West Visayas State University

College of Arts and Sciences

Iloilo City
Chapter 1
Chapter One consists of seven parts: (1) Background and of
the Study; (2) Theoretical Framework; (3) Research Paradigm; (4)
Statement of the Problem; (5) Significance of the Study; (6)
Definition of Terms; and (7) Delimitation of the Study.
Part one, Background of the Study, gives the basis and
explanation for choice of the problem.
Part two, Conceptual/Theoretical Framework, gives the basis
and the variables considered in conducting the study.
Part three, Research Design, illustrates the Independent and
Dependent Variables of the study and their correlation.
Part four, Statement of the Problem, reveals the general and
specific statements of the problem sought after for the major
objectives of the study.
Part Five, Significance of the Study, discusses the benefits
that may be derived from the results of the study and the people
who would benefit from them.
Part Six, Definition of Terms, deals with the conceptual and
operational meaning of the important terms used.

West Visayas State University

College of Arts and Sciences
Iloilo City
Part Seven, Delimitation of the Study, sets the limits and
scopes of the study.
Background of the Study
Biofuels

are

gaining

increased

public

and

scientific

attention, driven by factors such as oil price spikes, the need

for increased energy security, and concern over greenhouse gas
emissions from fossil fuels. It can be obtained from renewable
sources containing starch, sugar, or cellulose, such as potatoes,
corn,

corn

cobs

and

stalks,

grains,

and

wood.

One

of

main

problems with using crops or woods as feedstock is that they will

affect directly crop prices and will result in destruction of
forests. Therefore, seaweed or macroalgae as a solution for this
problem has been introduced recently. Some of advantages in using
seaweed

as

feedstock

productiveness.

It

include

also

has

simple
easier

cultivation
manufacturing

and

possible

process

(No

lignin removal) with a higher CO2 fixation ability.

Algae are considered as the only alternative to current
bioethanol crops such as corn and soybean as they do not require
arable land (Chisti, 2007; Hu et al., 2008; Singh et al., 2010c).
The arable land could be used efficiently to grow food crops
rather than biomass and oil seed crops for the production of
biofuels. Water filled areas that are not suitable for growing
2

West Visayas State University

College of Arts and Sciences
Iloilo City
food

crops

and

industrial

waste

water

can

be

used

for

the

cultivation of algal biomass without any compromise with land and

water resource for the production of bioethanol that will also
not adversely affect the food cost (Singh et al., 2011).
In addition, algae can be converted directly into energy,
such as biodiesel, bioethanol and bioethanol and therefore can be
a source of renewable energy. Given the premises, the researchers
want to determine if the brown algae,
Mermaid fan seaweed can be an alternative source of fuel which is
locally available in the area.
Theoretical Framework
Certain species of microalgae have the ability of producing
high

levels

of

carbohydrates

instead

of

lipids

as

reserve

polymers. These species are ideal candidates for the production

of bioethanol as carbohydrates from microalgae can be extracted
to

produce

fermentable

approximately

sugars.

500015,000

gal

It
of

has

been

estimated

ethanol/acre/year

that

(46,760

140,290 L/ha) can be produced from microalgae. This yield is

several orders of magnitude larger than yields obtained for other
feedstocks.

Blue-green

algae

including

Spirogyra

species

and

Chlorococum sp. have been shown to accumulate high levels of

polysaccharides both in their complex cell walls and as starch.
3

West Visayas State University

College of Arts and Sciences
Iloilo City
This

starch

accumulation

can

be

used

in

the

production

of

bioethanol (Harun et al., 2010; Eshaq et al., 2011). Harun et al.

have shown that the blue-green algae Chlorococum sp. produces 60%
higher ethanol concentrations for samples that are pre-extracted
for lipids versus those that remain as dried intact cells. This
indicates that microalgae can be used for the production of both
lipid- based biofuels and for ethanol biofuels from the same
biomass as a means to increase their overall economic value
(Jones and Mayfield, 2012).
With these, the study intends to investigate the potential
of the macroalgae named Mermaid Fan Seaweed (sci. name)in
producing bioethanol using two types of chemical pretreatment
procedure: acid hydrolysis and enzymatic hydrolysis.

Research Paradigm
Mermaid Fan Seaweed
using pretreatment
Independent
Variable
procedures:
a. Acid hydrolysis
b. Enzymatic
hydrolysis

Dependent Variables
Bioethanol actual yield
4

West Visayas State University

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Physical Characteristic
of bioethanol:
a.
b.
c.
d.
e.
f.
g.

pH
methanol content
specific gravity
density
water content
copper content
flash point for
flammability
h. Electrical
conductivity
i. Visual appearance

Standard for Bioethanol

based on ASTM (American
International Testing
and Materials, 2011)

Fig 1. Showing the relationship between the Independent variable

and the dependent variables.

Statement of the Problem

West Visayas State University

College of Arts and Sciences
Iloilo City
This study aims to determine the potential of
bioethanol production from mermaid fan seaweed, Padina sp
(complete sci name with genus and species). The study
specifically aims to answer the following questions:
1. What is the actual yield of bioethanol produced from Mermaid
Fan

seaweed

(sci

name)

using

the

following

chemical

pretreatment procedures?
a. Acid hydrolysis
b. Enzymatic hydrolysis
2. What are the physical characteristics of bioethanol produced
from different pretreatment procedure using the following
parameters?
3. Does the bioethanol produced is in compliance with standard
set by ASTM?
4. Is
there
significant
characteristics

of

difference

bioethanol

on

the

physical

produced

from

different

pretreatment?
Hypothesis
For the purpose of the study, the following hypothesis will be
advanced:
1. There

is

no

characteristics

significant
of

difference

bioethanol

pretreatment.
Significance of the study
6

on

produced

the
from

physical
different

West Visayas State University

College of Arts and Sciences
Iloilo City
The

following

will

benefit

the

Filipino

researchers,

students and those who are interested in seaweeds will have more
information
seaweed,

about

Padina

the
sp.

bioethanol
in

production

Miagao,

Iloilo,

of

Mermaid

Fan

Philippines.

