evaluation the combination between bacteria isolated from gastric juice of goat and cow rumen for sugarcane bagasse hydrolysys in vitro

MINISTRY OF EDUCATION & TRAINING CAN THO UNIVERSITY BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE SUMMARY BACHELOR OF SCIENCE THESIS THE ADVANCED PROGRAM IN BIOTECHNOLOGY EVALUATION

MINISTRY OF EDUCATION & TRAINING

CAN THO UNIVERSITY

BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE

SUMMARY BACHELOR OF SCIENCE THESIS

THE ADVANCED PROGRAM IN BIOTECHNOLOGY

EVALUATION THE COMBINATION

BETWEEN BACTERIA ISOLATED FROM

GASTRIC JUICE OF GOAT AND COW RUMEN

FOR SUGARCANE BAGASSE

HYDROLYSYS IN VITRO

SUPERVISOR STUDENT

Ass.Prof Dr TRAN NHAN DUNG Session: 34 (2008- 2013)

Can Tho, 2013

APPROVAL

Dr HO QUANG DO

Can Tho, May 2013

PRESIDENT OF EXAMINATION COMMITTEE

ABSTRACT

Thesis " Evaluation The Combination Between Bacteria Isolated From Gastric Juice Of Goat And Cow Rumen For Sugarcane Bagasse Hydrolysys In Vitro” was done to increase the degradation of bagasse in in vitro conditions Four bacteria strains of goat rumen, including DD9, DD5, DD7 and DD13, had been working together to investigate the cellulase activity With halo zone diameter 27mm and degraded DM rate 11.13%, the combination between DD9 and DD7 was the most effective treatment that was selected to make post experiments Sequencing results showed that the line DD9 similar to the max identity of 94% with Bacillus subtilis RC24 and the 92% max idetnity with Bacillus subtilis BA3_1A was the result of bacterial strain DD7 In vitro, the combination of three lines of bovine rumen bacteria (BM13, BM21 and BM49) and line DD9 in 1:3 ratios for the best output with DM digestibility was 21.27 %, cellulose and hemicelluloses were 8.17% and 10.22%, respectively

Keywords: Bacilus subtilis, hydrolysis, in vitro, rumen bacteria, sugarcane bagasse

CONTENTS

Page

ABSTRACT ii

CONTENTS iii

1 INTRODUCTION 1

2 MATERIALS AND METHODS 3

2.1 Materials 3

2.2 Methods 4

2.2.1 Studying on the components of sugarcane bagasse… 4

2.2.2 Screening of combination of 4 rumen bacteria strains isolated from gastric juice of goat 6

2.2.3 Identification of bacteria by PCR method 7

2.2.4 Studying on a combination between ruminal bacteria isolated from gastric juice of goat and cow in vitro 10

2.3 Experiment Design and Statistical Analysis 12

3 RESULTS AND DISCUSSIONS 13

3.1 Studying on the components of sugarcane bagasse 13

3.2 Screening of combination of 4 rumen bacteria strains isolated from gastric juice of goat 14

3.3 Identification of bacteria by PCR method 17

3.3.1 Strain DD9 17

3.3.2 Strain DD7 18

3.4 Combination of ruminal bacteria isolated from goat and

cow in vitro condition 20

3.4.1 Dry matter content 20

3.4.2 Cellulose content 21

3.4.3 Hemicellulose content 23

3.4.4 Lignin content 25

4 CONCLUSIONS AND SUGGESTIONS 27

4.1 Conclusions 27

4.2 Suggestions 27

1 INTRODUCTION

The demands of animal protein in people diet lead to development of the animal husbandry Based on industrial modernization, many households have invested large scale of infrastructure in rural areas Feeds are mainly base on natural grasses and crop residues The more the sufficient nutrients in each diet are supplied quantity and quality, the more the livestock productivity will increase To improve production in each household, many various methods for improvement of feed utilization have been studied for several decades (Jackson, 1978)

