mechanical digestion occurs in the mouth, enzymatic Lovastatin as one of the statin compound is a compound
digestion in the intestine, and fermentative digestion performed that is commonly used to lower cholesterol by inhibiting HMG-
by rumen microorganisms. Ruminants are polygastric animal CoA reductase enzyme in cholesterol synthesis process [8]. Here
which digestive process not only by his own body, but also is a scheme involving the formation of mevalonate HMG-CoA.
fermentation process by microorganisms in the stomach (rumen)
[28]. Methane production by microorganisms in the rumen
consist of bacteria, fungi, and protozoa [12]. Methane production
by methanogenic bacteria decrease the gross energy produce
from the fermentation of feed about 6 to 10%. Methanogenic use
a hydrogen as a source of energy to form methane [14].
Protozoa also plays a role as predator of rumen bacteria
thus reduced the use efficiency of nitrogen in the rumen. These
could be a negative effect of protozoa existence to protein
metabolism in ruminants [27]. Protozoa perform a symbiosis
with methanogenic bacteria to produce a methane. Therefore, a
decrease the number of protozoa is the appropriate mechanism to
reduce methane production. Ciliated protozoa are known to have
interspesies hydrogen transfer mechanism specific to have a
symbiotic relationship with the endo and ecto methanogenic
bacteria. Methanogenic bacteria that bind to ciliated protozoa
that mainly come from families Methanobacteriaceae [9].
The decreasing of methane is closely associated with the
presence of protozoa in the rumen. Symbiotic relationship
between methanogenic with ciliate protozoa shows how
important a role in preventing the accumulation of methanogenic
hydrogen via hydrogen transfer mechanism interspecies with Fig. 1. cycle mevalonate formation and working principle of the
ciliate protozoa which causes only slight hydrogen converted enzyme HMG-CoA
into methane [11].
Can be seen in the image above that the mevalonate
II. CHOLESTEROL SYNTESIS AND INHIBITOR compounds produced by reducing HMG-CoA [15]. HMG-CoA
LOVASTATIN is a compound that is soluble in water and can be metabolized
Bacteria and protozoa body structure has the form of cell another when the enzyme HMG-CoA reductase is inhibited [30].
membrane phospolipids (cholesterol) composed of charged HMG-CoA reductase is target of HMG-CoA reductase inhibitors
phosphate group and attached to the two-chain fatty acids [20]. (statins) to inhibit cholesterol synthesis. Statins inhibit the
Cholesterol formation occurs through four stages. The first stage synthesis of mevalonate are specific [15].
is the condensation of three acetate to form a six-carbon Lovastatin can be produced from several species of fungi
intermediates and mevalonate. The second phase is change the such as Aspergillus tereus, Aspergillus parasiticus, Aspergillus
mevalonate into active isoprene. The third stage occurs flavus, Aspergillus umbrosus, penicillium funiculosum,
polymerization of 5-carbon isoprene to form carbon 30 squalene. Trichoderma viridae, Trichoderma longibrachiatum,
The last stage is the cyclization of squalene to form four rings of Acremonium chrysogenum [23], Monascus pilorus [16],
the steroid nucleus through some reaction (oxidation, removal or Aspergillus fischeri [22][13], Monascus purpureus [16],
transfer of a methyl group) to form cholesterol [17]. The most Aspergillus niger [22], and Aspergillus terreus [21].
important stage in the formation of cholesterol is the formation
of mevalonate from HMG-CoA which occurs in the first stage. III. ASPERGILLUS TERREUS
This process involves the enzyme HMG-CoA reductase. HMG- References [24] shows that Aspergillus sp is one of a
CoA is an integral membrane protein of the endoplasmic group of fungi that have asexual spores Askomikota
reticulum smooth [19]. conidiophores found on the head. References [6] shows that
Lovastatin as one of the statin compound will affect Aspergillus terreus as one of the species of the fungus
populations in the rumen microorganisms that can cause changes Aspergillus sp can live optimally at temperatures between 35 and
in methane production. [15] Mevastatin or lovastatin did not 40oC and the maximum temperature for life is between 45 and
CISAK 2015 – Paper Number (Will be replaced with paper registrationor alphabetical number if the manuscript is accepted)
48oC. References [6] shows that Aspergillus terreus conidia
spores can germinate at a temperature of at least 11 to 13oC. In
addition, this type of fungus can tolerate an environment that has
a high salt concentration, therefore, this fungus can be found in
brackish water marsh and coastal areas.
Aspergillus terreus can be obtained by isolation using a
dry grapes. References [25] shows that Aspergillus sp.
commercially used as a producer of organic acids and secondary
metabolic compounds such as lovastatin. References [10] shows
that lovastatin production level of Aspergillus terreus with a rice
straw substrat about 260,8 mg per kg DM after 8 hours
fermentation.