The

fisherfolk will be equipped with idea about possible source of

livelihood by engaging in seaweed culture.
The policy makers (like barangay, municipal and provincial
local government units) of the coastal communities will also
benefit

from

this

study

in

prioritizing

the

livelihood

opportunities in their respective areas.

The

Local

Government

Unit

will

consider

the

culture

of

mermaid fan seaweeds thus increasing livelihood in the locality

if big companies will establish investments.
The students and teachers who are interested to conduct
similar topic would use the result of this study as a future
reference. This study could serve as their baseline information
on bioethanol production of Mermaid Fan seaweed, Padina sp.
Companies that are interested in bioethanol production will
find this study as an important basis. And also the samples used
will

be

considered

as

possible

raw

material

for

their

production of bioethanol and culture of the samples will be made

possible.
7

West Visayas State University

College of Arts and Sciences
Iloilo City
Definition of Terms
For a clearer understanding of this study, all important
words used were given their conceptual and functional meanings.
Bioethanol - a renewable energy source made by fermenting
the sugar and starch components of plants by-products mainly
sugarcane and crops like grain, using yeast (Ecosmartfire, 2014).
In this study, "bioethanol" refers to the alcohol product
produced through fermentation of Laminaria Pallida subjected to
different chemical pretreatment.
Laminaria Pallida refers to the species of brown algae
with bunch of funnel-shaped structures. Each funnel about 3-5cm
in diameter, with concentric circles of tiny hairs and a rolled
edge. The funnel is often torn at the edges. The bunch is usually
attached to a hard surface and spreads out like a beautiful
bouquet when submerged. The white tinge is from the calcium
carbonate incorporated in the blade(Seaweed Industry, 2014).
In this study, "Laminaria Pallida" refers to the species of
brown algae tested for bioethanol production.
Delimitation of the Study

West Visayas State University

College of Arts and Sciences
Iloilo City
This study aims to determine the potential of Mermaid fan
seaweed in producing bioethanol. Fresh samples will be collected
along the shores of Miag-ao, Iloilo.
The samples will be subjected to physical pretreatment
procedure by heating. The solution will be divided into two and
will be separately subjected into chemical pretreatement: acid
hydrolysis and enzymatic hydrolysis. Both hydrosilate will be
subjected into anaerobic fermentation to produce bioethanol.
The bioethanol will be subjected to distillation process to
determine the actual yield produced and will be compared which
chemical pretreatment yields more bioethanol. The products will
also be tested for physical characteristics using the following
parameters:
a.
b.
c.
d.
e.
f.
g.
h.
i.

pH
methanol content
specific gravity
density
water content
copper content
flash point for flammability
Electrical conductivity
Visual appearance
The physical and chemical pretreatment and fermentation as

well as testing for physical characteristics will be done on UPVCFOS, Miag-ao, Iloilo.

West Visayas State University

College of Arts and Sciences
Iloilo City

Chapter 2
Review of the Related Literature
Chapter Two, consists of the Review of Related Literature. the
concepts and studies related to the current study that will give
better understanding of the nature of the research, It includes
a) Seewed; b) Culture and Harvesting of Seeweed; c) Carbohydrates
and its Composition and other important substances in Seaweeds;
d)

Problems

with

fuels;

e)

Previous

10

research

on

macroalgae

West Visayas State University

College of Arts and Sciences
Iloilo City
biofuels production; f) Potential of macroalgae for biofuels;
g)Potential for Bio-ethanol Production in the Philippines.

Seaweed
Any of a vast group of simple multicellular plant forms
belonging to the algae group, and found growing in the sea,
brackish estuaries, and salt marshes, from near the high-tide
mark

to

depths

of

100200

m/300600

ft.

Many

seaweeds

have

holdfasts (attaching them to rocks or other surfaces), stalks,

and fronds, sometimes with air bladders to keep them afloat, and
are green, blue-green, red, or brown (Manivannan et al., 2008).
11

West Visayas State University

College of Arts and Sciences
Iloilo City
Worldwide, there are approximately 2,000 species of brown
macroalgae (Phaecophyceae), 5,500 reds (Rhodophyceae), and 1,200
greens (Chlorophyceae), most of which are marine species. Some
have traditionally been gathered for food, such as purple laver
(Porphyra umbilicalis, which is used by the Japanese to make
sushi), green laver (Ulva lactuca), and carrageenan (Chondrus
crispus). From the 1960s, seaweeds have been farmed, and the
alginates (salts) that are extracted are used in convenience
foods, ice cream, and animal feeds, as well as in toothpaste,
soap, and the manufacture of iodine and glass (Manivannan et al.,
2008).
The

vast

majority

of

seaweed

is

collected

for

human

consumption and for hydrocolloid production. Seaweed exploitation

in the Philippines is currently restricted to manual harvesting
of natural stocks. The majority of Asian seaweed resources are
cultivated.
The traditional markets for seaweed products sustain a much
higher

price

for

raw

material

than

that

likely

for

biofuel

production. Thus, seaweed species have to be explored which do

not compete for human consumption so that biofuel production
becomes more competitive (Huang, 2009).

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West Visayas State University

College of Arts and Sciences
Iloilo City
Seaweed as Macroalgae
Marine macroalgae or seaweeds are plants adapted to the
marine environment, generally in coastal areas. Seaweed has long
been

priced

as

an

excellent

source

of

minerals,

which

are

essential for good health (Manivannan et al., 2008).

