production of high protein fungal biomass (hpfb) in … of high... · declaration i hereby declare...

PRODUCTION OF HIGH PROTEIN FUNGAL BIOMASS (HPFB) IN SHRIMP POND WATER: EFFECTS OF AERATIONS

Christine Rika anak Renggu

Bachelor of Science with Honours (Resource Biotechnology)

2016

P. KMIDMAT MAKLUMAT AKADEMIK

UNIMAS

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ACKNOWLEDGEMENT

All glory and honour to God for His wonderful mercy and grace throughout the

research that now, I am able to complete my thesis. And a huge gratitude to my supervisor,

Dr Micky Vincent for his words of encouragement, supports and comments on ways to

improve better which contributed a lot to the success of this research.

Besides that, I would like to thank members from Microbiology Lab 2, all of the

Postgraduates; Fazidah binti Junaidi, Patricia Rowena Mark, Ennry Esut and Abel Hwang

for their guidance and being supporting for the past few months in the laboratory. Not to

forget my labmates, Nur Syafiqah binti Mohd. Nasir, Shaneezan binti Shahhin, Dhelvine bin

Edwin and Siti Nadzirah Azeni for being there in times of ups and downs, through all the

hardships that at the end we managed to finish our researches together successfully. Other

than that, to all the lecturers and staff members of Faculty of Resource Science and

Technology, University Malaysia Sarawak for their encouraging support towards my

research.

I would also like to thank my family members especially to both of my parents for

their prayers, moral and financial supports for me during this three years of my studies in

UNIMAS. Without them, I won't be able to pursue my studies at this level.

Last but not least, to all individuals that have been there helping and guiding me

throughout the research. Thank you so much.

I

DECLARATION

I hereby declare that this Final Year Project Report entitle "Production of High Protein

Fungal Biomass in Shrimp Pond Water: Effects of Aerations" is based on my original

work except for quotations and citations, which have been duly acknowledged. I also declare

that it has not been submitted in support of an application for another degree or qualification

to this or any other university or institute of higher learning.

ý (Christiii'e Rika anak Renggu)

