Project Title: Ethanol Production through Biological Pretreatment of Pig (Sus scofa domessticusa)

Manure using Oyster Mushroom (Pleurotus ostreatus)

Proponents: Xyra Aceveda

Project Cost:

Significance: This study may benefit Department of Energy since the findings may help in the

development of less toxic and economically sustainable source of energy. It may

help in meeting the demanded energy of the society. Lastly, for the future

researchers, this study can serve as their stepping stone in investigating in line

with this kind of research.

Objectives: The researcher intends to investigate bioethanol production potential of Pig (Sus

scofa domesticus) manure through biological pretreatment using Oyster Mushroom

(Pleurotus ostreatus). Specifically, this study aims to achieve the following goals:

1.) To perform delignification on the Pig (Sus scofa domesticus) manure

2.) To produce ethanol out of Pig (Sus scofa domesticus) biologically pretreated

using Oyster Mushroom (Pleurotus ostreatus)

Hypotheses: The research work is anchored on the following null and alternative forms of

hypotheses. The research has one set of hypotheses because the researcher attempts to

answer the goals stated in the statement of the problem.

1. Ho = Oyster Mushroom (Pleurotus ostreatus) has no a significant effect on Pig

(Sus scofa domesticus) manure.

2. Ha = Oyster Mushroom (Pleurotus ostreatus) has a significant effect on Pig (Sus

scofa domesticus) manure.

REVIEW OF RELATED LITERATURE

Philippines demand for fuel had been increasing as the population continues to grow with

rising living standards. The country primarily depends on coal with having the highest contribution to

energy mix at 44.5% in 2015. But coal as a energy source, can have significant consequences on the

environment. It is a inexpensive and is abundant in supply but on the other hand, burning of coal can

impost health hazards as it produces carbon dioxide. Thus, the look for more cleaner and renewable

energy is needed.

One of the promising renewable energy source is bioethanol. Ethanol, commonly known as ethyl

alcohol, is a clear, colorless liquid with high latent heat vaporization, high octane number and rating

and low emission of toxic compounds on its combustion. To improve vehicle emissions and power,

ethanol is being blended with gasoline which can reduce heavy dependence on oil producing

countries. It is produced via fermentation process of sugars or cellulose converting it to glucose.

When agricultural wastes is utilized to power the process of converting it into cellulosic ethanol, it

lowers the levels of life cycle greenhouse gas emissions. Another benefit of ethanol is that it comes a

renewable source, making it more practical and affordable.

The pig industry is among the top economic contributors of the agricultural sector. Pig industry

provide 60% of total meat consumption of Filipinos. Based on Swine Situation Report (2018), the

total pig population is 12.71 million heads which is significantly higher compared to the previous

year of 12.60 million heads. Abundant pile of manure is being produced by pigs, causing potential

damaging effects on the environment. Therefore, there is a need to efficiently utilize these by-

products to improve environmental performance of pig farms. According to Chen, S., et.al. (2003), a

grower type-pig manure contains 20.5 %DM hemicellulose, 13.9 %DM cellulose and 6.4%DM
lignin. The solid fraction of the manure is comprised of phosphoric and inorganic compounds (Lens,

P. et.al., 2004). Study showed the liquid fraction have nitrogenous compounds (ammonia,

ammonium compounds) and organic matter (Bertora, et.al, 2008).

Preliminary research using organic waste material as feed stocks for the production of ethanol was

being conducted and is capturing attention worldwide. Thus, this study aims to investigate the

bioethanol production potential of Pig (Sus scofa domesticus) manure through biological

pretreatment using Oyster Mushroom (Pleurotus ostreatus).

METHODOLOGY

I. Acquisition of Material

The manure will be collected near a piggy farm in Biñan, Laguna.

II. Treatment of Manure

Stir the manure. Then, the manure will be diluted with water. Next, we add 12 mL

sulfuric acid and 300 mL of CaO solution (20g of CaO diluted in 300 mL of distilled

water). Heat the treated manure into the oven for 90 minutes.

III. HPLC Analysis

The treated manure will undergo HPLC to quantify the cellulose and glucose content.

IV. Biological Pretreatment

Biological pretreatment will be carried out in a 500-mL Erlenmeyer flask. The fungi

Pleurotus ostreatus will be inoculated and be put inside the Erlenmeyer Flask. And will

be incubated without agitation at 28°C. After 12 days, the lignocellulosic substrate will

be througly washed and dried at 40°C in an oven for 24h.

V. Enzymatic Hydrolysis

The cellulase preparations produced by Trichoderma reesei (ATCC 26921) and βglucosidase

from almonds will be purchased. A typical hydrolysis mixture consisted of 0.2 g of the pretreated
 sample, 10 mL of 50 mM sodium acetate buffer (pH 4.8) supplemented with 40 μL of tetracycline

and 20 μL of cycloheximide, 30 FPU/g of cellulose, and 37.5 IU/g of β-glucosidase. The mixture

will be incubated at 50 °C in a rotary shaker at 150 rpm for 96 h. Samples will be taken from the

reaction mixture and centrifuged for 10 min at11180 x g, then stored at -20 °C until further use.

Then, it will be subjected to HPLC for the cellulose and glucose content.

VI. Statistical Analysis

All degradation data and sugar yields were subjected to analysis of variance (ANOVA).

Multiple comparisons among different pretreatment methods were performed with

Tukey’s test with a significance level of 0.05.