Seaweeds are also called macroalgae. This distinguishes them
from microalgae (Cyanophyceae), which are microscopic in size,
often unicellular, and are best known by the blue-green algae
that sometimes bloom and contaminate rivers and streams (McHugh,
2003). Seaweeds are any of a large number of marine plants and
protists in the category of benthic algae. They are macroscopic,
multicellular, and macrothallic (McHugh, 2003)
The FAO Guide to the Seaweed Industry provides an excellent
overview of the seaweed resource and markets worldwide (McHugh,
2003). It enumerates a worldwide survey performed in 1994/5,
listed 221 species of seaweed collected for human applications
(145 for food and 101 for hydrocolloid extraction).
Types and Classification of Seaweed
According to McHugh (2003), seaweeds can be classified into
three broad groups based on pigmentation: brown, red and green.
Botanists

refer

to

these

broad

13

groups

as

Phaeophyceae,

West Visayas State University

College of Arts and Sciences
Iloilo City
Rhodophyceae and Chlorophyceae, respectively. Brown seaweeds are
usually large, and range from the giant kelp that is often 20 m
long, to thick, leather-like seaweeds from 2-4 m long, to smaller
species

30-60

cm

long.

Red

seaweeds

are

usually

smaller,

generally ranging from a few centimeters to about a metre in

length;

however,

sometimes
classified

red

purple,
by

seaweeds

even

botanists

are

brownish
as

not
red,

always
but

Rhodophyceae

red:

they
because

they

are

are

still

of

other

characteristics. Green seaweeds are also small, with a similar

size range to the red seaweeds.
There are a very large number of species around the world,
belonging to several phylogenic groups. Broadly, three types of
seaweeds are defined according to their pigments e.g. the brown
seaweeds (e.g.:

Laminaria,Fucus, Sargassum), the red seaweeds

(e.g. Gelidium, Palmaria, Porphyra) and the green seaweeds (e.g.

Ulva,Codium). With the exception of green seaweed, terrestrial
and marine plants have little in common. This partly explains the
unique

chemical

composition

observed

in

seaweeds.

The

marine

environment also induces the production of unique chemicals to

resist the environmental stresses plants are subjected to (Pons
et., 1981)
Green Seaweed

14

West Visayas State University

College of Arts and Sciences
Iloilo City
Green seaweeds, in particular Ulva spp are being researched
as potential renewable fuel feedstock (McHugh, 2003; Bruton et
al., 2009). According to the study of Bruton et al. (2009) the
moisture content of green seaweed is even higher than that of
brown

seaweeds,

and

it

has

similarly

high

ash

content.

The

species is attracting interest as an energy resource due to the

comparatively

high

level

of

accessible

sugars,

specifically

starch. It also has high cellulose content. In these respects it

resembles

some

suggesting

it

of
is

the

properties

compatible

with

of
a

terrestrial

cellulosic

and

plants,
starch

fermentation process. There is some potential for manipulation of

the components in favor of energy production. The high sulphate
content will cause high yields of H 2S during fermentation, which
is a fermentation inhibitor.
Brown Seaweed
The

various

brown

seaweeds

have

since

the

early

20th

century, been used for industrial applications, and now attention

is turned in many regions with brown seaweed resources to the
production of energy (Bruton et al., 2009).
There are several species of brown seaweed and one of them
is the Mermaid fan seaweed, Padina species. This is a bunch of
funnel-shaped structures. Each funnel is about 3-5cm in diameter,
15

West Visayas State University

College of Arts and Sciences
Iloilo City
with concentric circles of tiny hairs and a rolled edge. The
funnel is often torn at the edges.
The bunch of Mermaid fan seaweed (Appendix H, Plate 1) is
usually

attached

to

hard

surface

and

spreads

out

like

beautiful bouquet when submerged. The color is visibly golden

brown, sometimes with a bluish or whitish tinge. The white tinge
is

from

the

blade. Padina is

calcium
the

only

carbonate
brown

incorporated

seaweed

known

to

in

the

incorporate

calcium.
The same study stated that the composition of brown seaweeds
varies according to species, location, salinity and season so it
is usual to give either an average or range of values. Brown
seaweeds have high moisture content, typically around 85%, and
high ash content, typically around 25% (Bruton et al., 2009).
Red Seaweed
The main uses of red seaweeds are as food and as sources of
two hydrocolloids: agar and carrageenan. Useful red seaweeds are
found in cold waters such as Nova Scotia (Canada) and southern
Chile; in more temperate waters, such as the coasts of Morocco
and Portugal; and in tropical waters, such as Indonesia and the
Philippines. Red seaweed is not of prime interest to biofuel
production at the present times.
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West Visayas State University

College of Arts and Sciences
Iloilo City

Culture and Harvesting of Seaweed

The most common system in the Philippines to obtain seaweed
biomass is by harvesting natural stocks in coastal areas with
rocky

shores

seaweed

is

and
a

tidal

system.

significant

The

resource.

natural

population

Depending

on

of

water

temperature, some groups will dominate, like brown seaweeds in

cold waters and reds in warmer waters.
In 1995 about 3.6 million tons wet weight were collected
globally from natural stocks (Lithothamnion not included). This
was about 48% of the total global seaweed biomass harvested with
the balance produced by aquaculture. More recent numbers (FAO,
2006) give about 1 million tons harvested annually from natural
stocks, making up only 6% of the global resource, with over 15
million tons produced by aquaculture.
Culture
Naturally growing seaweeds are often referred to as wild
seaweeds, in contrast to seaweeds that are cultivated or farmed.
There is possibility for seaweed biomass generation through
cultivation

(Richmond,

2009).

