POTENTIALITY OF SELECTED SEAWEED FOR THE PRODUCTION …jestec.taylors.edu.my/Special Issue...

Journal of Engineering Science and Technology Special Issue on SOMCHE 2014 & RSCE 2014 Conference, January (2015) 30 - 40 © School of Engineering, Taylor’s University

30

POTENTIALITY OF SELECTED SEAWEED FOR THE PRODUCTION OF NUTRITIOUS FISH FEED USING SOLID

STATE FERMENTATION

N. N. ILIAS, P. JAMAL*, I. JASWIR, S. SULAIMAN, Z.. ZAINUDIN, A. S. AZMI

Biotechnology Engineering Department, Kulliyyah of Engineering, International Islamic

University Malaysia, Jalan Gombak, 53100, Kuala Lumpur, Malaysia

*Corresponding author: [email protected]

Abstract

The rising cost of imported fish feed ingredients have called for an intensive

research towards the production of high nutritional fish feed using cheap and

natural nutrient sources. In this study, seaweed and palm kernel cake were used

as alternative protein ingredients in fish feed and the enrichment of its

nutritional value was achieved by using solid state fermentation. Following the

fermentation process, the nutrient compositions of the three types of seaweeds:

Caulerpa lentillifera (Chlorophyta), Eucheuma cottonii (Rhodophyta) and

Sargassum fulvellum (Phaeophyta) were compared. Solid state fermentation

was conducted using fungus (Phanerochaete chrysosporium) and yeast

(Candida utilis) to improve the bioprotein production. The comparison between the nutrient compositions before and after the fermentation showed that

fermentation process can increase the nutrient value of the bioprotein. Results

of the analysis showed that Sargassum fulvellumis the best type of seaweed

with the highest total protein, 52.28 mg/g, high percentage improvement of ash

after fermentation, 61.31% and contains high amount of carbohydrate, 445.89

mg/g which are very important in fish diet. Hence, Sargassum fulvellum can be used as an alternative protein ingredient in the fish feed since it was rich in

nutritional compounds compared to the other types of seaweed tested.

Keywords: Seaweed, Solid state fermentation, Phanerochaete chrysosporium,

Candida utilis, Fish feed.

Potentiality of Selected Seaweed for the Production of Nutritious Fish Feed . . . . 31

Journal of Engineering Science and Technology Special Issue 3 1/2015

1. Introduction

Several studies are directed to an efficient and cost-effective supplementary fish

feed because of the importance of fish as a protein source for human and

animal’s diet. Aqua feed is the major determinant that influences the successful

growth and intensification of aquaculture production. According to Pereira et al.

[1], fish feeding constitutes over 50% of operating cost in intensive aquaculture,

where protein is the most expensive dietary source. Fish meal is one of the

major ingredients in making fish feed, considering its high importance as a

source of protein. However, it is an expensive feed ingredient and the supplies

often vary unpredictably due to the overfishing or large-scale transient oceanic

changes [2].

The use of terrestrial plants like leguminous plants and oil seed meals as an

alternative source in fish feed has been limited due to the presence of anti-

nutritional compounds, nutritional profile that differs from fish requirement as

well as to the palatability problem [3]. Nowadays, various types of seaweeds

have become the centre of attention due to the possibility of these aquatic plants

as an alternative source of protein for cultured fish. This is also because of the

high content of protein and high production rate [4]. Seaweeds are macroscopic

marine algae. There are about 6000 species of seaweeds that have been

identified and grouped into three different classes which are green

(Chlorophyceae), red (Rhodophyceae) and brown (Phaeophyceae) algae [5].

Various types of seaweeds had been used in Asia since ancient time as a source

of human food, fertiliser, animal feed, herbicides as well as fungicides [6]. In

Malaysia, seaweed has become an economically important natural source with

resource mobilisation of about RM 21.16 billion in the year of 2002. Total

export of dried seaweed from Sabah in 2002 was 1750 MT by weight and

RM14 million by value [7].

Palm kernel cake (PKC) is one of the by-products that was obtained from

mechanically pressed nuts of the palm fruit [8]. The cost of imported fish

ingredients like soybean meal, corn flour and fish meal affects the local fish

farmers where the profits obtained are not sufficient enough to cope with the

Nomenclatures

FeSO4.7H2O Iron (II) sulphate heptahydrate

KCl Potassium chloride

KH2PO4 Potassium dihydrogen phosphate

MgSO4.7H2O Magnesium sulfate heptahydrate

NH4H2PO4 Ammonium dihydrogen phosphate

Abbreviations

AOAC The Association Of Analytical Communities

CU Candida utilis

MARDI Malaysian Agricultural Research and Development Institute

MT Metric tonnes

PC Phanerochaete chrysosporium

PKC Palm Kernel Cake

YEPG Yeast Peptone Glucose media

32 N. N. Ilias et al.


cost required in maintaining aquaculture enterprise. Therefore, the use of

locally available feed ingredients like PKC, which can be easily obtained at a

low cost, is very beneficial and profitable in fish feed production. Several

studies have already been carried out by using PKC as a substrate in solid state

fermentation for various purposes such as in the production of lipase [9] and

xynalase [10].