Aspergillus terreus can be obtained by isolation of fungi
from dry grapes. Aspergillus terreus by the description can be
derived from a variety of sources, among others, on food, plants,
drinks [3], soil [31][2][29], sponges [30], wine [17], river sand
sediment [4]. References [6] shows that isolation fungus
Aspergilllus found in many fruits, especially grapes. References
[5] shows that nine types of wine obtained isolates of Aspergillus
sp. are 212 isolates.
[3].
Fig. 2. isolation of the Aspergillus terreus
IV. FERMENTED FORAGES (SILAGE)
Silage is animal feed forage (grasses and legume) is
stored in the form of fresh undergo a process ensilase. Making
silage aims to address the shortage of feed in dry season or when
livestock grazing is not possible done. The fermentation process
was able to increase the value of digestibility of rice straw, even
an increase in quality that can replace the elephant grass.
Therefore, the provision of rice straw has disilase or fermented
can increase consumption and animal body weight higher than in
control animals who ate the rice straw without treatment [28].
References [10] shows that addition of fermented rice
straw extract containing lovastatin at 10 and 20 mg levels
significantly reduced the total gas and CH4 production.
References [15] shows that addition of lovastatin contain
concentration level more than 10 nmol inhibited the in vitro
growth and production of CH4 of strains Metanobrevibacter and
increase feed efficiency.
C. CONCLUCION
Based on the literature riview, addition of Aspergillus
terreus as a lovastatin producer in fermented feed can reduced
methan production in the rumen by inhibited the cholesterol
synthesis from cell membrane of protozoa. However, further
research to understand the digestibility of the fermented feed in
vitro and in vivo.
ACKNOWLEDGEMENT
This work has been completed, thanks to our teacher
and guidiance during this study Prof. Lies Mira Yusiati from the
Faculty of Animal Science Universitas Gadjah Mada.
REFERENCES
[1] Aydinalp, C. and M.S. Cresser. 2008. The Effects of Global Climate
Change on Agriculture. American-Eurasian J. Agric and Environ. Sco., 3
(5): 672-676
[2] Afzal, H., S. Shazad, and S. Q. Un Nisa. 2013. Morphological
identification of Aspergillus species from the soil of larkana district
(Sindh, Pakistan). Asian J Agri Biol 3:105-117
[3] Aryantha, I. N. P., S. Widayanti, dan Yuanita. 2004. Eksplorasi Fungi
Deuteromycetes (Aspergillus sp. dan Penicillium sp.) penghasil senyawa
anti kolesterol lovastatin. Fakultas MIPA Institut Teknologi Bandung.
Bandung.
[4] Chen, H., G. Daletos, M. S. A. Aziz, D. Thomy, H. Dai, H. B. Oesterhelt,
W. Lin, and P. Proksch. 2015. Inducing secondary metabolite production
by the soil-dwelling fungus Aspergillus terreus through bacterial co-
culture. Phytochemistry Letter 12: 35-41
[5] Culebras, P. V. M. and D. Ramon. 2007. An ITS-RFLP method to identify
black Aspergillus isolates responsible for OTA contamination in grapes
and wine. Journal of Food Microbiology 113:147-153.
[6] Domsch, K. K. H., W. Gams, and T. H. Anderson. 1980. Compendium of
Soil Fungi Vol.1. Academic Press. Massachusetts, United States.
[7] Domsch, K. K. H., W. Gams, and T. H. Anderson. 2007. Compendium of
Soil Fungi. Eching. Germany. 672
[8] Gazzero, P., M. C. Proto, G. Gangemi, A. M. Malfitano, E. Ciaglia, S.
Pisanti, A. Santoro, C. Laezza, and M. Bifulco. 2012. Pharmacological
Actions of Statins: A Critical Appraissal in the Management of Cancer.
Pharmacological Reviews. 64:102-146.
[9] Hegarty, R.S. 1999. Reducing rumen metane emissions through
elimination of rumen protozoa. Australian Journal Agricultural Research
50: 1321-1328.
[10] Jahromi M. F., J. B. Liang, Y. W. Ho, R. Mohamad, Y. M. Goh, P.
Shokryazdan, and J. Chin. 2012. Lovastatin-enrich rice straw enhances
biomass quality and suppresses ruminal metanogenesis. BioMed Research
International. Hindawi Publishing Corporation.
[11] Jordan E., D. Kenny, M. Hawkins, R. Malone, D.K. Lovett & F.P.
O’mara. 2006. Effect of refined soy oil or whole soybeans on intake,
CISAK 2015 – Paper Number (Will be replaced with paper registrationor alphabetical number if the manuscript is accepted)

metane output, and performance of young bulls. Journal of Animal [33] Zain, M. E., A. A. Razak, H. H. El-Sheikh, H. G. Soliman, and A. M.