JUNE 2016

11

Pusat Khidmat Maklmnat Akademik UNIVERSPfI MALAYSIA SARAWAK

TABLE OF CONTENTS

TITLE

ACKNOWLEDGEMENT

DECLARATION

TABLE OF CONTENTS

LIST OF ABBREVIATIONS

LIST OF TABLES

LIST OF FIGURES

LIST OF APPENDICES

ABSTRACT

CHAPTER 1 INTRODUCTION

1.1 Introduction

PAGE

I

II

III

V

VI

VII

IX

I

2

1.2 Problem Statement 6

CHAPTER 2 LITERATURE REVIEW

2.1 Rhizopus oligosporus 7

2.2 Shrimp pond water 9

2.3 Submerged Fermentation (SmF) 11

2.4 High Protein Fungal Biomass (HPFB) 13

2.5 Water Aeration 14

CHAPTER 3 MATERIALS AND METHODS

3.1 Materials 16

3.2 Methods 17

3.2.1 Sample collection 17

3.2.2 Substrate pre-treatment 17

3.2.3 Rhizopus oligosporus culture preparation 17

III

3.2.4 Growth of Rhizopus oligosporus in shrimp pond 17

water at different rate of aerations

3.2.5 Analysis of data

3.2.5.1 Dry Cell Weight Analysis 18

3.2.5.2 Nitrate Analysis 19

3.2.5.3 Nitrite Analysis 21

3.2.5.4 Phenol-Sulphuric Analysis 23

CHAPTER 4 RESULT AND DISCUSSION

4.1 Propagation stage 24

4.2 Accumulation stage 28

4.3 Phenol-sulphuric analysis 41

4.4 Nitrate analysis 45

4.5 Nitrite analysis 49

CHAPTER 5 CONCLUSION AND RECOMMENDATION 53

REFERENCES 55

APPENDICES Appendix I: Propagation stage of R. oligosporus 59

Appendix II : Accumulation stage of R. oligosporus 60

Appendix III : Phenol-sulphuric analysis data 64

Appendix IV : Nitrate analysis data 66

Appendix V: Nitrite analysis data 68

IV

LIST OF ABBREVIATION

R. oligosporus

mg

ml

YMB

L

vvm

g

hrs

rpm

Zn

NaCl

nm

Dý

HPFB

SmF

%

Rhizopus oligosporus

milligram

millilitre

Yeast Malt Broth

Liter

volume per medium

grams

hours

rotation per minute

Zinc

Sodium Chloride

nanometer

Degree Celsius

High Protein Fungal Biomass

Submerged fermentation

Percentage

V

LIST OF TABLES

Page

Table 1 Annual Malaysian white shrimp production. 2

Table 2 Concentration of wet biomass and dry biomass weight during 27

propagation stage of R. oligosporus.

Table 3 Wet biomass during accumulation stage of R. oligosporus for 38 0.00 vvm, 0.50 vvm, 0.75 vvm and 1.00 vvm.

Table 4 Dry biomass during accumulations stage of R. oligosporus for 39

0.00vvm, 0.50 vvm, 0.75 vvm and 1.00 vvm.

Table 5 Data collected of phenol sulphuric analysis. 43

Table 6 Concentration of Nitrate for 6 days for 0.00 vvm, 0.50 vvm, 47 0.75 vvm and 1.00 vvm aerations.

Table 7 Concentration of Nitrite for 6 days for 0.00 vvm, 0.50 wm, 51

0.75 wm and 1.00 vvm aerations.

VI

LIST OF FIGURES

Page

Figure 1 Rhizopus oligosporus viewed under light microscope. 7

Figure 2 Shrimp pond located at Kuala Santubong, Kuching. 9

Figure 3 Shrimp pond farm. 10

Figure 4 Set-up of submerged fermentation. 11

Figure 5 Dry biomass obtained after accumulation process. 13

Figure 6 Water aeration on shrimp pond farm. 14

Figure 7 Propagation of R. oligosporus in YMB. 19

Figure 8 API Nitrate Kit used to test concentration of nitrate. 19

Figure 9 Colour changes observed by using API Nitrite Kit as the 20

concentration of nitrate decreases.

Figure 10 API Nitrate Kit used to test concentration of nitrite. 21

Figure 11 Colour changes observed by using API Nitrite Kit as the 22

concentration of nitrite increases.

Figure 12 Phenol sulphuric analysis used to determine the total 23

carbohydrates present in the sample. Figure 13 Propagation stage of R. oligosporus from Day 1 until Day 6.25

Figure 14 Propagation graph of R. oligosporus wet biomass against day. 27

Figure 15 Propagation graph of R. oligosporus dry biomass against day. 27

Figure 16 The accumulation stage of R. oligosporus for 0.00 vvm. 29

(A) Day 1; (B) Day 2; (C) Day 3.


(D) Day 4; (E) Day 5; (F) Day 6.


(A) Day 1; (B) Day 2; (C) Day 3.


(D) Day 4; (E) Day 5; (F) Day 6.

vu


(A) Day 1; (B) Day 2; (C) Day 3.


(D) Day 4; (E) Day 5; (F) Day 6.


(A) Day 1; (B) Day 2; (C) Day 3.


(D) Day 4; (E) Day 5; (F) Day 6.

Figure 24 Wet biomass weight graph of R. oligosporus at different days 39

during accumulation.

Figure 25 Dry biomass weight graph of R. oligosporus at different days 40

during accumulation. Figure 26 Phenol-sulphuric analysis colour changes at different day. 42

Figure 27 Phenol-sulphuric analysis graph of concentration against day. 44

Figure 28 Nitrate analysis colour changes at different day. 46

Figure 29 Nitrate analysis graph of concentration versus day. 48

Figure 30 Nitrite analysis colour changes at different day. 50

Figure 31 Nitrite analysis graph of concentration versus day. 52

vin

LIST OF APPENDIXES

Appendix A

Appendix B

Appendix C

Appendix D

Appendix E

Appendix F

Appendix G

Appendix H

Appendix I

Appendix J

Appendix K

Appendix L

Appendix M

Appendix N

Appendix 0

Appendix P

Appendix Q

Appendix R

Page

Wet biomass for propagation stage of R. oligosporus from 60

Day 0 to Day 6.