Solid state fermentation (SSF) is defined by Couto and Sanroman [11] as ‘a

fermentation process conducted on non-soluble material that has a role as both

physical support as well as a source of nutrients in the absence or near absence

of water’. Jecu [12] has reported that the highest possibility for SSF method to

be successful is when fungi were used. This is due to the ability of fungi to

grow in nature on solid substrates like seeds, piece of wood, roots, stems

and some animals’ dried parts. The usage of co-culture system (fungi and

yeast as the fermentative microorganisms) was reported to be beneficial as it

can enhance the protein synthesis through their synergistic effect on solid

matrix as well as improving the protein content and the digestibility of the

substrate [13,14].

Nutrition plays an important role in efficient aquaculture production as it gives

high influences not only to the production costs, but also to the fish health, growth

and production of waste. Specific nutritional requirement of a particular fish

should be known before the formulation of any fish feed in order to produce

nutritious and cost-effective diets.The use of various seaweeds and other aquatic

plants are getting more attention as possible alternative protein sources for

cultured fish due to the high production rate and protein content [4]. In order to

further improve the nutritional value of seaweed, this experiment was conducted

with the aim of evaluating the nutritional compounds of few seaweeds and

selecting one of them with the potentiality of producing nutritious fish feed using

solid state fermentation process involving Phanerochaete chrysosporium and

Candida utilis.

2. Materials and Methods

2.1. Preparation of raw materials (substrates)

Three types of seaweeds (Caulerpa lentillifera, Eucheuma cottonii and

Sargassum fulvellum) were used as the substrate in the production of high

nutritional fish feed. These seaweeds were collected from the near-shore waters

of Port Dickson, Negeri Sembilan. It was thoroughly washed and was dried

in an air forced oven at 60 °C for 24 hours. Then, the dried seaweed was

grounded to the size of 2 mm and was kept in an airtight container at 4 °C

for further use. The other substrate that was used in this study is Palm kernel

cake (PKC). PKC was obtained from a local palm oil plantation (Sime Darby,

Pulau Carrey, Banting, Selangor). It was dried at 60 °C for 72 hours in order to

reduce the moisture content to approximately 5% and was grounded to the size

of 2 mm [13].



2.2. Microorganisms

Two different types of microorganisms were used in this study. They were

Phanerochaete chrysosporium (PC) (PC 2094) and Candida utilis (CU) (FTCC

8100). PC strains were sourced from the Laboratory stock of Bioenvironmental

Laboratory, IIUM while CU cultures were bought from MARDI, Serdang. CU

stock cultures were maintained on nutrient solution containing YEPG media:

yeast extract 0.3%, peptone 0.5%, glucose 1%, malt extract 0.3% and distilled

water. The ingredients were thoroughly mixed, and was then kept in culture

tubes and sterilised at 121 °C for 15 minutes. The sterilised slants were

inoculated with these microorganisms. They were then be incubated at 32 °C to

obtain luxuriant growth. PC was subcultured fortnightly for subsequent use.

2.3. Inoculums preparation

Spores of PC were then harvested from 7-days old plates and were washed with

25 mL of sterile distilled water. The filtered spore suspension was collected in

250 mL Erlenmeyer flask and maintained at 7.5×109 spores/mL. For CU, 1-2

loopfull of cell suspension was added into the YEPG media in 250 ml conical

flasks that was then incubated at 30 °C and 150 rpm for 24 hours. Then, the

inoculums were stored at 4 °C for further use.

2.4. Solid state fermentation

About 4 g of seaweed was added into a 250 mL flask containing 2 g of

accurately weighed palm kernel cake (PKC) in order to obtain the biomass. The

fermentation medium (pH 4) containing the following pre-optimised mineral

concentration (g/L): 47.9 sucrose, 5 KH2PO4, 6.1 MgSO4.7H2O, 5 KCl, 4.4

NH4H2PO4, 3 peptone and 3 FeSO4.7H2O were then added into the flask [13].

The substrate moisture was adjusted to 70% using distilled water and growth

media and was then autoclaved at 121°C for 15 minutes. After that, the

specified amount of inoculums was added and the mixture was incubated at 32

°C in an incubator for 6 days. After the fermentation, the biomass was

harvested. Flasks containing the fermentation media and substrate (without

undergoing fermentation process) were also prepared to act as a control for

comparison purposes.