Only

few

genera

have

been

commonly cultivated for many years. The main genera cultivated

17

West Visayas State University

College of Arts and Sciences
Iloilo City
include: Laminaria, Porphyra, Undaria, Gracilaria, Euchema, Ulva
and

Chondrus.

The

seaweed

harvested

from

natural

stocks

has

decreased significantly, while cultivated seaweed has sharply

increased. The overall amount of seaweed harvested has almost
doubled in the last 10 years to 15 million wet tons (FAO, 2006).
Over half of cultured seaweeds, or 7.4 million wet tons, are
brown

Laminaria

sp,

mainly

L.

japonica.

The

global

industry

turnover also increased from US $6.2 billion in 1994 to US $7.2

billion in 2006. Values have not been inflation-adjusted, but the
trend is of increased volume and static turnover. It reflects the
significant

cost-reduction

brought

about

by

cultivation

practices. There is a much larger amount of seaweed available and

mechanized operations have improved productivity allowing lower
market prices (Edwards et a;., 2008)
There are potential economic advantages and opportunities
for developing aquaculture facilities in conjunction with off
shore wind farms (Yokoyama, 2007). Anchorage of long-lines, ropes
and rafts has been a major problem for pilot seaweed cultivation
projects with numerous reports of structures being swept away by
tides and currents. Sharing infrastructure with a wind farm or
other offshore enterprise would seem to make economic sense from
planning,

design

and

operation

18

points

of

view.

The

right

West Visayas State University

College of Arts and Sciences
Iloilo City
conditions for cultivation of seaweed would need to be present.
Previous studies outline this concept in depth.
Harvesting
Producing significant amounts of biofuel from natural stocks
involves the harvesting and processing of large volumes (millions
of

tons)

of

seaweeds.

To

harvest

higher

amounts

may

not

be

sustainable. If for any reason hydrocolloid production decreases

or

ceases,

part

of

the

unused

seaweed

resource

could

be

redirected towards biofuel production and extraction of other

marine natural products from the feed stock or highly abundant
seaweed species.
Manual harvesting has been used since the preindustrial age.
This is still used for harvesting natural stocks of Ascophyllum
nodosum and Fucus species, as they are located in the intertidal
zone on the shore. At low tide, terrestrial vehicles can access
the shore and seaweeds are accessible for manual harvesting. This
method

of

emergence

harvesting

was

of

scale

large

also

used

for

application

Laminaria,
for

but

the

hydrocolloids,

stimulated the development of mechanical systems. Trawlers are

used in Norway to cut the large size adult canopy, leaving the
small size seaweed attached to the rocks. Re-growth is stimulated
by the increased light reaching the small size seaweeds. The
19

West Visayas State University

College of Arts and Sciences
Iloilo City
trawler system is operated from a boat. Using either a dredge or
the Scoubidou allows one man in a boat to collect several tons of
seaweed per day. This is a significant improvement over manual
harvesting.

These

two

examples

are

the

most

widely

known

mechanized harvesting techniques currently used for industrial

applications (FAO, 2006).
Another primarily natural source is the drift seaweeds. Some
reports suggest as much as 20% of L. hyperborean stocks are
washed

up

on

shore

every

year

in

Ireland.

The

location

and

seasonal availability of these resources are unpredictable. It

has traditionally been collected by coastal communities on a
small

scale

to

use

as

fertilizer

or

soil-conditioner.

When

collected on the foreshore drift seaweeds are considered a waste

product. Developing an application for a waste has very positive
connotations.
The green seaweed found in the shore of Boracay although
this is not a large quantity can contribute to local energy
production.
enterprise

However,
based

on

it

can

wastes

be
which

difficult
are

to

build

desirable

to

local

eradicate

pollution but culturing the species for possible carbohydrates

production is one good option. The drift seaweed biomass provides
an opportunity, as and when it is available, to be integrated in

20

West Visayas State University

College of Arts and Sciences
Iloilo City
a broader process using other type of biomass raw materials
(Palligarnai et al., 2008)
Problems with fuel
Over the last few decades, the negative impacts of fossil fuel on
the environment and consequent global warming, progressive demand
for energy, inevitable depletion of the worlds energy supply,
and the unstable oil market (such as the energy crisis of the
1970s) have renewed the interest of society in searching for
alternative fuels (1; 2). The alternative fuels are expected to
satisfy several requirements including substantial reduction of
greenhouse gas emission, worldwide availability of raw materials,
and capability of being produced from renewable feedstocks (3).
Production

of

fuel

ethanol

from

biomass

seems

to

be

an

interesting alternative to traditional fossil fuel, which can be

utilized as a sole fuel in cars with dedicated engines or in fuel
blends (Pelagiaresearchlibrary.com, 2014)
Although CO2 is the most important greenhouse gas (GHG),
several studies show that it is important to consider other GHGs
as well. The continued use of fossil fuels to meet the majority
of

the

worlds

energy

demand

is

threatened

by

increasing

concentrations of CO2 in the atmosphere and concerns over global

21

West Visayas State University

College of Arts and Sciences
Iloilo City
warming. The combustion of fossil fuels is responsible for 73% of
the CO2 production.
The heightened awareness of the global warming issue has
increased interest in the development of methods to mitigate GHG
emissions. Much of the current effort to control such emissions
focuses

on

advancing

technologies

that:

(i)

reduce

energy

consumption, (ii) increase the efciency of energy conversion or

utilization, (iii) switch to lower carbon content fuels, (iv)
enhance natural sinks for CO2, and (v) capture and store CO2.
Reducing use of fossil fuels would considerably reduce the amount
of CO2 produced, as well as reduce the levels of pollutants. As
concern

about

global

warming

and

dependence

on

fossil

fuels

grows, the search for renewable energy sources that reduce CO2
emissions becomes a matter of widespread attention. To reduce the
net contribution of GHGs to the atmosphere, bioethanol has been
recognized

as

potential

alternative

to

petroleum-derived

transportation fuels (Biotek.lu.se, 2014)