Dry biomass for propagation of R. oligosporus from 60

Day 0 to Day 6.

Wet biomass for accumulation stage of R. oligosporus from 61

Day 0 to Day 6 for 0.00 vvm.







Dry biomass for accumulation stage of R. oligosporus from 63



Day 0 to Day 6 for 0.50 vvm. Dry biomass for accumulation stage of R. oligosporus from 64



Day 0 to Day 6 for 1.00 vvm. Phenol-sulphuric analysis data for 0.00 vvm from Day 0 to Day 6.65

Phenol-sulphuric analysis data for 0.50 vvm from Day 0 to Day 6.65



Nitrate analysis data for 0.00 vvm from Day 0 to Day 6.67




ix

Appendix S Nitrite analysis data for 0.00 vvm from Day 0 to Day 6.69

Appendix T Nitrite analysis data for 0.50 vvm from Day 0 to Day 6.69

Appendix U Nitrite analysis data for 0.75 vvm from Day 0 to Day 6.70

Appendix V Nitrite analysis data for 1.00 vvm from Day 0 to Day 6.70

X

Production of High Protein Fungal Biomass in Shrimp Pond Water: Effects of Aerations

Christine Rika anak Renggu

Resource Biotechnology Faculty of Resource Science and Technology

University Malaysia Sarawak

ABSTRACT

As the agricultural industries are developing over the years, the discharged of untreated waste materials are also on the rise. One of the examples is the discharge of unwanted wastes from shrimp aquaculture. High levels

of toxic materials such as nitrogen and phosphorus lead to pollutions towards the environment. Due to this matter, shrimp pond water was treated by using Rhizopus oligosporus to treat the organic matters present in the substrates by using submerged fermentation method. Based on this research, different rate of aerations were used to test the growth of R. oligosporus. Different parameters included were 0.00 vvm, 0.50 vvm, 0.75 vvm and 1.00 vvm rate of aerations. 1.00 vvm aerations produced the highest dry biomass of 4,60 g/L followed by 0.75 vvm, 0.50 vvm and 0.00 vvm. The highest rate of aerations had the highest production of biomass as the microorganism was able to grow with sufficient oxygen provided. The analysis that were done in this research were phenol-sulphuric analysis, nitrate and nitrite analysis. For the phenol-sulphuric analysis, 1.00 vvm aeration had the highest amount of reduction with 93.6% compared to the other three parameters. Other than that, both nitrate and nitrite analysis also showed that 1.00 vvm had the lowest concentration of 0.52 mg/L and 0.00 mg/L respectively. This proved that R. oligosporus was able to treat the shrimp pond water after days being introduced to the substrate. Moreover, the high protein fungal biomass produced could be used as animal feeds which is highly in demand at the global food industry other than reducing rate of environmental pollutions.

Keywords: Rhizopus oligosporus, shrimp pond water, aerations, submerged fermentation, high protein fungal biomass (HPFB)

ABSTRAK

Sektor perindustrian pertanian semakin membangun dari tahun ke tahun yang menyebabkan sisa buangan

juga meningkat sejak kebelakangan ini. Salah satu contoh sisa buangan ialah dari air kolam udang. Tahap bahan toksik yang tinggi seperti nitrogen dan fosforus menyebabkan berlakunya pencemaran terhadap alam sekitar. Oleh sebab itu, air kolam udang telah dirawat dengan penggunaan Rhizopus oligosporus untuk

membantu merawat bahan organik yang terdapat dalam substrat tersebut menggunakan cara fermentasi

substrat cecair. Berdasarkan kajian ini, jumlah pengudaraan yang berbeza telah digunakan untuk mengkaji pertumbuhan R. oligosporus. Parameter yang berbeza termasuklah 0.00 vvm, 0.50 vvm, 0.75 vvm dan 1.00

vvm tahap pengudaraan. 1.00 vvm pengudaraan telah menghasilkan biomas yang tertinggi iaitu 4.60 g/L yang diikuti oleh 0.75 vvm, 0.50 vvm dan 0.00 vvm. Pengudaraan yang tertinggi mempunyai produksi biomas yang tertinggi kerana mikroorganisma dapat membantu pertumbuhan dengan tahap oksigen yang mencukupi. Antara analisis yang digunakan dalam kajian ini ialah fenol-suNrik, nitrat dan nitrit. Bagi analisis fenol-

