A Study of Micro-flora Of Fruit Wastes in the Production of Ethanol and Vinegar

Thesis Submitted in Partial Fulfillment for the Award of the Degree in Doctorate in Philosophy in Microbiology

By

Catherine G, M.Sc, M.Phil., (Reg.No. MIC BIO 2009 AP 23)

Under the Guidance of

Dr. Vanitha N M, M.Sc, Ph.D., Associate Professor, Department of Microbiology,

St. Joseph’s College, Bangalore -560027

VINAYAKA MISSIONS UNIVERSITY SALEM, TAMIL NADU-636 308

INDIA 2014

CERTIFICATE

Certificate by the Guide

I, DR. VANITHA N M, M.Sc, Ph.D., certify that the thesis

entitled ‘A STUDY OF MICRO-FLORA OF FRUIT WASTES IN

THE PRODUCTION OF ETHANOL AND VINEGAR’,

submitted for the degree of Doctoral of Philosophy in

Microbiology, by CATHERINE G, M.Sc, M.Phil., Reg No. MIC

BIO 2009 AP 23, is a record of research work during the period

2009-2013, under my guidance and supervision and that this

work has not been the basis of any award for the degree,

diploma, associateship, fellowship or other titles in this

University or any other Universities or Institutions of higher

learning.

Signature of the Supervisor with

Designation

Place: Bangalore

Date:

Associate Professor, Microbiology

DECLARATION

DECLARATION

I, CATHERINE G, MSc, M.Phil., Reg No. MIC BIO 2009 AP 23, do hereby declare that the thesis entitled ‘A STUDY OF MICRO-FLORA OF FRUIT WASTES IN THE PRODUCTION OF ETHANOL AND VINEGAR’, submitted for the degree of Doctorate in Philosophy in Microbiology, is the original research work carried out by me during the period 2009-2013 under the guidance and supervision of Dr. VANITHA N.M., MSc., Ph.D., Assistant Professor, Microbiology, St. Joseph’s College, Langford Road, Bangalore. I further declare that this research work or any part thereof has not been the basis of any award for the degree, diploma, associateship, fellowship or other titles in this University or any other Universities or Institutions of higher learning.

Place: Bangalore Signature of the Candidate

Date:

ACKNOWLEDGEMENT

ACKNOWLEDGEMENT 

 

At the outset, I wish to thank the authorities of VINAYAKA MISSIONS UNIVERSITY, Salem, for kindly permitting me to register for my PhD and granting me permission to complete the same.

I express my sincere thank to Dr. K Rajendran, M A., Ph.D., Dean

(Research), for giving me the opportunity to carry out my research work in the

University.

I am most thankful and indebted to my respected guide Dr. Vanitha

N.M., Associate Professor, Microbiology, St. Joseph’s College, Langford

Road, Bangalore, for her encouragement, outmost patience, enthusiasm and

valuable guidance throughout the course of this work.

I extend my sincere gratitude to Dr. P Yashoda, Principal, S.S.M.R.V

Degree College for providing me the facilities to carry out my research work.

I wish to thank my sister Laura G K, who has been of immense help in

the completion of this work. Thanks also to John Sudhakar, my better half,

for the encouragement and support given.

Words are insufficient to express my gratitude to my beloved mother

and late father for inspiring, motivating and supporting me throughout my

educational career.

Place: Bangalore                                                Signature of the Candidate

Date: 

CONTENTS

 

 

Sl. No. CHAPTERS PAGE NO.

I. INTRODUCTION 1-13 II. REVIEW OF LITERATURE 15-27 III. MATERIALS AND METHODS 28-42

IV. RESULTS 43-56

V. DISCUSSION 57-61

VI. SUMMARY 62-64

VII. BIBLIOGRAPHY 65-81

VIII. APPENDICES 82-90

PUBLICATIONS

 

 

 

LIST OF TABLES

TABLE No.

TITLE

1 Isolation of fungi from pineapple peel.

2 Isolation of bacteria from pineapple peel.

3 Isolation of yeast from pineapple peel.

4 Isolation of fungi from jackfruit peel. 5 Isolation of bacteria from jackfruit peel.

6 Isolation of yeast from jackfruit peel. 7 Biochemical tests for bacteria isolated from Pineapple

peel.

8 Biochemical tests for bacteria isolated from jackfruit peel.

9 Initial parameters for pineapple fermentation 10 Initial parameters for jackfruit fermentation 11 Production of acetic acid in secondary fermentation

of pineapple peel ethanol 12 Production of acetic acid in secondary fermentation

of jackfruit peel ethanol

LIST OF FIGURES

Figure No.

TITLE

1a Hydrolysis of cellulose by bacteria from pineapple by CMC method

1b Hydrolysis of cellulose by yeasts from pineapple by CMC method

2a Hydrolysis of cellulose by bacteria from jackfruit by CMC method

2b Hydrolysis of cellulose by yeast from pineapple by CMC method

3a Estimation of cellulose by anthrone method for

Pineapple peel wastes 3b Estimation of cellulose by anthrone method for

Pineapple pulp wastes 4a Estimation of cellulose by anthrone method for

jackfruit peel wastes 4b Estimation of cellulose by anthrone method for

jackfruit pulp wastes 5a Estimation of glucose by DNS method for Pineapple

peel 5b Estimation of glucose by DNS method for Pineapple

pulp 6a Estimation of glucose by DNS method for jackfruit

peel (NEFP) 6b Estimation of glucose by DNS method for jackfruit

pulp (EFP) 7a Estimation of Ethanol by Dichromate Method using

Bacterial Isolates from Pineapple peel 7b Estimation of Ethanol by Dichromate Method using Yeast

Isolates from Pineapple pulp 8a Estimation of Ethanol by Dichromate Method using

Bacterial Isolates from Jackfruit peel (NEFP) 8b Estimation of Ethanol by Dichromate Method using Yeast

Isolates from Jackfruit pulp (EFP) 9a Production of acetic acid in secondary fermentation

of pineapple peel ethanol 9b Production of acetic acid in secondary fermentation

of jackfruit peel ethanol

LIST OF PLATES

Plate No. TITLE 1a Pineapple fruit 1b Jackfruit 1c Pineapple fruit peel 1d Jackfruit peel 2 Cellulomonas uda 3 Saccharomyces cerevisiae var ellipsoides 4 Acetobacter aceti 5 Acetobacter xylinum 6a Fungal isolates from pineapple peel on MRBA 6b Microscopic view of fungal isolates from pineapple

peel 7a Bacterial isolates from pineapple peel on NA 7b Microscopic view of bacterial isolates from pineapple

peel 8a Yeast isolates from pineapple peel on SDA 8b Microscopic view of yeast isolates from pineapple peel 9a Fungal isolates from jackfruit peel on MRBA 9b Microscopic view of fungal isolates from jackfruit peel 10a Bacterial isolates from jackfruit peel on NA 10b Microscopic view of bacterial isolates from jackfruit

peel 11a Yeast isolates from jackfruit peel on SDA 11b Microscopic view of yeast isolates from jackfruit peel 12a Biochemical test for bacteria isolated from pineapple

peel –Indole test 12b Fermentation of carbohydrates 12c Biochemical test for bacteria isolated from Jackfruit

peel –Indole test 12d Biochemical test for bacteria isolated from pineapple

peel – Simmon’s citrate test. 12e Glucose fermentation as biochemical test 12f Sucrose fermentation as biochemical test 13 CMC hydrolysis by bacteria from pineapple peel 14 CMC hydrolysis by yeast from pineapple peel 15 CMC hydrolysis by bacteria from jackfruit peel 16 CMC hydrolysis by yeast from jackfruit peel

LIST OF PLATES

Plate No. TITLE 17 Pineapple peel for fermentation 18 Pineapple pulp for fermentation 19 Jackfruit peel for fermentation 20 Jackfruit pulp for fermentation 21 Cellulose estimation by anthrone method in

pineapple 22 Cellulose estimation by anthrone method in jackfruit 23 Sugar estimation by DNS method in pineapple 24 Sugar estimation by DNS method in jackfruit

25 Ethanol estimation by Dichromate method in pineapple

26 Ethanol estimation by Dichromate method in jackfruit

27 Distillation of ethanol 28 Vinegar fermentation in pineapple peel 29 Vinegar fermentation in jackfruit peel

Abbreviations

ATCC – American Type Culture Collection

Cms- Centimeters

CMC - Carboxy Methyl Cellulose

° C – Degrees Centigrade

DMC - Direct Microbial Conversion

DNS - Dinitro Salicylic Acid

EFP - Edible Fleshy Part

ED – Entner Duodroff

HC- Hydrolysis Capacity

IMViC – Indole Methyl Red Vogesprauskaer Citrate

L – Liter

MRBA - Martin’s Rose Bengal Agar

mm – millimeter

µm – micrometer

ml – milliliter

Mins – Minutes

NCIM – National Collection of Industrial Microorganisms

NEFP - Non Edible Fleshy Part

OD - Optical Density

PDA - Potato Dextrose Agar

Rpm – Revolutions per Minute

SDA - Sabouraud’s Dextrose Agar SSF - Simultaneous Saccharification and Fermentation  

INTRODUCTION

Generating waste material is an irrefutable component of human

society. Several sectors that produce solid wastes include industries,

agriculture, forestry and municipalities. The accretion of solid wastes and the

throw-away attitude result in several environmental issues, health problems

and safety hazards. It also prevents sustainable growth with regard to

resource revitalization and salvage of waste materials. An outlook intended at

promoting larger sustainable improvement and resource recovery has

influenced solid waste management exercises. It is steadily becoming

implemented through various strategy guidelines at nationwide levels in a

large number of developed and developing countries. Many procedures,

guidelines and directives aimed to diminish waste generation and encourage

waste recovery have been laid down as per the waste management hierarchy

in which waste avoidance, reclaim, recycling and energy recovery are planned

to lessen the amount of waste remaining for final protected disposal, (Isa, et

al, 2004.)