Previous research on macroalgae biofuels production
The interest in macroalgae for biofuel lies in their high
carbohydrate (e.g. polysaccharide) content. Macroalgae were first
proposed as a possible source of energy by Howard Wilcox in the

22

West Visayas State University

College of Arts and Sciences
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late 1960s, who presciently considered their production not only
as a solution to the energy crisis but also for global warming
(Wilcox, 1975). Much research was carried out by the US during
the 1970s and early 1980s to develop open ocean macroalgae
farms to produce a substitute for natural gas, an energy source
then considered in the USS to be approaching depletion. The
Marine Biomass Program, supported between 1979 and 1985 with over
$50 million by the U.S. Dept. of Energy (about twice the budget
of the 1980- 1996 U.S. DOEmicroalgae Aquatic Species Program) had
as its ultimate objective to replace the entire U.S.
Potential of macroalgae for biofuels
Macroalgae are again being considered as a biofuel feedstock
for similar reasons as thirty years ago: because they are thought
to have very high biomass yields (though still this remains to be
established) and, perhaps most importantly, because they dont
compete with agricultural crops for land, water resources, and,
potentially,
cultivation
problems,

fertilizers.
of

seaweeds,

mainly

the

Herein
which

very

high

we

neglect

suffers
water

from

the

land-based

several

exchange

inherent

and/or

mixing

required for high productivity cultivation, in addition to CO2

supplementation. However, land-based cultivation is already a
commercial

process

for

some

seaweed
23

species,

and

may

be

of

West Visayas State University

College of Arts and Sciences
Iloilo City
interest

for

biofuels

undoubtedly

their

cultivation.

Compared

production

greatest
to

in

some

potential

microalgae,

locations,

is

in

macroalgae

though

open
have

ocean

major

advantage: their macroscopic nature allows for ready and low

costharvesting.
difficulties

of

Against

these

working

in

advantages

the

sea,

must

even

be

placed

near-shore,

the

which

imposes significant costs and risks, as already experienced by

the earlier U.S. Marine Biomass Program. The three most commonly
mentioned

fuels

that

could

be

derived

from

macroalgae

are

methane, ethanol, and butanol.

The interest in macroalgae for biofuels was recently reinitiated, mainly in Japan, Korea and Europe, but at a relatively
low level of funding initially. In Japan, Tokyo Gas studied the
production of biogas from seaweed that was collected from biomass
naturally

deposited

on

beaches

after

storms

and

high

tides

(Huesemann et al., 2010). However, several factors - the small

amounts and sporadic nature of such harvests, the sand and dirt
collected along with such biomass, and the transportation costs
make such schemes impractical. Thus most attention has focused on
off-shore cultivation of local seaweeds, with a popular candidate
species

being

Laminaria

japonica,

the

most

common

seaweed

currently used for food and chemical production, and already

24

West Visayas State University

College of Arts and Sciences
Iloilo City
considered thirty years ago for such applications (Tseng, 1981;
Chynoweth, 2002). In Ireland, Laminairia ssp and Ulva ssp, are
being considered because of their relatively high carbohydrate
content (Bruton et al. 2009). Ulva can be readily digested to
methane gas and seem to lack epiphytes, e.g. microalgae growing
on the surface of the seaweed leaves, which can interfere with
their production (Chynoweth 2002). Other species also evaluated
for fuel production in the 1980s, include Gracilaria tikvahiae (a
red algae species), notable for its high yields in on-shore
cultivation tests (Hanisak 1987).
Another interesting macroalga is Sargassum, notable because
it is one of the few species that is found free floating in the
open ocean (Chynoweth 2002). The possibility of growing Sargassum
in

the

open

reasonably

ocean

plausible

is

intriguing,

approach

to

but
its

there
mass

is

presently

cultivation.

no

Their

cultivation was already discussed over forty years ago, and the
concept proposed (but not published) was to release propagules of
Sargassum into a marine current, near an upwelling zone, and then
harvest the plants downstream, after a few weeks of growing in
the ocean current. Of course, this intriguing concept is at this
point entirely hypothetical.

25

West Visayas State University

College of Arts and Sciences
Iloilo City
Because of limited suitable near-shore areas, many already
being used extensively for commercial macroalgae cultivation, the
key concept for cultivation of macroalgae for biofuels remains,
as

before,

some

type

of

open

ocean

cultivation

technology.

However, the design of such systems also remains, as before,

mainly hypothetical, with no design apparent at present that
could be scaled-up or deployed beyond a near-shore environment.
An earlier analysis for the U.S. Biomass Program of such systems
pointed

out

the

many

inherent

essentially

insurmountable

engineering difficulties of such concepts (Ashare et al., 1978).

In what follows we thus must per-force assume that an open ocean
cultivation technology will eventually prove to be technically
and economically feasible, and that the macroalgae productivities
will be high enough to justify such efforts. The remaining issues
are then harvesting and conversion to fuels.
Algae are considered as the only alternative to current
bioethanol crops such as corn and soybean as they do not require
arable land (Chisti, 2007; Hu et al., 2008; Singh et al., 2010c).
The arable land could be used efficiently to grow food crops
rather than biomass and oil seed crops for the production of
biofuels. Water filled areas that are not suitable for growing
food

crops

and

industrial

waste

26

water

can

be

used

for

the

West Visayas State University

College of Arts and Sciences
Iloilo City
cultivation of algal biomass without any compromise with land and
water resource for the production of bioethanol that will also
not adversely affect the food cost (Singh et al., 2011). In
addition, algae can be converted directly into energy, such as
biodiesel, bioethanol and biomethanol and therefore can be a
source of renewable energy.
Bioethanol from algae holds significant potential due to
their low percentage of lignin and hemicellulose as com- pared to
other lignocellulosic plants (Harun et al., 2010). While having
low