In recent years, there has been an increasing trend towards more efficient utilization of agro-industrial by products, such as sugarcane bagasse, as raw material for new industrial applications Bagasse waste has been used in large quantities by the sugar and ethanol industries, mainly as fuel for sugar mills However, the remained bagasse is still pollutant to the environment Thus, a suitable mean for treatment of this residue

is an important objective to be pursued

Using microorganisms for enzymatic treatment of sugarcane bagasse is one of prospective strategies A large amount of cellulase enzyme can be produced by ruminal bacteria In 2012, TH Truemilk factory applied successfully using some kind of by-products of agricultural to feed beefs Breeding cows by sugarcane bagasse can be applied in some countries such as Japan and Korea Goats are ruminants with cows but there have many more outstanding features than Goats

have the ability to adapt to the harsh conditions of food and climate One of the reasons for that is the activity of microorganisms in the rumen of goats (Ho Quang Do and Nguyen Thu Thuy, 2013) To help beef enhance nutrient absorption from food, the thesis was conducted to study the possibility of bagasse degradation of goat rumen bacteria

combined with cow rumen bacteria in vitro conditions

2 MATERIALS AND METHODS

2.1 Materials

Equipments: water bath, incucell incubator (Germany),

Velp Raw Fiber Extractor (Italia), Orion 420A pH meter (USA), Pbi-international autoclave (Germany), Rotary shaker GFL 3005 (Germany), Sequencer ABI 3130 (USA)

Chemicals: Ethanol 95%, sulfuric acid H2SO4 98%, NaOH, acetone, sodium sulphite anhydrous (Na2SO3), n-octanol (C8H18O), disodium ethylenediaminetetraacetate (EDTA,

C10H14N2Na2O8), sodium lauryl sulfate neutral (C12H25NaO4S), cetryltrimethylammonium bromide technical grade (C19H42BrN), Tris-HCl (pH8), Isopropanol, Proteinase K …

Sample

Sugarcane bagasse: after collecting from Hau Giang

Sugar Factory, sugarcane bagasse were desiccated at 70ºC in 30-45 minutes, then ground by Reetsch Miill with holes 0.2mm

Microorganism: Three bacteria including BM13,

BM21 and BM49 were isolated from rumen of cow (Do Thi Cam Huong, 2012); and four ruminal bacteria species isolated from goat: DD9, DD5, DD7 and DD13 (Laboratory of Enzyme Technology)

Ruminal fluid of cow: collected at the farm, located on

Phu Thanh Town, Tra On District, Vinh Long Province

2.2 Methods

2.2.1 Studying on the components of sugarcane bagasse

Objective: to evaluate general of composition of

sugarcane bagasse

a) Dry matter content:

Dried crucibles for weight balance at 70ºC Tarred and

recorded the weight of each crucible

Added 2g sample into each crucible Put these crucibles

with lids into preheated oven at 70ºC Tarred and recorded the

weight of each crucible

Calculated dry matter (DM) content:

weight of residue – weight of crucible

b) Hemicellulose content (Neutral Detergent Fiber-

NDF, and Acid Detergent Fiber-ADF)

Acid Detergent Fiber-ADF

Added 100ml of acid detergent solution with 0.5 grams

of sample, and some drops of n-octanol Heated and refluxed for

60 minutes

Filtered and washed 3 times with boiling water, then

twice with acetone

Dried at 70ºC and weigh them

Calculated acid detergent fiber (ADF)

weight of residue

weight of sample (Van Soest and Robertson, 1979)

Neutral Detergent Fiber- NDF

Added 100ml of neutral detergent solution with 0.5grams of sample, 0.5 grams of sodium sulfite and some drops

of n-octanol Heated and refluxed for 60 minutes

Filtered and washed 3 times with boiling water, then twice with acetone

Dried at 70ºC and weigh them

Calculated neutral detergent fiber (NDF):

weight of residue

weight of sample (Van Soest and Wine, 1967)