The growing population of the world has resulted in consuming more of

the resources available. In the present situation, too much of the resources

are being wasted. Globally, much environmental and human health related

impacts are associated with waste. Wastes come from several sources and

have the maximum possible impact on the environment. This is because of

the quantity and structure of material produced or the technique by which they

are managed. Important wastes include construction and demolition waste,

commercial and industrial sectors, biodegradable waste, mining and

quarrying, hazardous waste, forestry, agriculture, municipalities, electrical and

electronic equipment waste and mixed wastes. The mounting of these solid

wastes present a challenge for a suitable and safe disposal of wastes, (Foyle

et al., 2007).

Biodegradable wastes comprises of food waste, manures, paper and

pulp waste, agriculture wastes, agro wastes, sewage sludge and slurries.

Most countries on a daily basis generate millions of tonnes of organic wastes

which are mainly kitchen wastes and account to more than 80 percent of all

wastes generated regularly. Most of these wastes are converted into compost

by spreading on to land. While some countries like UK have a means of

sending these biodegradable wastes to landfill to generate methane gas.

However, methane gas has 20 times more Global Warming Potential (GWP)

than CO2 , (Mor. et al., 2006).

Many metropolitan cities have an uncontrolled, open and poorly

managed dumping area usually located on the outskirts of the urban area.

This has been commonly practiced since time immemorial, giving rise to

serious environmental degradation. More than ninety percent of MSW in cities

and towns are unswervingly disposed off on land in a sub-standard manner.

This kind of dumping activity has led to accumulation of heavy metals and

their rapid leaching into the coastal waters, resulting in metallic poisoning of

aquatic life. In bigger towns and cities away from coasts, the availability of

land for waste disposal is very limited, (Siddiqui et al., 2006; Sharholy et al.,

2006). In India, the two foremost innovative mechanisms of solid waste

disposal being implemented comprise of - composting (vermi-composting and

aerobic composting) and waste-to-energy (WTE) (pelletisation,

biomethanation and incineration), (Gupta et al., 1998)

Municipal solid waste management is one of the foremost

environmental nuisances of Indian cities. Inappropriate management of

municipal solid waste (MSW) causes health hazards to the residential

populations. Different studies have revealed that ninety per cent of MSW is

disposed in an unscientific method in landfills and open dumps, thereby

causing inconvenience to community health and the surroundings, (Shekdar

et al., 1992). The MSW quantity is likely to mount considerably in the near

upcoming as the country attempts to achieve the status of an industrialized

nation by the year 2020, (Shah and Sharma, 2004; CPCB, 2005).

The consumption of fossil fuels has increased steadily due to the

increase in population and industrialization of many countries. As per the

studies done by Campbell and Leherree (1998), the annual global oil

production would decline drastically by 2050 leading to severe consequences

of insufficient accessibility and high cost of oil. Till the time clean electricity

turns out to be readily available for vehicles that run on electricity and have

cheap sustainable growth in battery technology. Therefore, bio-ethanol-from-

biomass course is the most feasible choice.

In the last three decades, much research work has been to search for

cheap and substitute sources of energy. Bio-fuels are energy fuels obtained

from plant sources and include biodiesel and bio-ethanol, (Naik et al., 2010).

Biogas is a useful source of energy obtained by using solid waste.

Many developed countries use biogas for vehicles as fuel and making of

electricity or heat, (Sims et al., 2003). Obtaining biogas from activated sludge

is conventional method. At present it has been largely produced by using

municipal solid waste (MSW) and manures. The carbohydrates, lipids and

proteins in MSW can be easily degraded by microorganisms. Whereas, other

types of materials like lignocelluloses and keratin cannot be degraded easily,

(Buffiere et al., 2006)

At present a major segment of bioethanol produced across the world is

by using different chemical and biological processes. These processes can

be additionally categorized into first and second-generation technologies

depending upon the plant biomass used, (Naik et al., 2010). First generation

techniques made use of crops like maize, sugarcane and fruits- which are

easily converted to ethanol, where as second-generation techniques produce

ethanol from cellulose biomass.

Bioethanol used for production of gasoline can reduce vehicle carbon

dioxide emission by 90%, (War and Singhs, 2002). Bio-ethanol signifies an

important, renewable resource of energy produced through fermentation of

simple sugars using bacteria and yeasts, (Zsolt Szengyel, 2012). The

production of bioethanol, mainly derived from plant cellulose biomass, has

been the most striking goal for public and private sector industries, (Sun Y and

Cheng J, 2002). Owing to their enormous potential in substituting different

bulk products of the petrochemical industry, cellulose and its derivatives

emerge to contribute a prime portion of the feed stocks in the sustainable

production of eco-friendly fuels, minimising the net CO2 emissions in the

atmosphere, (Naik et al., 2010) and moreover cellulosic ethanol does not

compete with food sources, (Naik et al., 2010).

Lignocellulose wastes (LCW) refer to mainly plant biomass wastes that

consist of cellulose, hemicelluloses, lignin, and many other inorganic

materials, (Sjöström, 1993). They may be assembled into diverse categories

such as wood residues (sawdust and paper mill junk), grasses, waste paper,

agricultural residues (including straw, fruit peelings, cobs, stalks, nutshells,

non-food seeds, bagasse, domestic wastes, food industry residues and

municipal solid wastes, (Qi, et al., 2005; Roig, et al., 2006; Rodríguez et al.,

2008). The ligno-cellulosic biomass signifies the leading renewable reservoir

of potentially fermentable carbohydrates on earth, (Mtui and Nakamura,

2005). However it is frequently lost either as pre-harvest and post-harvest

agricultural losses or as wastes from food processing units. Because these

wastes are abundant and can be renewed easily, Many researchers are

making use of LCW for producing many value-added products, (Pandey et al.,

2000; Das and Singh, 2004). The chief recovery products include reducing

sugars, ethanol, carbohydrates, enzymes, furfural, protein and amino acids,

lipids, organic acids, degradable plastic composites, phenols, activated

carbon, cosmetics, resins, medicines, foods and feeds, methane, bio-

pesticides, bio-promoters, secondary metabolites, surfactants, fertilizer and

other miscellaneous products, (Tengerdy and Szakacs, 2003; Mtui, 2007;

Ubalua, 2007; Galbe and Zacchi, 2007; Demirbas, 2008).

Cellulose or β-1-4-glucan is a linear polysaccharide polymer of glucose

made of cellobiose units, (Delmer et al., 1995) (Morohoshi, N. 1991).

Cellulose exists in waste streams in the form of lignocelluloses, or partly

purified in the form of papers or pure cellulose such as cotton, or mixed with

other materials like citrus wastes, (Talebnia, et al., 2008).

Cellulose biomass is obtained from agricultural wastes, fruit and

vegetable processing, forestry, household and municipal wastes, (Malherbe

and Cloete, 2002). Agricultural and forest remains could be used as a low-

priced and plentifully available resource for carbohydrate fermentation into

sustainable bioethanol, (Fujita et al., 2002; Edwards and Doran-Peterson,

2012).

These polymers can be biologically degraded by several enzymes like

cellulases, amylase, keratinase, protease and lipase, before any fermentation

degradation for ethanol or biogas production begins. However, these polymers

should be easily available to the enzymes if biodegradation has to be done.

Pre-treatment of ligno-cellulosic substances is a well known process for

ethanol production. The pre-treatment, helps to have better digestibility for

both ethanol and biogas production.

Pre-treatment

Although, many physical, chemical and microbial pre-treatment methods

for improving bioconversion of cellulosic materials have been reported, (Tang et

al., 1996; Kumakura, 1997; Wu, 1997; Depaula et al., 1999; Kansoh et al., 1999).

One of the primary drawbacks for the aggressive production of ethanol has been

the towering cost of both pre-treatment and the hydrolysis steps involved,

(Himmel M E, et al., 2007).

The main purpose of pre-treatment is to remove structural and other

obstructions related to composition for successive degradation and in increasing

the digestability rate of enzyme hydrolysis and boost yields of proposed products,

(Mosier et al., 2005; Hendriks and Zeeman, 2009).