lignin

content,

macroalgae

contain

significant

amount

of

sugars (at least 50%) that could be used in fermentation for

bioethanol production (Wi et al., 2009). However, in certain
marine

algae

such

as

red

algae

the

carbohydrate

content

is

influenced by the presence of agar, a polymer of galactose and

galactopyranose. Current research seeks to develop methods of
saccharification to unlock galactose from the agar and further
release glucose from cellulose leading to higher ethanol yields
during fermentation (Wi et al., 2009; Yoon et al., 2010).
Seaweeds are classified into three groups: green, brown, and
red,

and

they

contain

various

types

of

glucans

which

are

polysaccharides composed of glucose, though the concentration of

these glucans is known to be relatively low. Seaweed was proposed
27

West Visayas State University

College of Arts and Sciences
Iloilo City
as one of the most promising biomass materials that can be easily
converted to ethanol, since seaweeds are known to contain a low
concentration of lignin or no lignin at all. Three types of
seaweed in- cluding sea lettuce, chigaiso, and agar weed were
used as representatives of green, brown, and red seaweeds, respectively,
ethanol

and

methods

(bioethanol)

for

from

obtaining

these

high

seaweeds

concentrations

were

investi-

of

gated

(Yanagisawa et al., 2011). An ethanol yield of more than 3% was

obtained from these seaweeds.
Bioethanol from algae holds significant potential due to
their low percentage of lignin and hemicellulose as com- pared to
other lignocellulosic plants (Harun et al., 2010). While having
low

lignin

content,

macroalgae

contain

significant

amount

of

sugars (at least 50%) that could be used in fermentation for

bioethanol production (Wi et al., 2009). However, in certain
marine

algae

such

as

red

algae

the

carbohydrate

content

is

influenced by the presence of agar, a polymer of galactose and

galactopyranose. Current research seeks to develop methods of
saccharification to unlock galactose from the agar and further
release glucose from cellulose leading to higher ethanol yields
during fermentation (Wi et al., 2009; Yoon et al., 2010).

28

West Visayas State University

College of Arts and Sciences
Iloilo City
Brown

algae

as

seaweed

is

evolutionarily

diverse

and

abundant in the worlds oceans and coastal waters. The seaweed

industry has an estimated total annual value of 5.5 to 6 billion
US$, with 7.5 to 8 million tons of naturally grown and cultivated
seaweed

harvested

worldwide.

Seaweed

is

mainly

used

in

food

products for human consumption, which generates approximately 5

billion US$ per year, with the remainder used for production of
extracted hydrocolloids, fertilizers, and animal feed additives
(Adams et al., 2009;McHugh et al., 2003).
Brown

seaweed

has

high

content

of

easily

degradable

carbohydrates, making it a potential substrate for the production

of liquid fuels. The carbohydrates of brown seaweed are mainly
composed of alginate, laminaran, mannitol, fucoidan and cellulose
in small amounts (Horn et al., 2000)
Potential for Bio-ethanol Production in the Philippines
Biogas

production

is

long-established

technology

and

previous trials have indicated that anaerobic digestion (AD) of

seaweed is technically viable (Bruton et al., 2009). It should
initially

be

possible

to

incorporate

seaweed

resources

into

existing AD plant to allow for smaller quantities and seasonal

availability. This is the closest process to commercialization

29

West Visayas State University

College of Arts and Sciences
Iloilo City
for conversion of macroalgae to energy, though there is still a
need to reduce the cost of the raw material by at least 75% over
current levels.
Alcoholic fermentation is more difficult. The lack of easily
fermented sugar polymers such as starch, glucose or sucrose means
there is little point in pursuing standard sugar fermentation
processes. The polysaccharides that are present will require a
new commercial process to break them down into their constituent
monomers prior to fermentation, or else a direct fermentation
process

will

have

to

be

developed.

Promising

work

has

been

initiated in Ireland and elsewhere to isolate marine lyases which

would do this efficiently. Theoretically up to 60% of the dry
biomass
process.

in

Laminaria

Ulva

spp

sp.

are

could

also

of

be

fermented

interest

due

with
to

the

right

their

starch

for

alcohol

content (Pons et al., 1981).

The

competitiveness

of

macroalgal

biomass

fermentation must be viewed in the context of other available

cellulosic biomass such as wood, straw and dry organic waste
which

are

also

potential

ethanol

feedstock.

There

is

much

speculation that integrated bio-refinery solutions would allow

sufficient

scale

to

macroalgae.

The

only

enable

economic

industrial

30

production

product

of

of

fuel

from

significance

from

West Visayas State University

College of Arts and Sciences
Iloilo City
macroalgae

is

hydrocolloids.

Extraction

of

energy

from

waste

streams is a valid commercial bio-refinery concept. If the cost

of seaweed permits, a dual production of ethanol and biogas is
also possible. There are many other opportunities for extraction
of high-value niche products from seaweeds. Each would have to be
assessed on commercial terms and demonstrate the feasibility for
co-production of energy alongside the higher-value product, with
particular

attention

to

whether

the

scale

of

operation

is

appropriate (Pons et al., 1981).

Chapter 3
Research Design and Methodology
Chapter Three consists of three parts; namely, (1) Purpose
of the Study and Research Design; (2)

Methodology; and (3) Data

Analysis Procedure
Part One, Purpose of the Study and Research Design, presents
the research design used, restates the purpose of the study, and
describes the plan employed in the conduct of the study.
Part Two, Method, presents the subject of the study and
sampling techniques. It also describes the data-gathering

31

West Visayas State University

College of Arts and Sciences
Iloilo City
instruments; the material used, and delineates the procedures
followed in the study.
Part three, Statistical Analysis Procedure, reports on the
procedure for the statistical analysis used in the study.