2.5. Culture harvesting

Biomass (residue after the six days of fermentation process) was harvested by

drying in hot air oven at 60 °C for 48 hours and cooled in desiccators until weight

equilibrates. The biomass was then grinded for further analysis.

2.6. Proximate analysis

Proximate compositions of bioprotein after the fermentation were evaluated using

several types of analysis. Ash content was determined using the method of AOAC

Official Method 923.03 [15]. Crude protein was determined using the Lowry



method (Folin-Phenol Reagent) Lowry et al. [16] while total carbohydrate was

identified using the method used by Dubois et al. [17] and Krishnaveni et al. [18]

with slight modifications. The total reducing sugars was analysed by using the

method used by Miller [19].

3. Results and Discussion

3.1. Analysis of total protein content

One of the major growth promoting factors in fish feed is protein. Protein

including enzymes, hormones and immunoglobins are needed by normal bodily

functions and the deficiency of this nutrient can affect the protein synthesis and

lead to the reduction in weight and other symptoms to the fish [20]. Study done

by Al Mahmud et al. [21] showed that the protein requirement of fish was

influenced by several factors, including fish size, water temperature, water

quality, rearing environment, the genetic compositions and also feeding rates of

the fish. In present study, Fig. 1 shows the comparison between the total protein

content (mg/g of substrates) of the three types of seaweed namely S. fulvellum, C.

lentillifera and E. cottonii. From the graph, the protein content after the

fermentation using only seaweed as a substrate showed that C. lentillifera from

the group of green seaweed has the highest amount of protein content which was

(31.714 mg/g) compared to the other two types of seaweed that contains about

similar amount of protein content (14.751 mg/g). This result was identical with

the study reported by Matanjun et al. [22] which showed that green seaweed has

the protein content (10.41%) higher than red (9.76%) and brown seaweed

(5.40%). Figure 1 also shows the comparison between the methods of addition of

seaweed whether it was added during the fermentation along with PKC or after

the fermentation process. For all three types of seaweed, it was observed that

when the seaweed was added during the fermentation together with PKC as

substrate, the amount of protein content was higher compared to the addition at

the end of the fermentation process. This shows that the role of seaweed as

substrate is very important in order to make sure it works well with the PKC to

produce nutritive bioprotein. This finding has been supported by a study done by

Al Azad et al. [23] which has reported that the use of seaweed as substrate during

the fermentation process is a promising way in preparing a feed supplement for

finfish larvae.

Other than that, the addition of PKC as the substrate along with the seaweed

showed better results compared to the usage of the three types of seaweed only as

substrate during the fermentation. This is due to the high nutritional value of PKC

that contains an abundant amount of nutrients like protein (14.5-19.6%),

carbohydrate (34.9-52.5%) and fibre (13.0-20.0%) [24]. The comparison between

the protein content before (control) and after the fermentation was also compared

where the maximum amount of the protein content after the fermentation was

obtained from S. fulvellum followed by C. lentillifera and E. cottonii. It was also

observed that the total protein contents got increased from 37.53 mg/g to 52.28

mg/g and 35.16 mg/g to 36.06 mg/g for S. fulvellum and E. cottonii respectively.

Our results were in agreement with the study done by Igbabul et al. [25], where

the fermentation process has improved the protein content of the fermented



Mahogany Bean (Afzelia africana) flour. This is due to the extensive hydrolysis

of the protein molecules to simpler components like peptides and amino acids

caused by the increase in microbial biomass during the fermentation process.

Similar observations were reported by Azoulay et al. [26] which showed that the

fermentation process has improved the protein content of cassava from 8.75%-

20.6%. On the other hand, the protein content of green seaweed, C. lentillifera

was reduced after the fermentation. A similar result was reported by Osman [27]

which stated that the protein content was not significantly changed after the

fermentation process and a gradual and insignificant decrease in the amount of

protein was observed after the 20 hours of fermentation.

Fig. 1. The amount of protein content (mg/g of substrates)

for three types of seaweeds (Mean ±±±±SD; n=3).

3.2. Analysis of total ash content

Ash content is the indication of the amount of mineral elements present in the

bioprotein produced after the fermentation process [28]. According to Craig and

Helfrich [29], the minimum amount of ash content required for fish is about 8.5%.