Pre-treatment of cellulose unshackles the structure and removes the

secondary interaction amidst glucose chains. There are many pre-treatment

techniques on hand to disrupt the cellulosic biomass structure; these include

physical, chemical and biological:

1. Physical pre-treatment involves many methods like the mechanical

comminution by combining chipping, grinding and milling, Pyrolysis, steam

explosion, Ammonia fibre explosion (AFEX) and CO2 explosion. Since all these

methods aid in reducing the substrate size, so any damage done to the substrate

gets it ruptured and become more exposed to enzymatic hydrolysis, (Cadoche

and Lopez,1989), (Fan et al, 1987), (McMillian,1994), (Vlasenko et al.,1997) and

(Zheng et al,1998).

2. Chemical pre-treatment methods includes: Ozonolysis, acid hydrolysis,

alkaline hydrolysis, oxidative delignification and organo-solve process, (Vidal and

Molinear, 1988), (Esteghlalian et al, 1995), (Berlin et al., 2006), (Bjerre et al,

1996), (Azzam, 1989) and (Thring et al, 1990). The use of dilute acid, alkaline,

organic solvents and other chemicals helps to break open or enhances the

digestibility of the substrate for enzymatic hydrolysis.

3. Biological degradation is achieved chiefly by fungi like brown rot, soft rot and

white rot. Most efficient among these is white-rot basidiomycetes and also

certain actinomycetes (Lee, 1997). Studies have shown that several fungal

enzymes can degrade lignin, including lignin peroxidase, Mn-dependent

peroxidase and laccase, (Lee, 1997).

There are various possible methods for this process, and the most

commonly used methods can be classified into two groups: chemical hydrolysis

and enzymatic hydrolysis, (Taherzadeh and Karimi, 2007).

Hydrolysis

Hydrolysis is the process of breakdown of cellulose into cellulo-biose and

glucose, which can be accomplished either by enzymes or by acid /alkaline

hydrolysis, (Qian Xiang et al, 2003).

Compared to chemical i.e., acid or alkaline hydrolysis the cost

effectiveness of enzymatic hydrolysis are low. Depending on the composition of

the preliminary material, different pre-treatment method have been developed in

order to prepare it for the subsequent step of enzyme hydrolysis,(Sun Y, Cheng

J, 2002). This is because the enzymatic hydrolysis is frequently executed under

mild conditions, around pH 4.7 and temperatures from 45 to 50 °C. This method

does not interfere with corrosion either, (Sun Y., Cheng J., 2002). Although

enzymatic hydrolysis is an efficient technique and releases almost all the

carbohydrates in fruit peels, the method is hindered by a slow de-polymerization

reaction rate, (Talebnia F. et al., 2008).

Many enthusiastic efforts have been persistent on the expansion of cost-

effective and robust biocatalysts for the hydrolysis of cellulose to fermentable

sugars. The conversion of cellulose to ethanol requires two steps: degradation of

biomass to fermentable sugars, catalyzed by cellulolytic enzymes and

fermentation of the simple sugars to ethanol by yeasts or bacteria. The

enzymatic conversion of cellulose to glucose is a fabulous prospect, because of

the many alternatives that the molecule glucose holds as the ‘precursor’ of

diverse value-added products.

The enzymatic hydrolysis is achieved by two approaches:

1. Direct Microbial Conversion (DMC) in which the pre-treated material is

incorporated with enzymes like cellulases produced by microorganisms.

Fatma H. et al (2007), made use of the fungus Trichoderma reesei for

enzymatic hydrolysis. This method of hydrolysis helps convert cellulosic

biomass to ethanol in which the required enzymes and ethanol are

produced by utilizing a single microbe. However, DMC has its own

drawbacks and is not the foremost process of alternatives today because

of inadequacies in the search for microorganisms that can both produce

cellulases and other enzymes at the requisite elevated levels and also

generate ethanol at the required high concentrations.

2. Simultaneous Saccharification and Fermentation (SSF) combines the

cellulases enzymes and fermenting microbes in one vessel. This is a kind

of a one-step process of sugar production and species worthy of research

attention because of its wider potential use in nutrition and its potential to

increase local incomes when grown in agro-forestry and home garden

systems. It is locally sometimes of high value, (Rehm and Espig, 1991)

and has proved valuable when introduced to other parts fermentation into

ethanol. The difficulty is that the cellulases and fermenting organism have

to function under identical condition and this lowers the sugar and ethanol

yields.

The resulting product of the wastes– ethanol or biogas is obtained by the

enrichment of the difficult biodegradable materials.

Each year, huge quantities of fruit are wasted for the reason that the

surplus cannot be consumed straight away by the market and because some fruit

does not compete with the market requirements i.e, second or third quality fruit.

Even though some substitute to direct consumption have by now been put into

practice (purées, jams, fruit juices, nectars, fruit concentrates, etc) a huge

amount of fruit is still left in the harvest ground to either rot or get collected and

disposed as solid wastes, (Grewal et al., 1988).

These exercises generate both an ecological and an economic dilemma,

since a bulky amount of unprocessed organic matter have to be recycled and

involves capital for man power, agrochemicals and machinery for processing

both the fruit that is consumed and the fruit that is disposed off.

Developing countries lying in the tropical zones produce an enormous

portion of tropical fruits. Most of these fruits are consumed fresh while a major

fraction is discarded due to poor storage conditions and inadequate processing

techniques. With regard to increase the utilization of tropical fruits in the Indian

subcontinent and reduce the accumulation of solid wastes an effort to utilize

cellulosic wastes of tropical fruits for the production of ethanol and vinegar has

been considered as an attractive alternative to retrieve fruits wasted during post

harvest.

The jackfruit tree, (Artocarpus heterophyllus .Lam), is basically a

domesticated tree of tropical and sub-tropical regions of Southeast Asia. It has

been cultivated in India since early times. It was probably taken by Arab traders

to the East African coast, and now it has spread all over the tropics. It can also

be found throughout the Pacific, primarily in home gardens, where it is a favourite

multipurpose plant. The fruit often takes on the role of a secondary staple food

and also contributes to the livelihood of the poor. The durable timber of the tree

is known to age from cream to an orange or red-brown colour. The broad leaves

serve as important silage for live stock animals. Similarly, the bark, seeds, roots,

leaves, and fruit are attributed with medicinal properties.

Jackfruit is regarded as the largest fruit of the world. It is fairly widely

cultivated in suitable climates, (Morton, 1965). It is a fleshy fruit with lot of fiber

content and recognized easily by its large-size. The pulp serves as a staple

starchy diet. The composition of jackfruit wood has been determined,

(Komarayati, 1995). It contains 56% cellulose, 28.7% lignin and 18.64%

pentosan. Jackfruit is rich in pro-vitamins- A (β – Carotene), Vitamins D & C and

seven types of Vitamin- B along with minerals like Potassium, Phosphorus,

Calcium etc., Shamsudin, R., 2009, has reported the importance of all parts of

the tree having medicinal value. However, only 32 to 41 per cent of the fruit is

edible, while the remaining 60-70 percent inedible part is waste as reported by

Hardin et al., 2004. Moreover, Crane et al., 2002, have also reported that the

large fruits are easily susceptible to insects, pests & molds, thereby reducing the

shelf life of the fruit to 2-3 days.

Annual production of jackfruit in India is 1,436,570 Metric Tonnes, (Ghosh,

1996), AEC (2003). Mature trees yield up to 200-250 fruits, each fruit can weigh

upto 55 kgs, (Ghosh, 1996).

Thirty five species of insect pests have been recorded on jackfruit from

India. Major pests can be shoot and trunk borer, brown weevil, mealy bug and

jack scale, these are those found to be associated generally with jackfruit,

(Tandon 1998). Major diseases of jackfruit are: leaf spots, die-back, fruit rot and

pink Disease, (Rawal and Saxena, 1997) (Soepadmo, 1992) Azad (2000), IPGRI

(2000).

Pineapples (Ananas comosus L.) originated from the tropical region of

South America. There are many varieties of pineapples. Each of the varieties,

varies significantly in both shape and taste. In every variety there are local types.

All pineapples are self-sterile and free of any seeds. The seeds are inseminated

through external sources. The pineapple is actually a xerophyte and can

therefore endure long periods of dry spell. Pineapples are consumed as fresh

fruit or processed as dried fruits, canned fruits and as juice.

Pineapple (Ananas comosus) is an important commercial fruit crops of

India. Annual world production is 14.6 million tones. India stands at the fifth

position as the largest producer having an annual production 1.2 million tones.

(Database of National Horticulture Board, Ministry of Agriculture, Govt. of India,

2002).

Pineapples waste have the possibilities for recycling to obtain important

raw material, change it into a useful product of a higher value, food or feed after

biological treatment and even as raw material for other industries, (Kroyer, 1991).

One instance is pineapples waste that is converted to bioethanol production,

(Hossain et al., 2008). The wastes contain valuable components such as:

sucrose, glucose, fructose and other nutrients, (Sasaki et al. 1991; David et al.,

2008). Pineapple waste is a material rich in sugars and lingo-cellulosic

components, (Prados D. et al, 2010). Added to this, the conversion of

pineapples waste to useful products such as ethanol production can help to clean

the environment from wastes, also it is economically useful, when the wastes are

converted to valuable product.