Science 84: 2418-2425. Khalil. 2009. Influence of growth medium on diagnostic characters of
[12] Knapp J. R., G. L. Laur, P. A. Vadas, W. P. Weiss, and J. M. Tricarico. Aspergillus and Penicillium species. African Journal of Microbiology
2013. Enteric metane in dairy cattle production: Quantifying the Research 3:280-286.
opportunities and impact of reducing emissions. J. Dairy Sci. 97: 3231-
3261.
[13] Latha, P. M., Chanakya P., and Srikanth M. 2012. Lovastatin production
by Aspergillus ficheri under solid state fermentation from coconut oil
cake. Nepal Journal of Biotechnol. Vol (2)
[14] McDonald, P., R. A. Edward, J. F. D. Greenhalg, & C. A. Morgan. 2002.
Animal Nutrition. 6th Edition. Scientific and Tech John Willey & Sons.
Inc, New York.
[15] Miller, T. L. and M. J. Wolln. 2001. Inhibitor of growth of metane-
producing bacteria of the ruminant foretomach by hydroxymethylglutaryl-
ScoA (HMG-CoA) reductase inhibitor. J. Dairy Sci. 84: 1445-1448.
[16] Miyake, T., K. Uchitomi, M. Y. Chang, I. Kono, N. Nozaki, H. Sammoto,
and K. Inagati. 2006. Effect of the Principal Nutrient on Lovastatin
Production by Monascus pilorus. Biosci. Biotechnol. Biochem. 70 (5):
1154-1159.
[17] Moss, A.R. 1993. Methane. Cholombe Publications. United Kingdom.
[18] Narasimhan, Banu, and M. Asokan. 2010. Genteic variability of
Aspergillus terreus from dried grapes using RAPD-PCR. Advance in
Bioscience and Biotechnology 1:345-353.
[19] Nelson, D. L. and M. M. Cox. 1996. Lehninger Principal of Biochemistry
4th Ed. W. H. Freeman.
[20] Pommerville, J. 2011. Alcamo’s fundamental of microbiology 9th ed.
Jones and Bartlett Publisher. Canada
[21] Prabhakar, M., K. Lingappa, V. Babu, S. Amena, N. Vishalakshi, and D.
Mahesh. 2012. Characterization of physical facors fr ptimum lovastatin
production by Aspergillus terreus KLVB28mu21 under solid state
fermentation. Appl Sci. 27: 01-05.
[22] Rasyid, S. A., D. Ibrahim, and I. N. P. Aryantha. 2013. Effect of cultural
conditions on lovastatin production by Aspergillus niger using
combination of rice bran and brown rice as substrate. Appl Biology and
Pharmaceutical tech. 4:150-156.
[23] Samiee, S. M., N. Moazami, S. Haghighi, F. A. Mohseni, S. Mirdamadi,
and M. R. Bakhtiari. 2003. Screening of Lovastatin Production by
filamenteus Fungi. Iranian Biomedical Journal. 1:29-33.
[24] Samson, R. A., S. W. Peterson, J. C. Frisvad, and J. Varga. 2011. New
Species in Aspergillus Section Terrei. Studies In Mycology 69:39-55.
[25] Sayyad S. A., B. P. Panda, S. Javed, and M. Ali. 2007. Optimization of
nutrient parameters for lovastatin production by Monascus purpureus
MTCC 369 under submerged fermentation using response surface
methodology. Appl Microbiol Biotechnol (73).
[26] Schaechter, Moselio. 2011. Eukaryotic Microbes. Academic Press.
Californiathe soil of larkana district (Sindh, Pakistan). Asian J Agri Biol
3:105-117
[27] Steinfeld, H., P. Gerber, T. Wassenaar, V. Castel, M. Rosales and C.
deHaan. 2006. Livestock’s Long Shadow. Food and Agriculture
Organization of the United Nations. Rome.
[28] Sugoro, I dan I Yunianto. 2006. Pertumbuhan Protozoa dalam Cairan
Rumen Kerbau yang Disuplementasi Tanin secara In Vitro. Jakarta: Pusat
Aplikasi Teknologi Isotop dan Radiasi - Batan.
[29] Suwandi. 1997. Peranan Mikroba Rumen pada Ternak Ruminansia.
Bogor: Balai Penelitian Ternak Ciawi.
[30] Tobert, Jonathan. 2003. Lovastatin and beyond: the history of the HMG-
CoA reductase inhibitors. Laboratories Rahway. New Jersey. USA. 2:517-
526.
[31] Yadav, Maera, and K. D. S. Yadav. 2006. Enzimatic characteristics of
ligninperoxidases from Penicillium citrinum, Fusasrium oxysporum and
Aspergillus terreus using n-propanol as substrate. Indian Journal of
Biochemistry & Biophysics 43:48-51.
[32] Yin, Y., Q. Gao, F. Zhang, and Z. Li. 2012. Medium optimization for the
high yield production of single (+)- terrein by Aspergillus terreus strain
PF26 derived from marine sponge Phakellia fusca. Process Biochemistry
47:887-891.