Maximization of ethanol yield and adsorption of heavy metal ions by fruit peels

Group: 1-124

Leong Qi Dong 4S216Soh Han Wei 4I324Aman Mangalmurti AOSKara Newman AOS

Introduction

Depletion of non-renewable fossil

fuels

Heavy metal water

contamination of water is rampant

in many countries

Heavy metal ions accumulate

inside organisms and affect the

ecosystem

Introduction

Conversion to biofuel,

ensuring continual

energy supply

Biosorption in removal of

heavy metal ions by fruit peel wastes

Fruit peel waste

Introduction: Zymomonas mobilis

Ethanol Fermentation

Shorter fermentation time

(300%-400% faster than yeast)

higher ethanol yield (92%-94% versus 88%-90%

for yeast)

Microorganism used:

Zymomonas mobilis

Why Z. mobilis?

Nguyen, T., and Glassner, D. (2001 )

Objectives

To investigate the production of ethanol from fruit peels

To investigate the efficiency of adsorption of heavy metal ions by fruit

peelsTo determine the procedure which

maximises ethanol yield and efficiency of heavy metal ion

adsorption

Hypotheses Mango peels contain reducing sugars

that can be fermented to ethanol. Mango peels show different

efficiency levels of in the adsorption of copper, zinc and lead ions.

Experimental OutlinePreparation of fruit peel extract, microbe, heavy metal solution

Extraction of sugars

Ethanol Fermentation

Residue for Adsorption of Ions

Adsorption of Ions

Extraction of sugars

Ethanol Fermentation

• Mango Peels

Fruit

tested

• Pb2+

• Cu2+

• Zn2+

Ions

tested

Variables

Independent

•Heavy metal ion (Pb2+, Cu2+, Zn2+)•Sequence of procedures

Dependent•Initial concentration of reducing sugars in fruit peel extracts•Ratio of ethanol yield to sugar concentration•Amount of ethanol per g of fruit peel•Final concentration of heavy metal ions

Controlled•Mass of fruit peel used•Type of microorganism used (Z. mobilis)•Duration and temperature of fermentation

Apparatus

Centrifuge Centrifuge tube Spectrophotometer Glass rod Sieve Blender Dry blender Shaking incubator Oven Incubator Weighing Balance

Materials Zymomonas mobilis Glucose-yeast medium Sodium alginate Calcium chloride solution Sodium chloride solution Fruit peel Cuvettes Deionised water Dinitrosalicylic acid Acidified potassium chromate

solution Lead (II), Copper (II), Zinc (II) ion

solutions Copper (II) and Zinc (II) reagent kits

Ethanol FermentationPreparation of Z. mobilis, Extraction of Sugars, Fermentation, Determination of Yield

Methods

Growth of Z. mobilis

Immobilisation of cellsExtraction of sugars from fruit peelsEthanol fermentation by immobilized Z. mobilis cellsDetermination of ethanol yield with the dichromate test

Ethanol fermentation

Growth of Z. mobilis

Z. mobilis cells were inoculated in 20 ml GY medium (2% glucose, 0.5% yeast extract)

Incubated at 30°C for 2 days with shaking for growth to occur

Immobilisation of cells

Culture was centrifuged at 7000 rpm for 10 min

Cell pellets were

resuspended in 7.5 ml GY

medium.

Absorbance of the

cultures were taken at 600

nm.

7.5 ml of 2% sodium

alginate is added to the

cells.

Added dropwise to

0.1 M calcium chloride

solution to form beads.

Beads were rinsed in 0.85% sodium chloride solution.

Extraction of sugars from fruit peels

40 g of fruit peels were blended in 400 ml of deionised

water using a blender.

The extract was passed through a sieve to

remove the residue.

Suspension was

centrifuged at 7000rpm. Supernatant and residue

were collected.

Ethanol fermentation by immobilized Z. mobilis cells

200 Z. mobilis beads were added to 50

ml mango peel extract.

Control : 200 empty beads

were added to the same volume of

mango peel extract.