Pineapple fruits Ananas comosus belong to the class: Liliopsida and

family Bromeliaceae (Bartholomew, 2003). Its fruit are oval, six to eight inches

long with spiky hard leaves. The fleshy part of the pineapple is yellow to cream

coloured and juicy.

In addition to its nutritious uses, the pineapple has long served medical

purposes in folk medicine. It is used to stimulate appetite, useful as diuretic and

contraceptive and in the exclusion of internal worms. It has been used to avert

ulcers and enhance fat excretion. The enzyme bromelain is used in commercial

meat tenderizers, as a soft tissue anti-inflammatory and for topical debridement.

Pineapples have a tendency to get infected to a wide range of diseases

caused by pests, microbes and insects. The most likely and serious is the wilt

disease caused by the vector mealy bugs on the exterior of pineapples. Other

frequent pests that affect pineapple plants are thrips, ants, mites, mealy bugs,

scales and symphylids. Further additional microbial diseases include

anthracnose, bacterial heart rot, root rot, butt rot, pink disease, fungal heart rot,

black rot and yellow spot virus. Nematodes can become visible in pineapple

monoculture plantations. Trouble with nematodes occurs only on organic

plantations, due to the normal practice of fruit rotation.

Many fruit wastes are either used as cattle feed or dumped in open areas

wherein they get accumulated, it adds to environmental solid waste pollution.

Several methods for enumerating cellulose utilizing microbes have been

described which include liquid and solid media, (Teather and Wood, 1982) for

quantification of cellulose utilizing bacteria from fruit wastes. Ethanol production

from banana peels, (Manikandan et al., 2008) and pineapple peels, (Ban-koffi

and Han, 1990) were also investigated. The use of mango peel, as a source of

pectin and fibre production also has been reported, (Pandia et al., 2004).

Grohmann et al. (1994; 1995; 1996; 1998), previously reported ethanol

production from orange peel.

A hefty fraction of fruits are lost through spoilage. To overcome this

problem, producers trade their marketable surplus within a short period from the

time of harvest, at low prices. However, income can be generated appreciably if

the produce is stocked up correctly or processed, since fruit prices increases to

twofold or even threefold only a few months after the harvest, (Roy, 2000). An

alternative source of renewable energy i.e., ethanol could be produced from the

excess produce. Moreover, accumulation of excess of non-edible solid wastes

in the environment can be reduced. Thus, our farmers would be able to produce

higher commercial value products like ethanol and vinegar from their agro

wastes.

Vinegar is consumed worldwide as a food ingredient and preservative.

Chinese have traditionally produced vinegar from cereals like rice since more

than 3000 years, (Shi, 1999). Many different countries and different regions of

the world have their own local vinegar types. Vinegar consists of 4% acetic acid

and obtained during secondary fermentation of ethanol by acetic acid bacteria.

Vinegar is commonly used as a food ingredient and has medicinal

properties. Its physiological role like invigorating, regulator of blood pressure,

Diabetes mellitus, appetite stimulator, absorption of calcium during digestion has

been observed, (Ndoye et al., 2007).

In consideration of all the above points, the present investigation was

taken to study the hydrolytic ability, alcoholic fermentation and subsequent

acetification of the cultured and isolated cellulose degrading strains on tropical

fruit wastes like pineapple and jackfruit, by subjecting the raw material to acid

hydrolysis, microbiological and enzymatic pre-treatments for ethanol and vinegar

production, with its probable application to an industrial fermentation process.

The objectives of the study were to isolate, identify and study the cellulolytic and

fermentative microbes and utilization of the non-edible cellulosic part of

pineapple & jackfruit to produce ethanol and vinegar.

Review of Literature

Ethanol as Bio-fuel

Sagar (1995), stated that the rise in fuel costs has made researchers to seek

out substitute fuels for transportation. Even though alcohol based fuels can

be obtained from fossil fuels, alcohols originated from biomass have the

potential to reduce carbon footprints.

The ever-increasing concern regarding pollution occurring in the environment

from agriculture and industrial wastes has generated an interest in converting

the huge solid waste material into commercially valuable products like

enzymes, organic acids, ethanol etc. as stated by Zhang (2007).

Unal and Alibas, (2007), found that bio-fuels are more beneficial because it

comes from renewable resources. Its sustainability reduces green house gas

emission, regional development, social structure, agriculture and security

supply.

Ethanol is an alcohol basically prepared by fermenting plant based sugars

from biomass and agro-derived resources as observed by Ikilic and Yucesu

(2008).

As per Linde (2008), non-renewable sources are restricted with oil, natural gas

and coal reserves are expected to be depleted within approximately between

40 to 120 years.

According to EIA (2011) reports, the restricted resources and socio-economic

factors that have caused the oil price to mount sharply putting financial strain

on the global population. Human population have mostly become depended

on fossil fuels as the key energy source.

As cited by Pereira (2011), technological methods which enable the efficient

production of bioethanol have been continuously sought for both economic

and environmental reasons.

Agriculture Wastes

According to Baddi (2004), agriculture crop residues are among the most

important sources of the total biomass with significant production of food, feed

and fuel.

Ghofar (2005), stated that several processes have been designed to use

waste in many biotechnological processes in the production of fermentation

products or in biomass production. All these process depend on the of

degradation of waste.

Cellulose as Substrate for Hydrolysis for Fermentation

As per the studies of Fan (1987), cellulose contains a crystalline and lignified

structure that limit the ease of access and susceptibility of cellulose to

cellulolytic enzymes and hydrolytic agents.

Cellulose exhibits structural crystalline segment and amorphous component,

as mentioned by Saka and Ueno (1999).

The utilization of cellulose to the large quantity of cellulose and renewability

has generated a great deal of interest in utilizing cellulose wastes for the

making and recovery of several value-added products as studied by Pandey

(2000).

Mtui and Nakamura, (2005), found that the cellulosic biomass, which signifies

a major renewable pool of potentially fermentable sugars on earth is mainly

lost as pre-harvest and post-harvest agricultural waste and losses of food

processing industries.

Rubin (2008), found that the breakdown of cellulose components results in the

letting loose of long-chain polysaccharides, in particular cellulose and hemi-

cellulose. Further the subsequent hydrolysis results in the splitting of these

polysaccharides into their constituent sugar of 5- and 6-carbon chain.

Cellulose Degradation in Agriculture Waste

Demirbas (2006 & 2008), illustrated the importance of biomass-derived

alcohols produced from the fermentation of the polysaccharide cellulose.

Soto (2005), estimated the amount of ethanol yield by making use of an

equation to calculate the mass of ash obtained after fermentation of corn

mash. Glucoamylase 0.3% was added for simultaneous Saccharification and

yeast cells of 3x107 cells /g of mash was used for fermentation. The

observations confirmed that estimation by mass of ash for ethanol yield is

steady and applicable and could be used for solo fermentations.

Naureen (2006), utilized sugar cane bagasse as a substrate for ethanol

production, by saccharifying the media using four different strains of Bacillus

cereus. It was found that all the four strains of bacteria were able to grow at an

optimum temperature of 37°C and at a pH of 7 at 5% inoculum size.

Maximum yield of glucose 1411mg/100ml was obtained with strain 11a, upon

10 days of incubation period.

Lee (2010), studied the possibilities of ethanol production by using paper

sludge as the substrate. Two recombinant microorganisms viz.,

Saccharomyces cerevisiae RWB222 and Zymomonas mobilis 8b with xylose

fermenting ability was used. The process used was simultaneous

saccharification followed by co-fermentation. At 37°C and 30°C, 40g/L of

ethanol was obtained from Saccharomyces cerevisiae RWB222 and

Zymomonas mobilis 8b.

Naulchan (2010), studied the growth and batch fermentation of

Saccharomyces cerevisiae NP01 under normal gravity (NG) of total sugars of

240gL-1 and very high gravity (VHG) containing total sugars 280gL-1. Optimal

conditions employed for fermentation were 30°C, agitation rate of 100rpm,

having initial concentration of yeast cells as 1x108 . The substrate used for

the fermentation was sweet sorghum stem juice without addition of nitrogen.

The productivity, concentration and yield of ethanol obtained under NG

conditions was 2.92g L-1 h-1, 105.12g L-1 and 98.33% which was lower

compared to the theoretical yield under VGH condition.

Yuan (2011), devised a new strategy of producing ethanol by simultaneous

saccharification and fermentation of rice straw by employing non- recombinant

xylose and glucose fermenting yeasts – Pichia stipitis and Saccharomyces

cerevisiae. The two yeasts were sequentially applied by inactivating

Saccharomyces cerevisiae first through heat inactivation at 50°C for 6 hours,

during which a theoretical yield of 85% ethanol was achieved from the

conversion of lignocellulosic biomass. Full conversion of glucose and xylose

was reached within 80 hours by inactivating Saccharomyces cerevisiae.

21.1g/l of ethanol was formed from 10% (w/w) of the pre-treated lime and CO2

neutralised rice straw.