sulfurik, penggudaraan 1.00 vvm telah mencatatkan tahap reduksi yang tinngi sebanyak 93.6% berbanding dengan tiga parameter yang lain. Selain itu, kedua-dua analisis nitrat dan nitritjuga menunjukan bahawa 1.00

vvm mempunyai tahap konsentrasi yang rendah iaitu 0.52 mg/L dan 0.000 mg/L. Hal ini membuktikan bahawa R. oligosporus berpotensi untuk merawat air kolam udang selepas beberapa hari bertindakbalas dengan

substrat. Selain itu, biomas kulat berprotein tinggi (HPFB) yang dihasilkan dapat digunakan sebagai bahan

mentah makanan haiwan ekoran daripada permintaan yang tinggi dalam industri pemakanan di peringkai

global selain mengurangkan pencemaraan alam sekitar.

Kata kunci: Rhizopus oligosporus, air kolam udang, pengudaraan, fermentasi substrat cecari, biomas kulat berprotein tinggi (HPFB),

1

CHAPTER 1

INTRODUCTION

1.1 Introduction

Shrimp aquaculture is one of Malaysian's important agricultural as the industry is highly in

demand both national and international market (Ismail and Abdullah, 2013). Many coastal

areas are now developed into aquaculture ponds due to the increase of protein needs and

also act as an alternative for reducing landing of captured shrimp (Nyanti et al., 2011).

According to Ghee-Thean et al. (2016), most consumers preferred white meat such as

shrimp and fish products compared to red meat which is ruminant-based products. The

Ministry of Agriculture Malaysia (2003) stated that the Malaysian government has taken an

initiative to promote the brackish water shrimp culture with a target of RM 4.3 billion of

shrimp production in year 2010 (Islam et al., 2014).

Based on Table 1, it shows the statistics of white shrimp production from year 2002 until

year 2012. The number of white shrimp produced increased gradually from year 2002 to

year 2010. Meanwhile, it started to show a decreased from year 2011 onwards.

Table 1 Annual Malaysian white shrimp production. (Source Annual Fisheries Statistics, 2002-2012. )

Year White shrimp Pond size Productivity Productivity (metric tons) (ha) (metric (metric

tons/ha/year) tons/ha/year 2002 844.46 246.89 3.420 1.710 2003 803.59 192.21 4.181 2.091 2004 5117.49 1201.59 4.259 2.130 2005 11497.80 2391.37 4.808 2.404 2006 18600.59 2733.71 6.804 3.402 2007 23737.40 4808.63 4.936 2.468 2008 37544.31 4691.23 8.003 4.002 2009 52926.42 4823.63 10.972 5.486 2010 69084.10 5132.85 13.459 6.730 2011 60322.01 5828.71 10.349 5.175 2012 48991.81 5349.17 9.159 4.580

2

However, the shrimp aquaculture also has its own consequences as increase in market

demands brought up negative impacts especially towards the environments (Nyanti et al.,

2011). Paez-Osuna et al. (2003) and Primavera (2006) both stated that the negative impacts

include the damage on coastal ecosystem followed by reduce amount of fisheries products

and increase in surrounding pollutions caused by discharged of shrimp ponds.

Shrimp aquaculture creates many negative impacts due to illegal pond sediment disposal,

pond drainage during the harvesting and also large loading of oxygen-consuming organic

matters (Dierberg et al., 1996). The development of shrimp agriculture also lead to shortage

of clean water supply in which becomes a crucial matter as the surrounding becomes acidic

due to release of heavy metals from the sediments in shrimp pond water (Kautsky et al.,

2000).

On the contrary, few studies had been done to treat any unwanted disposal of agricultural

wastes. One of the ways is by using fungi treatment. Lennartson et al. (2014), mentioned

that the Rhizopus genus has also been investigated for treatment of industrial wastewater.

The wastewaters such as shrimp pond water could be used as organic sources and aid in the

production of animal feed. Anupama and Ravindra (2000), stated that the bioconvension of

agricultural and industrial wastes to a rich protein food has been giving a positive results in

producing a cheaper product and yet reducing wastes pollutions.