Set-ups were incubated with

shaking at 30°C for 2 days for ethanol

fermentation.

Beads were removed and the extracts

are distilled to obtain

ethanol.

Determination of ethanol yield with the dichromate test

2.5 ml of acidified potassium dichromate solution was added to 0.5 ml of distillate.

Samples were placed in a boiling water bath for 15 min.

Absorbance was measured at 590 nm using a spectrophotometer

Concentration of ethanol was read from an ethanol standard curve.

Adsorption of heavy metal ionsDessication of peel, Preparation of ion solution, Adsorption, Determination of final ion concentration

Preparation of Fruit Peel

Fruit peel residue was dried in an oven.

0.2g fruit peel powder was added to 10ml 50ppm of each ion

solution (test). No peel

was added in the control set-up.

Adsorption and Determination of final ion concentration

Mixtures were placed on a

rocker for 24 h at room

temperature.

Peel powder was removed

by centrifugation.

Using reagent kits for copper (II) and zinc(II)

and AAS for lead (II) the

final concentration

of ions in solution was determined.

Data analysis

•µmol of ethanol per g of fruit peelEthanol yield

•% of heavy metal ions adsorbed•(Final-Initial)/Initial x 100%•t-test to determine if difference between the control and the test is significant

Heavy metal ion

adsorption efficiency

Experimental Results

Maltose standard curve

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.50.0

0.5

1.0

1.5

2.0

2.5f(x) = 0.810149142857141 x − 0.0690952380952379R² = 0.996917803558814

Maltose standard curve

Maltose concentration / µmol/ml

Abso

rban

ce a

t 530

nm

Ethanol standard curve

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.60.00.10.20.30.40.50.60.70.80.91.0

f(x) = 0.639193642178717 x − 0.00343050978871884R² = 0.999628260592362

Ethanol standard curve

Concentration of ethanol / %

Abso

rban

ce a

t 590

nm

First sequenceSugar Extraction First, Ethanol Fermentation, Ion Adsorption

Dichromate test to determine ethanol concentration

1 20.19

0.20

0.21

0.196

0.205Ethanol yield from mango peels

Conc

entra

tion

of e

than

ol /

%

Ethanol / g of initial fruit peel

1 20

50

100

150

200

67.0 70.4

µmol of ethanol per g hydrated fruit peel

Round number

conc

entr

atio

n of

eth

anol

µm

ol /g

Adsorption of ions (1st round)

Copper Zinc Lead0

10203040506070

46.5

63.2

36.2

9.4

26.3

1.9

Adsorption of ions by mango peels (1st round)

Control without peelsTest with mango peels

Fina

l con

cent

ratio

n of

ions

/ pp

m

Adsorption of ions (2nd round)

Copper Zinc Lead0

10

20

30

40

50

60

45.953.8

32.5

4.4

15.8

0.5

Adsorption of ions by mango peels (2nd round)

Control without peels

Test with mango peels

Fina

l con

cent

ratio

n of

ions

/ pp

m

t-test analysis

Ion t-test p value to show difference

between control and test Round 1 Round 2

Copper 0.000399 0.000229Zinc 0.00037 0.00101Lead 0.00001 0.000405

All differences were significant as p < 0.05

Adsorption of ions (Summary)

Copper Zinc Lead0

20

40

60

80

100

120

85.164.5

96.6

A comparison of the efficiency of adsorption of ions by mango peels

Ion

adso

rbed

by

man

go

peel

s / %

Second sequence Ion Adsorption First, Sugar Extraction, Ethanol Fermentation

Adsorption of ions (1st round)

Copper Zinc Lead0

10

20

30

40

50

6048.6 51.2

55.3

15.0

33.3

4.3

Adsorption of ions by mango peels (1st round)

Control without peels

Test with mango peels

Fina

l con

cent

ratio

n of

ions

/ pp

m

Adsorption of ions (2nd round)