Cellulose from press cakes of Jatropha oilseeds, a by-product of biodiesel

plant was biodegraded by Mohit S. Mishra (2011). The press cakes were

converted to ethanol by utilizing the methods of acid pre-treatment. The

hydrolysis was done by adding dilute sulphuric acid and heating the mixture to

a high temperature with an incubation time of 72 hours, to hydrolyse cellulosic

component into simple sugars. Further, fermentation of the hydrolysed

product was done using Saccharomyces cerevisiae to produce 80% ethanol.

New cellulose-hydrolytic bacteria for the production of cellulase by monitoring

the cultural setting using cellulosic feedstock were screened by Kanokphorn

(2011). Five cellulolytic bacteria were isolated and identified by 16S rRNA

sequencing as Cellulomonas sp. The optimal condition for cellulase

production was maintained at pH 6 and a temperature of 35°C and 4% of

wastepaper as substrate. Cellulomonas sp. strain TSU-03 showed maximum

performance of xylanase at 1860.1 U mg-1 and endoglucanse at 388.5 Umg-1

Further, the wastepaper hydrolysate was employed as substrate for producing

ethanol using Saccharomyces cerevisiae. After 48 hours of cultural conditions

and fermentation (SHF) process, 12.5 g L-1 of ethanol was produced.

Cellulose Degradation in Fruit Waste

Lebaka (2011), was able to produce ethanol from the wastes of Mango fruit

processing industries which basically included mango peel and stones.

Saccharomyces cerevisiae CFTRI 101 was used as the strain for

fermentation. It was observed that the ethanol obtained from the extract of

mango peel was 5.13 percent (w/v). Cellulose degradation of mango peel is

difficult and requires pre-treatment. In this experiment pectinase 1% (v/v)

enzyme (Triton chemicals) was used to aid degradation.

Balasubramanian (2011), utilized spoiled fruits like grape, apple, pineapple,

banana, orange and papaya for ethanol production. Microorganisms were

isolated from the peels, pulp and juice and used for cellulose conversion.

Most important microbe involved during fermentation was Lactobacillus.

Debajit (2011), studied the possibilities of utilizing fruit wastes- rotten banana

and apple pomace for production of ethanol. The substrate mixture of 400g

was diluted to 1000ml to which 50g of sucrose and 10g of Saccharomyces

cerevisiae was added. After 36 hours of fermentation, 200 ml of 48% ethanol

was obtained from a total volume of 1500 ml fermented broth.

Junaidi (2011) optimised the conditions for synthesizing cellulose from the

bacterium Acetobacter xylinum by using pineapple wastes as the substrate. At

an optimal condition of 30°C temperature, pH 5.15, and pineapple substrate

concentration of 83.32 per cent, the cellulose obtained was 3.4368g. Thus it

was proven by FT-IR spectroscopy that bacteria are able to synthesis

cellulose of high purity, good elasticity and mechanical strength with a high

water holding capacity.

Priscila (2013), made a comparative study of Banana waste’s peel,

pseudostem and fruit for cellulose conversion to glucose for ethanol

production by simulation of hydrolysis through dilute acid using a conversion

reactor. Upon analysis of energy consumption for every molecule of sugar

obtained, it was found that for 2 per cent H2SO4, 15 minutes residence time in

the reactor and 120°C temperature, the cellulose to glucose conversion for

banana pseudostem was 36 per cent. However it was also noticed that

hydrolysis of banana fruit was unattractive as per the energy consumption

analysis per sugar molecule.

Products of Agriculture Wastes as Substrates

The possibilities of producing significant levels of α-amylase and

glucoamylase on SSF media consisting of a mixture of wheat flour, wheat

bran, corn flour, sweet potato and potato, using Saccharomycopsis capsularis

was studied by Soni (1996). Higher enzymes product of 35.9 U/g for α-

amylase and 33.8 U/g of glucoamylase were formed after 10 days incubation

at 28°C. The yield was further increased to 72 and 52 U/g for α-amylase and

glucoamylase by addition of yeast extracts, and inorganic salts like CaCl2

.H2O and CoCl2.

Cavaco - Pola, (1998), mentioned how cellulases have fascinated many

researchers owing to the variety of their application in textile industry.

Bhat (2000), researched on the uses of microbial sources of enzymes like

cellulases, hemicellulases and demonstrated the colossal potential in

biotechnology. Cellulases and related enzymes are used in animal feed,

textile and laundry, agriculture and research, pulp and paper industries, food,

brewery and wine. Thus, there is a growing demand for these enzymes

especially for research on cellulases and related enzymes.

Abdel-Naser (2000) worked on two strains of yeasts – Saccharomyces

bayamus and Saccharomyces cerevisiae on substandard date’s juice as

substrate for ethanol production. Three concentrations of dates juice were

prepared ie., 13.5%, 18% and 22.5%. It was found that S.bayamus was highly

active than S.cerevisiae and gave maximum yield of ethanol at a

temperature30°C, pH 3.5, sugar concentration of 18% at an incubation period

of 84 hours. Ethanol of 93% concentration was obtained after the distillation

of fermented dates juice. Addition of di-Ammonium phosphate of

concentrations 1, 2 and 3g/L had no effect on the efficiency rate of

fermentation.

David Pimentel (2005), was able to produce two value added products viz.,

ethanol and bio-diesel using different sources of cellulose agriculture wastes.

For ethanol production corn, switch grass and wood was used as the

substrate. For biodiesel soybean and sunflower were used as the substrate

source. Fossil energy of 29 percent for corn, 50 percent for switch grass and

57 percent for wood was required more than the ethanol fuel produced. The

fossil energy required for the output was 29 percent soybean and 27 percent

for sunflower. However, to produce bio-diesel fossil energy was 119% which

is a negative output.

Caritus (2006), studied the production of two enzymes: β- glucosidase and

CM-cellulases by using the fungi Aspergillus niger on the substrate Garcinia

kola (bitter kola) pulp waste. The substrate was pre-treated with 2.5 M

Sulphuric acid to obtain 200% and 500% of cellulases after 120h and 96h

after hydrolyzing.

Anbuselvi (2009), studied the scope of degrading coir wastes to obtain value

added product like manure or Biofertilizer. The degradation was brought about

by making use of three different species of cyanobacteria viz., Phormidium sp,

Oscillatoria sp and Anabaena azollae sps., isolated from marine source. It was

found that the three isolated species were able to degrade coir waste of upto

89% of lignin and 92% of hemi-cellulose. The presence of sugars and alcohol

was also detected in the end products. The manure of coir waste showed

good soil conditioner and having maximum water holding capacity. The

manure was also useful in increasing the nitrogen content of the soil, because

of the presence of nitrogenase in cyanobacteria.

Claudio (2010), made use of the surplus fruits like persimmon and strawberry

to produce vinegar. The processes used were alcoholic fermentation and

acetification. The procedure was performed by inoculating native

microorganisms and compared with commercially available wine yeast for

alcoholic fermentation. Upon comparing the results, it was found that ethanol

obtained from strawberry mash fermentations was higher than that of

persimmon mash. Moreover, acetification of persimmon mash (30 days) was

more rapid than in strawberry mash (70 days).

Kulanthaivel (2011), converted the lignocelluloses of fruit wastes into ethanol

by utilizing - Phoma Sp., a fungus. At pH 5, the Phoma Sp. showed maximum

performance for both laccase and cellulases with highest mycelia growth.

Upon fermentation by Saccharomyces cerevisiae the percentage of ethanol

obtained was 2.4 %( v/w) for every 100g of fruit waste.

Vikash (2012), studied the bioconversion of fruit peel and vegetable wastes

into viable product. For fruit peel conversion, papaya peel, banana peel and

apple peel extracts having natural fermentable sugar content between 14.6 –

15.2 w/v were used. Ethanol produced after 36 hours of fermentation was

5.90 – 4.94%. Sugar content of vegetable ie., turnip peel extract was low

compared to fruit peel extract. The alcohol content formed was 1.5% (w/v).

Other than alcohol production, the yeast biomass generated was compared

and found to be higher than commercially available SCP of food and feed

grade.

Microorganisms in Fermentation

Fermentation

According to Lee (1997), the use of bacteria or fungi in the fermentation of

glucose as the sole source of carbon and energy is a well documented and

developed procedure. The yeast Saccharomyces cerevisiae and a facultative

bacterium Zymomonas mobilis are the microorganisms used for industrial

ethanol production.

Yeasts

Linden and Hahn-Hägerdal (1989), researched in yeast physiology and

revealed that many strains in the genus of Saccharomyces cerevisiae can

tolerate far higher ethanol concentration than assumed.

Thomas and Ingledew (1992), found that among many microorganisms being

exploited for ethanol production, the genus of Saccharomyces cerevisiae still

remains as the prime species. Yeast are mostly used for commercial ethanol

production because it ferments glucose to ethanol as a virtually sole product

and it is known for its high ethanol tolerance, rapid fermentation rates and

insensitivity to temperature, substrate concentration and most importantly

social acceptability usually without any conditioning or genetic modifications

that risk making the modified strains lose some of their original traits.