Research Design
This

quantitative

study

will

investigate

the

bioethanol

produced from Mermaid fan seaweed using two chemical pretreatment

procedure: acid hydrolysis and enzymatic hydrolysis.
The

independent

variable

employed

in

the

study

is

the

Mermaid fan seaweed subjected to acid hydrolysis and enzymatic

hydrolysis. The dependent variables will be actual yield and
physical characteristics of bioethanol.Each testing will be done
in three trials with triplicates.
Methodology
Preliminary Activities
32

West Visayas State University

College of Arts and Sciences
Iloilo City
Gathering of Raw Materials. Mermaid fan seaweed samples will
gathered during the month of March, 2015 along the shores of
Miagao, Iloilo. An approximately 10 kg. of wet samples will be
gathered

and

will

be

identified

preliminarily

using

Simpsons

(2012). The algae will be further confirmed by an algae expert

from UPV-CFOS. The material will be stored in a room temperature
prior to experiment.
Physical pretreatment. Following the procedure by Sahu et
al. (2011), the algae will be soaked in water overnight followed
by

placing

the

sample

in

pressure

cooker

raising

to

high

pressure for 30 seconds to 1 minute and reducing it to normal.

The sample will be allowed to keep boiling for 2 hours. This is
to

physically

destroy

the

components

of

the

cell

wall

of

material. The solution will be divided into two flasks and will
be subjected separately to chemical pretreatments.
Experiment proper
Chemical Pretreatments
cid hydrolysis. 25 mL of 5% H2SO4 (sulfuric acid) will be
poured into a bottle glass containing 300 mL seaweed solution,
which will boiled using stirrer hotplate for 30 to 120 minutes.
The hydrolysis time will be measured after the acid boiled. The
hydrolysis temperature will be maintained at 100 degrees Celsius.
33

West Visayas State University

College of Arts and Sciences
Iloilo City
The solution will be then adjusted to pH 5 by adding dropwise of
0.1 M NaOH (Sudarmadji et al., 1984).
Enzymatic hydrolysis. A separate 300 mL of seaweed solution
which had undergone physical pretreatment will be obtained and
will be subjected to enzymatic hydrolysis. The solution will be
added with cellulase of about 5 mL per 100 mL of solution. The pH
of the solution will be adjusted by adding 50mM phosphate buffer
to pH 5. The set-up will be incubated at 30C for 4 days with
occasional shaking through an incubator shaker about 100 RPM.
Fermentation of Hydrolisate
The

hydrolisate

from

acid

hydrolysis

and

enzymatic

hydrolysis will be separately sieved and placed separately in an

improvised

container

designed

for

anaerobic

fermentation.

Overnight precultured yeast of about 10% (v/v) will be added as a

starter. The fermentation will be conducted at room temperature
(28-30C) for 7-15 days.
Test for reducing sugars. Monitoring of reduced sugar
on the fermentation set-up will be done through DNS method.
Liquefied

slurry

centrifuged

at

will

10,000

be
rpm

transferred
for

10

into

min.

falcon

Supernatant

tube
will

and
be

transferred to fresh tube and reducing sugars will be estimated

by DNSA reagent (Dinitrosalicylic acid) as described by Miller
34

West Visayas State University

College of Arts and Sciences
Iloilo City
(1959).

DNSA

reagent

will

be

prepared

by

dissolving

1g

of

dinitrosalicylic acid, 200 mg crystalline phenol and 50 mg of

sodium sulfite in 100 ml of 1% NaOH. After addition of DNSA
reagent

to

the

standard

solution

and

test

solution,

whole

reaction mixture will be incubated in boiling water bath for 15

min and will be followed by addition of 40% of Rochelle salt in
solution to stabilize color of reaction mixture. Subsequently
observance will be measured at 510 nm.
Data gathering procedure
Bioethanol actual yield through Distillation
About 100 ml of ethanol will be placed in a volumetric
flask. Add sodium chloride powder so that the solution becomes
almost saturated with NaCl. About 50 ml of ether will be added
and will be shaken for 2-3 min. It will be settled and the lower
layer will be transferred to the distillation flask. About 20-30
ml of saturated sodium chloride solution will be added to the
petroleum ether layer and gently shake. Mixture will be allowed
again to settle and transfer the aqueous layer to the
distillation flask. It will be mixed gently and make the solution
just alkaline with NaOH solution. The distillation assembly will
be connected via condenser to the volumetric flask. It will be
distilled gently. The distillate will be collected in the
35

West Visayas State University

College of Arts and Sciences
Iloilo City
volumetric flask almost to the mark. The contents will be placed
to room temperature.
The specific gravity of the alcohol will be confirmed. The
percent by volume will be determined (volume/gram) to identify
the actual yield through distillation.
Physical Characteristics of Bioethanol
pH. The pHe is a measure of the acid strength of a fuel
ethanol and is a predictor of the fuel ethanols corrosion
potential. Ethanol obtained from fermentation procedure utilizing
acid hydrolysis and from enzymatic hydrolysis will be tested for
pH using pH meter. The test will be done in three trials with
triplicates. The value will be recorded

and will be compared to

the standard pH for bioethanol.