Since 500 years ago, the Rhizopus genus has been used in full-scale industrial applications

especially in the warm and moist Southern Asia compared to colder Northern Europe

(Lennartson et al., 2014). One long-time species in use is the fungi Rhizopus oligosporus in

tempeh, a dish from fermented soybeans (Jennessen et al., 2008).

3

According to Mitra et al. (2012) and Rasmussen et al. (2014), R. oligosporus can be used

for the removal of organic compounds from thin stillage. Fungal cultivation also assist in the

recycling of water and to reduce water evaporation that is low in energy cost. The fungi that

have been growing in thin stillage can then be harvested, dried and used as animal feed

(Nitayavardhana et al., 2013).

Mitra et al. (2012) and Ramussen et al. (2014) also stated that thin stillage acts in providing

nutrients such as organic compounds and micronutrients that makes it an ideal feed stock for

fungal cultivation such as lactic acid. Animal feed is important in the global food industry

in order to increase products which originated from animals throughout the world.

Moreover, Jaganmohan et al. (2013) stated that research on high protein fungal biomass

(HPFB) production in animal feed has been done due to shortage of essential amino acids

for synthesizing of protein in animals in the early days. Increasing of protein synthesis can

help in providing nutrients and enhance feeding efficiency for the animal. Submerged

fermentation is one of the methods that can help in production of HPFB. It can aid in the

agricultural industry in terms of animal feed. At the end of the submerged fermentation

process, it also can help in reducing the production cost (Zhang et al., 2008).

In this research, Rhizopus oligosporus was used and grown in shrimp pond water by using

submerged fermentation method to treat and reduce organic matters present in shrimp pond

at the same time producing HPFB.

4

t'usat Khidmat Nlaklumat Akademik UNIVERSITt MALAYSIA SARAWAK

The specific objectives of this research are:

1. To determine the effects of different rate of aeration on R. oligosporus growth.

2. To investigate the effectiveness of shrimp pond water treatment using R. oligosporus.

3. To identify the optimal aeration rate for growth of R. oligosporus in shrimp pond water.

5

1.2 Problem Statement

There are many organic materials that are present in shrimp pond water that leads to

contamination of water. Due to this matter, this research is conducted in order to remove this

unwanted materials from shrimp pond water. R. oligosporus was used to reduce

contaminating substances and produced HPFB to be used as animal feed. We investigated

the growth rate of R. oligosporus for the production high protein fungal biomass (HPFB) in

shrimp pond water that acts as medium to provide nourishment for the R. oligosporus at

different levels of aeration.

6

CHAPTER 2

LITERATURE REVIEW

2.1 Rhizopus oligosporus

Rhizopus oligosporus belongs to Rhizopus n icrosporus group that associated with food

fermentation, pathogenesis or unwanted metabolite production like rhizoning and

rhizotoxins (Jennessen et al., 2008). Jennessen et al. (2008) stated that R. oligosporus has a

higher defect in spore formation process with a high proportion of 10-31% larger and

irregular spores compared to the other natural Rhizopus taxa. Their optimum growth are at

42 °C and pH of 4 (Medwid et al., 1984). R. oligosporus is one of the fungal species that

have the ability to produce several of proteases enzymes compared to bacteria. This cause

the R. oligosporus been used widely in the fermentation of vegetal substrates (Kovac el al.,

1996). For example, it is traditionally used in producing tempeh which is a fermented food

based on soybeans.

Figure 1 Rhizopus o/igosporus viewed under light microscope. (Magnification: I000X)

7

According to Hesseltine (1965), R. oligosporus is the main principle mold responsible

species for fermentation of soybeans. Handoyo et al. (2006) stated that R. oligosporus in

cultured soybeans are hydrolyse protein to amino acids and peptides by secreting several

proteolytic enzymes. Meanwhile, the oligosaccharides of Rhizopus will be hydrolysed to

monosaccharides. As for the phytic acid, it will be degraded into inorganic phosphate in the

process of cultured soybeans. The synthesis of the enzymes hydrolysed contribute to the

development of desirable texture, flavour and aroma of the product (Kovac et al., 1996).