Madigan (2000), researched on the key metabolic pathway concerned in the

ethanol fermentation of yeast and concluded that it is glycolysis, through

which single molecule of glucose is metabolized to form two molecule of

pyruvate under an anaerobic condition, the pyruvate is further reduced to

ethanol along with the liberation of CO2. Theoretically, the yield for ethanol is

0.511 and for CO2 it is 0.499, whenever 1 g glucose is metabolized. Also, the

two ATPs produced in glycolysis are used to induce yeast cell biosynthesis,

along with a wide range of energy-needing reactions under the anaerobic

condition.

Zymonomas mobilis

Swings and Deley (1977), originally discovered this microorganism in

fermenting sugar-rich plant saps, e.g., in the traditional pulque drink of Mexico,

palm wines of tropical African, or ripening honey. Zymomonas mobilis

catabolises only three sugars, D-glucose, D-fructose and sucrose as energy

and sole carbon sources.

Conway (1992), pointed to Zymomonas mobilis as having more advantages

over S. cerevisiae with regard to ethanol production and tolerance. Z. mobilis

is a gram-negative and an anaerobic bacterium producing ethanol from

glucose via the Entner-Doudoriff (ED) pathway.

Jia Jia Wu (2012), studied the culturable microbial biodiversity present in

Shanxi vinegar, Chinese aged vinegar made from a mixture of cereals

subjected to solid state fermentation. The indigenous group of microbes

isolated were 47 yeast isolates dominating during alcoholic fermentation, 28

lactobacilli isolates and Acetobacter isolates dominating during bacterial

fermentation of vinegar. Gluconobacter oxydans was isolated during the initial

stages of fermentation of both alcoholic and vinegar fermentation.

Jackfruit Waste

Rahman (2001), studied on the benefits of adding jackfruit juice of

concentrations 0.5, 10 and 15% in milk for yoghurt preparation. The result of

the studies indicated that the yoghurt sample containing 5% jackfruit juice

showed better performance. The total solid content was increased, but fat,

ash and protein content decreased. Yeast cells were found to be higher in

Jackfruit yoghurt than plain yoghurt.

Babitha (2006), carried out solid state fermentation of the substrate jackfruit

seed powder for the production of Monascus pigment, using the fungus

Monascus.purpureus LPB97. Results showed that over a wide pH range,

particle size of jackfruit powder of 0.4 and 0.6mm, supplementing with

monosodium glutamate and without addition of external source of carbon was

suitable for pigment production.

Thitipong (2011), separated and purified the pre-biotics from the extracts of

jackfruit seed by the method of crystallization at temperature between 55-

64°C. At a mixing speed of 100rpm and temperature of 58°C, 83.5% of non-

reducing sugar obtained.

Dushyantha (2011) isolated lactic acid bacteria from the jackfruit

Phyllosphere, perianth lobes and juice. The isolates were further

charecterized and compared with the standard lactic acid bacterial strain.

Jackfruit juice from the bulbs was ameliorated at 18° Brix and adjusted to

0.5% acidity. Isolated starter cultures were added at 5% inoculum rate and

supplemented with nitrogen source. The strain JFL1 showed higher fermenting

ability with superior quality of beverage produced with maximum residual

sugar content of 2.18%.

Andri (2012), investigated the use of clarified jackfruit juice for wine

fermentation by monitoring addition of yeast and preliminary sugar

concentrations. Jackfruit juice of 14 % w/w sugar concentration and 0.5 to 2.0

% w/v Baker’s yeast ie., Saccharomyces cerevisiae was fermented under

anaerobic condition for 14 days at 30°C to obtain 12.13% v/v of ethanol of

definite jackfruit aroma. The report of sugar and ethanol analysis showed that

elevated yeast inoculums and preliminary sugar concentrations inhibited

growth of yeasts.

Pineapple waste

Nigam (2000), was able to continuously produce ethanol using pineapple

cannery waste as the medium of growth for immobilized Saccharomyces

cerevisiae ATCC 24553, a respiratory deficient strain. Highest ethanol yield of

42.8g /l/h was obtained at 30°C, pH 4.5 and dilution rate of 1.5h-1. The

maximum rates of specific sugar uptake and ethanol productivity of the

immobilized cells were 2.6 sugar g-1 dry wt. cell h-1 and 1.2g ethanol g-1 dry

wt. cell h-1.

Seyram (2009), made use of pineapple peeling for the fermentation of ethanol

and vinegar. The alcoholic fermentation was carried out using the yeast strain

Saccharomyces cerevisiae (LAS01) at 30°C. Secondary fermentation with

ethanol as the substrate source was carried out using the bacterium –

Acetobacter sp. Isolated from a previously prepared pineapple wine. At 20°

Brix containing Saccharomyces cerevisiae yeasts concentration of 106 cells

were incubated for four days for alcoholic fermentation. This was followed by

seeding of Acetobacter sp. of 106 cells were inoculated at pH 2.8, Brix 5.3

and incubation for 23-25 days, yielded vinegar from a pH 4.4 to 2.9.

Hossain (2010), found that rotten pineapples waste could be used to produce

bioethanol for running petrol engine by the process of fermentation using

commercial yeast, Saccharomyces cerevisiae. The optimal parameters

required for the yield of bioethanol were adjusted as 8.7% substrate

concentrations for rotten pineapple waste, pH 4 and 30°C, the bioethanol

obtained was 3 g/l. The anhydrous ethanol was found to have no dangerous

element. Based on ASTM standard, it had acceptability as a transportation

fuel.

Several reports suggest the possibilities of ethanol and vinegar production

from fruit processing wastes as reported by Ethiraj and Suresh, (1990).

Gullo et al, (2005), reported that the production of vinegar has some sugar in

fruit juice and that the concentration of ethanol is not a self limiting factor for

the growth of acetic acid bacteria.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MATERIALS AND METHODS

I. Raw Materials used.

a. Pineapple fruit wastes was obtained from the dump yard of Kalsipalyam, K R Market, Bangalore, while for the fermentation, ripe pineapple fruit were procured from Hopcoms, a common vegetables and fruits outlet of Lalbagh Farmers Association, Bangalore. (Plate-1)

b. Jackfruit pulp and wastes was obtained from a plantation in the Makali area of Neelamangala Taluk of Rural Bangalore and used both for isolating microbes and fermentation studies. (Plate- 2)

II. Isolation of Microbes

For isolation of microorganisms, 1g of the rotten fruit (pineapple and Jackfruit) was serially diluted to six-fold using sterilized distilled water and 0.1ml of the diluted sample was inoculated on MRBA, Nutrient Agar and Saboraud’s dextrose agar media and incubated for 24 hours. (Appendix- II) (Appendix- III) and (Appendix- IV)

III. Assessment of Bacteria and Yeasts for Attributes of Cellulose Degradation

From amongst bacterial isolates, only bacilli were chosen for the studies,

along with the yeast.

Each bacterial and yeast isolate was further screened by using pure cellulose

powder of Hi-media grade. Carboxymethylcellulose hydrolysis capacity (HC-

value) method was used for assessing cellulose degradation.

The composition of the carboxymethylcellulose media (CMC) (Hi-media) (gL-1)

is:

Ingredients Composition (g/L)

K2HPO4 0.5

MgSO4 0.25

Cellulose Powder 1.88

Congo red indicator 0.20

Noble agar 20

Gelatin 2

Distilled water 1000 ml

pH 7

For bacterial isolation, the antibiotic cyclohexamide 100µg/ml was

added to inhibit fungal growth. Similarly, for yeast isolation the antibacterial

streptomycin sulphate 100µg/ml was added. The plates were incubated at

32°C for 48h, by streaking and growing each isolate on the cellulose Congo-

red agar medium.

The ratio of the diameter of clearing of zone around the colony has been

measured Hankin and Anagnostakis, (1977); Hendricks et al., (1995); Reese

et al (1950). The single colony showing significant clear zone was sub-

cultured and streaked on CMC media for 48hrs to measure the

carboxymethylcellulose hydrolysis capacity (HC value) (Hendricks et al.,

(1995). The isolates with high HC values were selected and maintained on

the respective maintenance medium at 4°C.

IV. Microorganisms Used

Positive cellulose degrading and ethanol producing bacteria

Cellulomonas uda and ethanol producing yeast – Saccharomyces cerevisiae

var ellipsoides were used to compare the cellulose degrading activity of the

isolates. Similarly, Acetobacter aceti and Acetobacter xylinum were used for

the vinegar fermentation; all of these microbial cultures were obtained from

NCL, Pune. They were used as positive cultures to investigate the cellulose

hydrolysis and fermentation studies on pineapple and jackfruits.

Microbial Strains NCIM. No. ATCC.No.

Cellulomonas.uda 2353 21399

Saccharomyces.cerevisiae.var. ellipsoides

3494 24702

Acetobacter aceti 2251 15973

Acetobacter xylinum 2526 -----

(Plate 3), (Plate 4), (Plate 5) & (Plate 6)

V. Maintenance of Positive strains, Bacterial and Yeast Isolates on Microbial Medium

The bacterial isolates were maintained on the tested enrichment media.