Standard requirement: pHe 6.5 9.0.
Methanol content. Ethanol must contain a minimum
concentration of methanol to protect the properties of
ethanol/gasoline blends.
The method will make use of spectrophotometric method. About
50 ml of sample in a simple still and distil will be obtained,
collecting about 40ml of distillate. Then, 1 ml of distillate
will be diluted to 5ml of distilled water and will be shaken
36

West Visayas State University

College of Arts and Sciences
Iloilo City
well. 1 ml of the solution will be obtaained, 1 ml of distilled
water (for blank) and 1 ml of each of the methanol standards in
to 50 ml stoppered test tubes and will be kept in an ice-cold
water bath. To each test tube, 2 ml of KmnO4 reagent will be
added and be kept aside for 30 min. The solution will be
decolorized by adding a little sodium bisulphite and adding 1 ml
of chromotropic acid solution. Mixture will be mixed well and be
added with 15ml of sulphuric acid slowly with swirling and place
in hot water bath maintaining 800C for 20 min. Color development
from violet to red will be noted. The mixture will be cooled and
the absorbance be measured at 575 nm. The values will be recorded
and will be compared to the standard.
Standard requirement: a maximum methanol concentration of
0.5% by volume.
Specific gravity. Specific Gravity is required for the
conversion of measured volumes to volumes at 15C (the standard
temperature). The specific gravity of an ethanol product may be
an indicator of contamination.
A clean and dry pyknometer will be weighed along with the
stopper at room temperature (W). It will be filled with the
ethanol to the brim and insert the stopper gently. The sample
will be weighed again (W1). Next, the ethanol will be removed and
37

West Visayas State University

College of Arts and Sciences
Iloilo City
will be filled with water in the same manner as described above.
The weight will recorded (W2). The specific gravity will be
solved as follows:
Specific gravity = W1 W2
W2 W
Standard for the specific gravity of ethanol is 0.787.
Density. Density is a fundamental physical property. It is a
required measurement to convert measurements from mass % to
volume %.
Standard for the density of ethanol is 0.789 g/cc.
Copper content. To the ethanol, 15 ml concentrated nitric acid
and 5 ml concentrated sulphuric acid will be added. About 10 ml
of water is first added to the sample before adding nitric acid.
The mixture will be mixed well and the set-up will be connected
to the extractor and water condenser. The mixture will allowed to
stand at room temperature overnight in order to prevent foaming.
The mixture will then be heated, first by means of a soft flame.
The flame will be removed from digestion apparatus as necessary
to minimize escape of nitrogen oxides from the top of condenser.
Maintaining full heat, the tap will be turned through 900 so that
liquid distils into the receiver. Temperature of the vapour in
the digestion flask at this stage will not exceed 120C. Then the
38

West Visayas State University

College of Arts and Sciences
Iloilo City
tap will be turned through further to 900 (what is this?) so that
the distillate (mainly water) drain off through another receiver.
Turn the tap in such a way that liquid distils into the receiver
B. Heating will be intensified in such a way that nitric acid
distills into the receiver. If the solution begins to darken add
a few millilitres of nitric acid from the receiver with the help
of the double bore stopcock. The digest will be transferred
quantitatively with the help of redistilled water into a beaker
of 100 ml. Adjust the pH of the solution with ammonium hydroxide
(25% m/v) to approximately 3. The solution will be transferred
and obtained into a separation funnel of 50 ml with the help of
redistilled water. The solution should not exceed 30 ml. Pipette
I ml of ammonium pyrrolidine dithiocarbamate and 5 ml of
methylisobutylketone. Shake for 1 minute. The solution will be
subjected to spectrophotometer.
Standard copper content is 0.10 mg/kg.
Water content. About 20 mL methanol and 10 mL formamide are
placed in the titration vessel, warmed to 50C and titrated
volumetrically to dryness. The comminuted sample is then added
and

its

water

content

titrated

volumetrically

at

the

same

temperature. The water content will be measured using Coulometric

Karl Fischer Titration technique.

39

West Visayas State University

College of Arts and Sciences
Iloilo City
Standard water content is 1.0 0.5% by volume
Flash Point for flammability. This test will be carried out
using flash point apparatus. The cup in the apparatus will be
dried. About 50ml of each sample (ethanol produced) will be
transferred into the flash point cup. The cup will be fixed into
the position in the apparatus assembled with thermometer, and the
apparatus

will

be

switched

on;

the

heat

is

controlled

by

steadily stirring the ethanol to maintain a uniform temperature

while

passing

small

flame

across

the

material

every

five

seconds. The temperature at which the vapour first flashes with a

blue flame will be recorded as the flash point of the sample,
after

each

test

the

cup

will

be

washed

and

dried

before

subsequent test.
Standard for flash point for bioethanol is 15-25 Celcius.

Electrical Conductivity Test. This test will be done to

identify

ion

content

on

the

ethanol.

Digital

electrical

conductivity meter will be used. About 20 mL of ethanol produced

from fermentation using different chemical pretreatment procedure
will be used for the test. The electrode will immersed to the
censor market point. The meter will be turned on to the required

40

West Visayas State University

College of Arts and Sciences
Iloilo City
calibrated

units

and

the

reading

will

be

taken

for

all

the

samples.
Standard electrical conductivity is 300 microsiemens/meter
(us/m).
Visual Inspection. Test for visual appearance of bioethanol
will also be used through observation. About 20 mL of ethanol
will be placed in a test tube. Ocular observation will be used to
check on the clarity of the ethanol. Below is the scoring to be
used for visual inspection:
Score
1
2
3

Description
blurry, not clear
moderately clear with occasional floating particles
bright and clear

Data Analysis
Descriptive Data Analysis. Mean and standard deviation will be
used to determine the actual yield and physical characteristics
of bioethanol produced.
Inferential data analysis. t-Test will be used to compare the
means of actual yield and physical characteristics of bioethanol

41

West Visayas State University

College of Arts and Sciences
Iloilo City
produced

using

acid

hydrolysis

hydrolysis pretreatment.

42

pretreatment

and

enzymatic