Hemicellulose content

% Hemicellulose = %NDF- %ADF

(Van Soest and Robertson, 1979)

c) Cellulose and Lignin content

Acid Detergent Lignin-ADL

Used the residue of acid detergent fiber determination Added 25ml of 72% sulfuric acid at room temperature during 3 hours, stirring every hour

Filtered and washed 3 times with boiling water

Dried at 70ºC and weigh them

Calculated acid detergent lignin:

% Cellulose = %ADF - %ADL

(Van Soest and Robertson, 1979)

2.2.2 Screening of combination of 4 goat rumen bacteria strains

Objective: evaluate the sugarcane bagasse degradation

ability of mixing of 4 goat rumen bacteria

Procedure:

a) Studying the sugarcane bagasse degradation productivity

- Injection of 5% (w/w) bacteria suspension with

107CFU/ml concentration based on experiment setting

- Bacteria were incubated in 3 days at 38ºC

- Dried the bagasse and weigh the mass

- Formula to definite the degradation output

Degradation output (%) = % DMmaterial - %DMtreatment

b) Studying the creating of halo zones in M1 medium

- 15µl bacteria solution of each treatment was spotted in the well with 5mm diameter on M1 medium

- The plates were incubated at 38ºC for 24 hours

- Cellulase activity was detected by staining the plates with Congo Red dye (0.1g/l) for 30 minutes, then washing with NaCl 1M solution (Teather và Wood, 1981; Wood và Bhat, 1988)

- Measure the halo zone diameter (mm)

2.2.3 Identification of bacteria by PCR method

Objective: To indentify of DNA sequences of bacterial

strains isolated from the rumen of goat

Procedure:

A protocol for extracting genomic DNA from bacterial cells was based on the CTAB method from Maniatis et al (1989) This protocol was followed:

- Bacteria were bred in 6ml LB medium for 16 hours

- Transferred 2ml to a 2.2ml centrifuge tube and centrifuged tubes at 13,000rpm for 10 minutes

- Decanted the supernatant in the tube

- Resuspended the pellet in 250μl TE 1X buffer by repeated pipetting

- Added 250μl of 1X TE buffer, 50 μl of 10% SDS and 5μl of ProteinaseK, mixed well (but gentle), and incubated 20 minutes in a water bath at 65°C

- Added 400μl of 10% CTAB, gently mix, and incubated at 65oC for 20 minutes

- Added 600μl Chloroform:Isoamyl alcohol (24:1), mixed well (but gentle), and centrifuged 13.000rpm for 10 minutes Transferred the upper aqueous phase to new tube (avoid interface)

- Added 600μl of isopropanol, mixed well (but gentle), and freezed cell suspension at -20ºC for 2 hours

- Washed DNA by 50μl of ethanol, centrifuged at 13,000rpm for 10 minutes

- Discarded the supernatant and dried the pellet either at

45ºC by a vacuum drier Dissolved DNA in 100μl 0.1X TE buffer Stored at -20ºC for further use

Table 1 The chemical components in the PCR reaction

(*Source: Maniatis et al., 1989)

Figure 1 The cycle of PCR reaction

(*Source: Maniatis et al., 1989)

DNA sequencing

- The PCR product was sequenced by the ABI3130

genetic analyzer, an automated DNA sequencing The sequence was identified by using the BLASTN program and comparing

the gene sequences of other bacterial strains on GenBank, in order to show the level of similarity matrix of strains

Screening the morphology of bacteria

- The bacterial colonies were ensuring by the growing pattern of bacteria on the plate and under microscopic observation

- Gram staining was tested following Cao Ngoc Điep and Nguyen Huu Hiep (2008)

2.2.4 Studying on combination ability of ruminal bacteria

isolated from goat and cow in vitro

Objective: To evaluate generally cellulose degradability of combination of ruminal bacteria isolated from

goat and cow in in vitro

Procedure:

- Preparation of ruminal fluid (cow): ruminal fluid was

collected through a permanent fistula of cow before the

breakfast The cows were fed with 100% para grass (Brachiaria

mutica) before 1 month The fluid was strained through four

layers of muslin cloth The carbon dioxide was dissolved in a solution The solution was kept in an incubator at 38oC

- Preparing buffer: added component of buffer solution

(Table 5) into round flat-bottomed flask The carbon dioxide gently bubbled into the solution until the liquid turn from opaque to almost transparent After 15minutes of warming in an incubator 38oC, the ruminal fluid and buffer solution were mixing

Table 2.The compositions of buffer in vitro

(*Source: Tilley and Terry, 1963)

- All experiments were arranged completely random

with 1 negative control, 1 positive control and 7 treatments

With an in vitro experiment (Menke and Steingass, 1988), the

components of each treatment were followed:

Table 3 Components of treatments

Component

(ml) DC- DC+ NT1 NT2 NT3 NT4 NT5 NT6 NT7

Ruminal Fluid 40 0 40 40 40 40 40 40 40 Suspension

DD7 (v/v) 0 0 0 0 6 3 0 9 4,5 Buffer 160 186 148 148 148 148 148 148 148

- Poured buffer solution and ruminal fluid into dark

bottle, then added sugarcane bagasse and bacteria Subsequently, clays were used to seal lip and mouth bottles which were put in incubator at 38oC After incubating 3 days, the samples (sugarcane bagasse) were collected and tested their components

Determination: content of the ingredients of the

samples in experiments: DM, cellulose and hemicelluloses

2.3 Experiment Design and Statistical Analysis

Raw data were analysed by Microsoft Excel 2007 and Statgraphic XV Software DNA sequencing was supported by BLASTN software All experiments were performed in triplicates

3 RESULTS AND DISCUSSIONS

3.1 Studying on the components of sugarcane bagasse

According to the Table 4, dry matter of bagasse was 98.41% after drying The result was similar to both the dry matter given in the research of Shakweer (2003) with 94.57% and the value of studying on bagasse of Do Thi Cam Huong (2012) with 93.69%

The cellulose content achieved 50.61% This result was likely to the result of Lu Vu Thao Vi (2012) with 56.32% cellulose However, the result was different with the research of Hamissa et al (1985) with 48,1% Sallam et al (2007) had the result of cellulose content about 36.5%

Hemicelluloses content was calculated from NDF and ADF value With Van Soest method, hemicellulose content contained 29.81% in bagasse According to researches of Lee and Koo (2003) and Akinfemi (2012), the hemicellulose contents were 24% and 15.98%, respectively

0.5grams of sugarcane bagasse were analysed by acid detergent solution Lignin content was 14.63% The results of Feng Peng et al (2009) with 20-30% of lignin percent in sugarcane According to Ilindra and Dhake (2008) with 21.1% lignin content, it was different with this lignin content of the bagasse

Table 4 The components of sugarcane bagasse

% DM

(m/m)

% Cellulose (m/m)

% Hemicelluloses (m/m)

% Lignin (m/m)

Due to analysis of fiber components in bagasse and comparison with other materials, the result was determined high cellulose and hemicelluloses, low lignin Therefore, sugarcane bagasse was an appropriate material to culture bacteria synthesized cellulose and by-products in breeding

3.2 Screening of combination of 4 goat rumen bacteria strains

Objective: To evaluate generally cellulase activities of

bacteria combination

The linear equation between optimal density at 600nm length wave and bacterial concentration of 4 goat rumen bacteria

Strain DD9: y = 0,823x + 5,505 R2 = 0,987 Strain DD5: y = 1,008x + 5,247 R2 = 0,987 Strain DD7: y = 3,362x + 4,714 R2 = 0,996 Strain DD13: y = 0,238x + 6,374 R2 = 0,995 Due to above equations, bacteria were isolated at

107CFU/ml content

a) The results of sugarcane bagasse degradation of 15 treatments by halo diameter method were shown in Figure 2