Ingredients Concentration (g/L)

Beef extract 10g/L

Peptone 10g/L

NaCl 5g/L

Agar 20g/L

Distilled water 1000ml

pH 7.3

Saccharomyces.cerevisiae.var.ellipsoids was maintained on the media

recommended by NCL. The composition is as follows:

Ingredients Concentration (g/L)

Malt Extract 3

Glucose 10

Yeast Extract 3

Peptone 5

Agar 20

Distilled water 1000

pH 6.6

The yeast isolates were maintained on SDA media.

Cellulomonas.uda was maintained on the medium as per the recommendation

of NCL. The composition is as follows:

Ingredients Concentration (g/L)

Beef Extract 10

NaCl 5

Peptone 10

Agar 20

Distilled water 1000ml

pH 7.0 - 7.5

Acetobacter aceti was maintained on the following media.

Composition Concentration

(g/L)

Tryptone 10

Yeast Extract 10

Agar 20

Glucose 10

CaCO3 10

Distilled water 1000ml

Adjusted the pH to 6.0. Steam the medium to melt the agar then add the following components:

Acetobacter xylinum was maintained on the following media

Ingredients Concentration (g/100ml)

Sorbitol 5

Yeast Extract 0.5

Agar 2

Distilled water 100ml

pH 6.2

VI. Identification of the isolates

The bacteria (bacilli) isolates from Pineapple and Jackfruit on CMC media

were recognized for cellulose degrading activity characterized and identified

(Halt et al., 1994). Similarly, three cellulose degrading yeasts isolates from CMC

were characterized and identified (Barnet et al., 1983).

Identification of bacterial & yeast isolates were done by microscopic

observation. The bacteria were also subjected to biochemical tests viz., IMViC,

(Appendix- V a & b) (Appendix- VI a, b &c) & (Appendix- VII) fermentation of

carbohydrates (Appendix- VIII a & b), using glucose, lactose, fructose, sucrose

and maltose and study of growth pattern. This was done to know the credibility

of bacterial isolates from both Pineapple and Jackfruit. (Madigan, et al.,1997).

VII. Collection, Preparation and Hydrolysis of Raw Material

Ripened Pineapple ‘Charlotte Rothchild’ variety and Jackfruit of ‘Beluva’ variety,

known for their soft textured bulbs were procured. The Pineapple and Jackfruit

were washed with tap water, immersed in a solution of 20% NaCl and 10% baking

soda to eliminate any adhered surface micro-flora. The Pineapple and Jackfruit

were skinned off the peel using a knife was disinfected with 70% ethanol. The

pulp of pineapple and edible fleshy part (bulbs) - (EFP) of Jackfruit were deseeded

aseptically within 24hrs of collection and stored at 4° C. The peel of pineapple and

the fleshy non edible part-(NEFP) of jackfruit are rich in cellulose fibers, but

generally discarded as waste or sometimes used as fodder were also used in the

study.

VIII. Preparation of Slurry

400g of peel and pulp of Pineapple and Jackfruit were crushed in an

electric grinder and diluted to 1000ml distilled and sterilized with hot water for 30

minutes.

IX. Acid pre-treatment

The mashed slurry was pre-treated with sulphuric acid at 30 ml of 4%

concentration and kept for 2 hours incubation period for hydrolysis of

fermentable sugars.

X. Microbial pre-treatment

The mashed substrate was inoculated with the 24 hr old culture of the

bacteria and yeast at the rate of 5 ml and incubated for 8 days on a rotary

shaker (130 rpm) for saccharification.

XI. Preparation of Inoculants

All the inoculants were previously prepared and maintained in a pre-

culture sterile broth of glucose for bacteria and sucrose for yeast, for 24hrs -

36hrs prior to inoculation in the main fermentation container. The inoculums

were maintained at OD660= 0.6.

XII. Chemical Analysis

Before fermentation could set in, the initial cellulose content, total

soluble solids - Brix, sugar content, pH, titrable acidity and volatile acidity were

performed for the pulp and peel mash. The pH was adjusted using citrate and

phosphate buffer. (Appendix- IX)

The mash were further mixed with 200 ppm Potassium-meta-bi-

sulphite and kept for 2-3 hours to remove/ suppress any remnants of wild

micro-flora. The mash was further supplemented with diammonium hydrogen

phosphate (0.5 g/l) as a source of nitrogen and phosphorus.

The hydrolysate from the pre-treatment was ameliorated to obtain

24°Brix or total soluble solids by diluting, using Abbe’s refractometer. About

250 w/v of the slurry of pineapple peel and jackfruit NEFP each were

introduced into separate reagent bottles and inoculated separately with the

yeast isolates and one culture strain-Saccharomyces.cerevisiae.var

ellipsoides.

Similarly, 250ml w/v of Pineapple peel and Jackfruit NEFP each were

introduced into different reagent bottles and then inoculated separately with

the three different cellulose degrading isolates and the one culture strain

bacteria – Cellulomonas uda.

XIII. Growth Studies of the Isolates

Eight conical flasks containing 100ml of growth medium for each

bacterial and yeast isolates were taken. Each flask was inoculated with a loop

full of the bacterial and yeast isolates along with the culture strains and

incubated in an orbital incubator shaker, Servewell, (India) at 140rpm at 29°C.

Absorbance of the growth medium was regularly noted at 530nm, at time

intervals of one hour after the third hour up to the eighth hour to record the

growth phase.

XIV. Fermentation

All the reagent bottles containing the fermented broth of pineapple and

Jackfruit peel and pulp were fermented anaerobically in a rotary incubator

shaker, at an agitation rate of 130rpm at 220 C for 96 hrs for pulp of pineapple

and EFP of Jackfruit and 21 days for peel of pineapple and NEFP of Jackfruit.

Similarly the fermentations were also carried out at 270 C, 320 C and 350 C to

know the optimum temperature yield. After complete fermentation each of the

fermented broth was filtered through double folded clean muslin cloth,

pasteurized and stored in a volumetric flask at 16°C. The clear supernatant

fermented broth was racked with fine Bentonite clay at 0.1 per cent (w/v) and

further drawn off into clean bottles and pasteurized at 63°C for 30 mins.

XV. ANALYTICAL METHODS

a. pH

pH of the samples were recorded using a pH digital meter of Analog

model (Biochem PM 79). Standard solutions of 4.0 and 7.0 were made using

buffer capsules (Merk) and used as reference to calibrate.

b. Total Titrable Acidity

Ten ml of the fermented broth was taken in 100 ml volumetric flask and

the volume made up. From this 10 ml of aliquot sample was taken in a 100 ml

conical flask and titrated against 0.1N sodium hydroxide solution using one to

two drops of phenolphthalein indicator.

Total titrable acidity was expressed as per cent citric acid (g/100 ml of

sample).

TV X Normality X 64 X100 X 100 % citric acid = ---------------------------------------------------------------------- Vol. of sample taken X Vol. of aliquot X 1000 X 10 X10

Tartaric acid & Acetic acid were determined as:

Volume of alkali x N of alkali x 7.5 Tartaric acid % = -------------------------------------------------

Wt of Sample

Volume of alkali x N of alkali x 6.0

Acetic acid % = ------------------------------------------------- Wt of Sample

c. Alcohol Content

The fermented broth of pineapple and jackfruit were fractionally distilled

at 72°C to recover ethanol. The distillate of ethanol was further estimated

using potassium dichromate method (Caputi et al, 1969). (Appendix)

d. Estimation of Reducing Sugar

Reducing sugar concentration was estimated at the end of 21 days

using U-V spectrophotometer (Elico SL- 150) at OD 540nm by the DNS method

(Miller G L, 1959).

e. Estimation of Cellulose Content

The concentration of Cellulose content was done by the Anthrone

method and results recorded at 630nm (Hedge and Hofreiter (1962).

f. Statistical Analysis

The estimations for experiments were completed in triplicates and the

standard deviation was < 0.2.

XVI. Preparation of Reagents

Estimation of Cellulose

1. Anthrone reagent

The reagent was freshly prepared by dissolving 200mg of anthrone

powder in 100 ml of 95 per cent sulphuric acid and chilled for two hours before

use.

2. Preparation of 67 per cent sulphuric acid

67ml of sulphuric acid was mixed in 33ml distilled water.

3. Acetic / Nitric acid reagent

150ml of 80 per cent acetic acid was mixed with 15ml of Conc. HNO3.

4. Preparation of standard stock cellulose solution

Standard stock solution was prepared by dissolving 100mg of Cellulose

(Hi Media) to 100ml in a volumetric flask.

Procedure

1g Sample was ground in a mortar and pestle to smoothness using cold

water. It was then mixed with 3ml acetic acid reagent using a vortex mixer.

The test tube containing the test sample was kept in a water bath for 30

mins. After cooling it was centrifuged at 2000rpm for 20mins. The

supernatant of acetone was decanted and discarded, while the residue of the

test sample was washed with distilled water. Next, 10 ml solution of 67

percent sulphuric acid solution was added and allowed to stand for one hour

in a beaker. The supernatant was decanted and washed with fresh cold water

for two times and again washed with 80 per cent ethanol at room temperature.