Copper Zinc Lead0

10

20

30

40

50

6050.4 51.6

32.4

19.8

31.7

5.8

Adsorption of ions by mango peels (2nd round)

Control without peelsTest with mango peels

Fina

l con

cent

ratio

n of

ions

/ pp

m

Adsorption of ions (3rd round)

Copper Zinc Lead0

10

20

30

40

50

60

49.0 51.2

31.9

15.1

24.4

7.2

Adsorption of ions by mango peels (3rd round)

Control without peelsTest with mango peels

Fina

l con

cent

ratio

n of

ions

/ pp

m

t-test analysis

Ion t-test p value to show difference

between control and test Round 1 Round 2 Round 3

Copper 0.0000000723 0.000485 0.000000139

Zinc 0.000542 0.002072 0.000619Lead 0.00000471 0.004109 0.00000225

All differences were significant as p < 0.05

Adsorption of ions (Summary)

Copper Zinc Lead0

102030405060708090

100

64.9

36.7

87.2

A comparison of the efficiency of adsorption of ions by mango peels

Ion

adso

rbed

by

man

go p

eels

/ %

Ethanol / g of initial fruit peel

1 2 30.0

50.0

100.0

150.0

200.0

63.8

150.8

45.5

µmol of ethanol per g hydrated fruit peel

Round number

conc

entr

atio

n of

eth

anol

µm

ol /g

Summary

Summary: Ethanol yield

1 20

50

100

150

200

67.0 70.4

µmol of ethanol per g hydrated

fruit peel

Round number

conc

entr

atio

n of

eth

anol

µm

ol

/g

1 2 30.0

50.0

100.0

150.0

200.0

63.8

150.8

45.5

µmol of ethanol per g hydrated fruit peel

Round numberconc

entr

atio

n of

eth

anol

µm

ol

/g

Ethanol fermentation 1st Ion adsorption 1st

Yield of ethanol with different sequence of procedures

Ethanol yieldExtraction of sugars first

Adsorption of ions first

Round 1

Round 2

Round 1

Round 2

Round 3

Mean ethanol concentration / %

0.196 0.205 0.062 0.227 0.070922

Total amount of ethanol / µmol

2679.2 2815.2 2553.4 6031.8 1818.2

Amount of ethanol / µmol per g of fruit peel

66.98 70.38 63.8 150.8 45.5Sequence 2 (Adsorption of ions followed by extraction of sugars) resulted in a higher yield of ethanol on average

Adsorption of ions with different sequence of procedures

Metal ion

Mean % of ions adsorbed

Extraction of sugars

first

Adsorption of ions

firstCopper ion adsorbed / % 85.1 64.9Zinc ion adsorbed / % 64.5 36.7Lead ions adsorbed / % 96.6 87.2Sequence 1 (Extraction of sugars followed by adsorption of ions) resulted in higher efficiency of adsorption of ions

Fourier transform infrared spectroscopy analysis of mango peel

O-H stretch

C-H stretch

FTIR analysis of mango peel after copper ion adsorption

Some changes in the 1000-1800cm-1

wavenumbers

FTIR analysis of mango peel after zinc ion adsorption

FTIR analysis of mango peel after lead ion adsorption

weakerC-H stretch

Adsorption of ions has resulted in changes in FTIR spectra

No significant change in the strength of O-H stretching

Weaker C-H stretch after lead ion adsorption Stretching of more bonds in between 1800-

1000 cm-1 after all three ion adsorption We believe that the carboxylic acid, ester and

lactone (1700cm-1) and alkene groups (1600cm-1) are responsible for adsorption.

Summary of FTIR analysis

Difficulty in standardising batch of mango peels for all tests performed May yield inconsistent results for each

repeat

Limitations

Applications

Cost-effective method of producing ethanol

Reduces reliance on

non-renewable fossil fuels

Using by-product waste

Low cost, viable method in wastewater

treatment

Further Work

Investigate the effect of pH of ion solution on adsorption

Investigate the production of ethanol and adsorption of ions on peels of other locally available fruits such as pineapple

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