Other than being used to ferment other substrates, it can also treat waste material (Jennessen

et al., 2008).

8

2.2 Shrimp pond water

Globally, shrimp agriculture has been developing steadily over the last few decades in

response to the increasing of the world market demand. The production system evolved from

extensive toward intensive with increasing inputs of high quality feed and water supply (Lin

ei al., 1995).

Figure 2 Shrimp pond located at Kuala Santubong, Kuching.

Nyanti et al. (2011), stated that the shrimp agricultural is one of the industries that are

developing to the creation of Aquaculture Industrial Zone in Malaysia. Nevertheless, the

increase in waste loads of uneaten feed and metabolic wastes from culture ponds, is causing

the surface and subsurface salinization of freshwater and the loading of solids which also

includes any oxygen-consuming organic matter (Dierberg el al., 1996).

9

Dierberg et al. (1996) also stated that between 1979 until 1993, about 16 to 32 % of total

mangrove area were destroyed due to shrimp agriculture. Based on Thakur et al. (2003), in

a traditional intensive shrimp culture, the deteriorated pond water is frequently exchange

with new water supply in order to maintain a desirable water quality of shrimp growth.

However, nitrogen wastes like ammonia and nitrite are produced in the system in which

exceed the assimilating capacity of receiving waters. This leads to the deterioration of water

quality that caused toxic environment for the shrimp growth. Dierberg el al. (1996) reported

that the abandonment of shrimp pond water caused the reductions of productivity of the

), in a closed system pond year by year. Nonetheless, in the study done by Thakur et al. (200)

nutrient, nitrogen and phosphorus accumulated within the system could be used to support

the growth of natural food organisms for shrimp growth.

Figure 3 Shrimp pond farm.

10

2.3 Submerged Fermentation (SmF)

Submerged fermentation (SmF) is one of a process which is used in maximizing the

productivity of microorganisms such as bacteria and fungi (Subramaniyam et al., 2012). In

submerged fermentation free-flowing liquid substrates is usually been utilized. Due to the

rapid utilization of nutrients, it also requires rapid supplementation (Mienda el al., 2011).

Vidyalakshmi et al. (2009), mentioned that a submerged culturing is an advantage because

it helps in easing of sterilization and in controlling the process system. According to Chisti

et al. (1999), submerged fermentation can be divided into oxygen requiring aerobic

processes and also in anaerobic processes which in absence of oxygen. He also stated that in

submerged fermentation systems, it can be carried out either as batch-wise, fed-batch

operations or continuous cultures.

0.45 µm syringe filter

Air pump

II

J

Peristaltic pump

Silicone Tubes

Shrimp pond water + Treatment

Air Stone

Figure 4 Set-up of submerged fermentation.

11

For batch processing, a batch is inoculated in a fermenter with microbes for certain amount

of time for product to be harvested. As for fed-batch fermentation, culture medium will be

added periodically to the inoculated fermentation batch. Product is then harvested after batch

period (Chisti et al., 1999).

Meanwhile, for continuous fermentation, sterile medium is fed continuously into the

fermenter and product will then withdraw continuously so that the fermentation volume

remain unchanged (Chisti et al., 1999). It is also said that solid-state fermentation offers

some advantages over submerged fermentation. Subramaniyam et al. (2012), mentioned that

SmF is usually used for the extraction of secondary metabolites that required in liquid form.

12

2.4 High Protein Fungal Biomass (HPFB)

Reaction between carbon dioxide (C02) in the water, air and sunlight via photosynthesis in

plant material produce carbohydrates which is the building blocks of biomass (McKendry,

2002) High protein fungal biomass are substances that contain a high concentration of

protein with essential amino acids for both human and animal (Moore et al., 2001).

According to Jennessen el al. (2008), R. oligosporus has been used to produce single cell

protein due to its ability to utilize carbon sources and produce a high protein yield. Other

than that, it also aid in the production of animal feedstock. As protein is one of the most

essential components needed for the growth and development of living things, the single cell

protein (SCP) provides an alternative yet convenience way for consumption (Suman et

a1., 2015).

Figure 5 Dry biomass obtained after accumulation process.

13

production of high protein fungal biomass (hpfb) in … of high... · declaration i hereby declare...

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