Hence, the ash content found in the three types of seaweeds (used in this study)

can be considered as high compared to the minimum requirement for fish with the

maximum percentage of ash was observed in E. cottonii (15.1%) followed by C.

lentillifera (14.97%) and S. fulvellum (11.27%) before the fermentation as

depicted in Fig. 2. The same results were also reported by Al Azad et al. [23]

where the ash content of C. lentillifera was higher than Sargassum sp.. However,

the results obtained from this study were slightly different from the study done by

Matanjun et al. [22] in which they found that the least ash content was shown by

C. lentillifera instead of S. polycystum. This might be due to the difference in the

seasonal and environmental growth conditions of the place where the seaweeds

had been collected. Although the ash content after the fermentation using S.

fulvellum was found to be the least among the other two seaweeds, the percentage

improvement from control (before the fermentation process) to the enriched

bioprotein was higher (61.31%) compared to C. lentillifera (51.17%) and E.

cottonii (57.02%), as shown in Table 1. This shows that fermentation process

really gives significant improvement for S. fulvellum.



Table 1.Percentage improvement in ash content (%) from

control to the enriched biomass (after fermentation).

Fig. 2. The amount of ash content (%) for the three types of seaweed (Mean ±±±± SD; n=3).

3.3. Analysis of carbohydrate and total reducing sugars

Soluble carbohydrate in fish feed is important not only as a source of energy, but

also because of its ability to increase the pellet integrity and stability, which is a

crucial characteristic for fish feed [20]. In this study, the maximum amount of

carbohydrate was observed after the fermentation of using green seaweed (C.

lentillifera). Contradict results were observed from the study done by Murugaiyan

et al. [30] which found that Sargassum sp. has the maximum carbohydrate content

(25.5±1.37%) followed by the other types of brown seaweed which is

Stoechospermum marginatum (15.8±0.8%). Green seaweed, Caulerpa taxifolia

and Caulerpa racemosa on the other hand showed a minimum carbohydrate

content which are (9.7±0.47%) and (8.5±0.84%) respectively. Nutrition status of

the cells has contributed to the variations of lipids and carbohydrate contents in

the seaweed. It was also observed from Table 2 that the amount of carbohydrate is

decreasing at the end of the fermentation process compared to the control (before

the fermentation process) and S. fulvellum was found to have the highest

carbohydrate content in the final bioprotein produced. This is due to the utilisation

of glucose by the microorganisms to support their growth [27].

Due to high carbohydrate content of S. fulvellum after the fermentation,

additional analysis about the total reducing sugars for S. fulvellum was conducted.

The results proved that the excellent growth of microorganisms has caused the

increment of the amount of total reducing sugars at the end of the fermentation

(day 6) from 56.5 mg/g to 345 mg/g due to the breaking down of the cellulose

Type of Seaweed Percentage Improvement of Ash(%)

Sargassum fulvellum 61.31

Caulerpa lentillifera 51.17

Eucheuma cottonii 57.02



since it has been metabolised by the microorganisms during the fermentation

process. This was clearly proven in Figs. 3(a) to (c) which show that the growth

of the P. chrysosporium and C. utilis were observed to be well-supported by S.

fulvellum compared to the other two types of seaweeds.

Table 2. Amount of carbohydrate content (mg/g of substrates)

for the three types of seaweed (Mean ±±±± SD; n=3).

Sargassum fulvellum Caulerpa lentillifera Eucheuma cottonii

Control 549.420 ± 15.21 623.044 ± 120.42 565.986 ± 17.68

PKC+Seaweed

(Fermentation) 445.886 ± 1.77 419.658 ± 17.68 258.145 ± 30.05

(a) (b) (c)

Fig. 3. Solid state fermentation of (a) Sargassum fulvellum

(b) Eucheuma cottonii and (c) Caulerpa lentillifera.

4. Conclusions

From all the results of analysis, S. fulvellum was found to be the best type of

seaweed in which the nutritional compounds were significantly improved after the

fermentation compared to C. lentillifera and E. cottonii. The protein content,

percentage improvement of ash and total carbohydrate of S. fulvellum were found

to be 52.28 mg/g, 61.31% and 445.89 mg/g of substrates respectively. The

amount of reducing sugars was also increased which shows that the substrate was

metabolised by the microorganisms during the fermentation process. It can also

be concluded that the solid state fermentation process has the ability to improve

the nutritional value of the seaweed, which is very important in the diet of fish

and can lead to the production of high nutritional fish feed. The nutritious fish

feed will then produce high quality and healthy fish that is also one of the

important sources of protein for human and animals consumption. Further study

can be done in narrowing the scope of the nutritional analysis by analysing the

amino acid content and fibre compositions of the seaweed which is also important

for fish.



Acknowledgements

Research was supported by a research grant FRGS13-025-0266 approved by the

Ministry of Higher Education (MOHE), Malaysia. The authors would also like

to thank the Research Management Centre and Department of Biotechnology

Engineering, IIUM, for supporting and providing the laboratories facilities for

this project.

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