The sample was desiccated and then weighed. 50 mg of dried powder was

dissolved in 100 ml of distilled water. 1 ml was taken into test tubes and

volume was made to 1 ml using distilled water. A blank reagent with1 ml of

distilled water was kept. In the same way, other standards were also kept in

order in a serial from 40 mg to 200 mg of glucose concentration. Next, 4 ml

of anthrone reagent was added to each test tube containing the sample further

kept for boiling in hot water bath for 9 minutes. The samples were cooled

quickly and the colour intensity of the standards and the samples were

recorded as 630 nm by means of a UV/Visible spectrophotometer- Elico SL-

150. The standard curve of cellulose was plotted on graph.

Estimation of Reducing Sugars

Reducing sugars were estimated by following the 3, 5, Dinitrosalicylic

acid method (Miller,1959).

Preparation of Reagents

DNSA

One gram of 3, 5 dinitrosalicylic acid (DNSA), 200 mg of phenol crystal

and 50 mg of Sodium sulphite was dissolved in a 100 ml of 1 per cent NaOH.

The mixture was stored at 4°C. Given that, the reagent deteriorates owing to

sodium sulphite, whenever pro-longed storage was required, sodium sulphite

was added only at the time of use.

Rochelle salt solution - 40%

Rochelle salt solution was prepared by dissolving 40 g of potassium

sodium tartarate in 100 ml of distilled water.

Preparation of stock solution of glucose

Standard stock solution was prepared at 1 mg/ml by dissolving 100 mg

of D-glucose in distilled water using a volumetric flask. The final volume was

made up to 100 ml with distilled water.

Procedure

Test sample of 0.5 ml from acid and biologically pre-treated was taken

and conveyed into test tubes and volume made to 1.0 ml with distilled water.

The reagent blank containing 1 ml of distilled water was also kept. In the same

way, standards were also incorporated ranging from 10 mg to 100 mg

concentration of glucose. 0.5 ml of DNSA reagent was added to each

sample, mixed well and kept on boiling water bath for 5 mins. The contents of

the sample tubes were then mixed with 1 ml of 40 per cent Rochelle salt

solution before cooling and the volume made upto 25 ml using volumetric

flask.

Absorbance in terms of optical density of the standard and the sample were

recorded at 510 nm using UV/visible spectrophotometer- Elico SL-150. The

standard curve of glucose was plotted on graph.

Estimation of Total Sugars

Preparation of reagent in hydrochloric acid and NaOH

The normality of hydrochloric acid (0.1 and 1N) was calculated based

on specific gravity and per cent purity.

0.1N of sodium hydroxide was prepared by taking 0.4 grams of sodium

hydroxide and dissolving it in 50 ml distilled water and the final volume made

upto 100 ml in volumetric flask. It was standardized by titrating against

standard acid in presence of phenolphthalein indicator.

Phenolphthalein indicator

1 g of phenolphthalein powder was dissolved in 70% of 50 ml alcohol. The

final volume was made upto 100 ml using distilled water.

Procedure

1 ml of the representative fruit sample from the acid treatment was taken

in test tubes. One ml of 1N HCl was supplemented to each tube and then

positioned in boiling water bath for 15 minutes. After cooling the tubes, one

drop of phenolphthalein indicator was added to each tube. 1N NaOH was

added till a pale pink colour appeared followed by addition of 0.1 N HCl till the

pale pink colour disappeared. Finally samples were mixed well and volume

was made upto 5 ml with distilled water. Total sugar was estimated by DNSA

method by drawing out 0.5 ml of representative fruit sample taken from the

tubes.

Estimation of Ethanol

The ethanol was estimated by colorimetric method as described by Caputi

et al. (1968).

Preparation of Reagent

Potassium dichromate solution

34 of K2Cr2O7 were dissolved in 500 ml distilled water. To this solution

325 ml of Conc. sulphuric acid was added. The volume was made up to 1000

ml with distilled water to give K2Cr2O7 of 0.23N.

Preparation of Standard stock solution

Hundred per cent pure analytical grade ethanol (789 mg/ml) was

prepared by dissolving 12.6 ml of ethanol in 100 ml distilled water, which

results in 100 mg/ml of standard ethanol. From these, different percentage of

ethanol was prepared in the range from 1% to 11%.

Preparation of silver nitrate solution

0.1 N of AgNO3 was prepared by adding 1.698gmol-1 in 100ml distilled

water.

Procedure

5 ml of representative fruit samples of the fermented broth was diluted

with 30 ml distilled water and transferred to a round bottom distillation flask of

250 ml, which was further connected to the condenser. The sample was

distilled at 72°C. The distillate of 5ml was collected in 25 ml of 0.23 N

K2Cr2O7 reagents, which was kept at receiving end.

Similarly standards (20-100 mg ethanol) were mixed with 25 ml of

K2Cr2O7 separately. To this mixture 2ml of AgNO3 was added to catalyse the

reaction. The distillate of samples and standards were heated in water bath at

600C for 20 minutes and cooled. A blank was prepared by adding 5ml water to

25ml K2Cr2O7 along with 2ml AgNO3.

The volume was made upto 50 ml with distilled water and the optical

density was measured at 520 nm using U-V spectrophotometer. The standard

curve was plotted by taking into consideration the concentration against

absorbance.

RESULTS

I. Isolation of Microbes from Pineapple Peel

The fungi isolated from the pineapple peel included Aspergillus niger, Botrytis

Sps., Curvularia, Penicillium, Trichoderma on MRBA media. ((Table 1) (Plate 6a

& 6b)

Twenty two colonies of bacteria were isolated on nutrient aga . Of which, six

isolates were bacilli, 15 cocci and one diplo-cocci. (Table 2) (Plate 7a & 7b)

Twelve colonies of yeasts were isolated on SDA media. (Table 3) (Plate 8a &

8b)

II. Isolation of Microbes from Jackfruit Peel as Source

The fungi isolated from the jackfruit peel included Sporotrichium Sps.,

Trichothecium, Geotrichum, Dreschlera. Sps, Fusarium. Sps, and Aspergillus.

Sps on MRBA media. . (Table 4) (Plate 9a & 9b)

Twenty three colonies of bacteria were isolated on nutrient agar media. Of

these, five isolates were bacilli, 16 cocci and two diplo-cocci. . (Table5) (Plate

10a & 10b)

Sixteen colonies of yeasts were isolated on SDA media. . (Table 6) (Plate 11a

& 11b)

III. Biochemical Tests for Bacterial Isolates from Pineapple Peel

Of the 22 bacterial isolates, 21 bacteria showed response and reactions to IMViC

tests. All the 22 bacteria fermented glucose, maltose and sucrose but were

unable to ferment lactose as the sole source of carbon. Among the three

cellulose degrading bacteria - PPP04, PPP13 & PPP16, was identified for the

studies.

Isolate PPP04 showed positive to Methyl red and Simmon’s citrate test.

Although it also showed positive for growth, acid and gas production to glucose

and maltose fermentation tests, it however it showed only growth for Sucrose

and lactose fermentation tests. The isolate PPP13 showed positive for VP test

and exhibited growth for all the four sugars used for the carbohydrate

fermentation test. The isolate PPP16 was positive for VP and Simmon’s citrate

test. It exhibited growth in all the four sugars used and produced acid during

glucose and maltose fermentation. Gas production was seen only in glucose

fermentation. (Table 7) (Plate 12a, b, c & d)

IV. Biochemical Tests of Bacterial Isolates from Jackfruit Peel

Of the 23 bacterial isolates, all 23 bacteria showed response reactions

to IMViC tests. All the 23 bacteria fermented glucose, maltose and sucrose and

were unable to ferment lactose as the sole source of carbon. Among the three

cellulose degrading bacteria – CKJB01, CKJB08 & CKJB17, was identified for

the study.

Isolate CKJB01 was positive to Indole test. It also showed positive for

growth and acid production to glucose and maltose fermentation tests. It however

showed only growth for sucrose and lactose fermentation tests, whereas the

isolate CKJB08 was positive to Simmon’s Citrate test and exhibited growth for all

the four sugars used for fermentation. Production of gas was observed in

glucose, sucrose and maltose for the carbohydrate fermentation. The isolate

CKJB17 was positive for Indole, Methyl red and VP test. It showed growth and

acid production for glucose, sucrose and maltose. In Lactose fermentation only

growth observed. (Table 8) (Plate 13a, b, c & d)

V. Isolation of Cellulose utilizing bacterial & yeast isolates from

pineapple and jackfruit peel, on the basis of Hydrolysis Capacity (HC)

value.

A. Hydrolysis of Cellulose by Bacterial Isolates from Pineapple

Peel. (Fig 1a) (Plate 14)

After an incubation period of 36 hours, the following results were recorded.

The radius of the zone of hydrolysis for the positive strain C.uda was 0.77cms

when grown on Carboxy methyl cellulose media, while the radius of the zone of

hydrolysis for the bacterial isolate PPP04 was 0.75cms. The radius of the zone of

hydrolysis for the bacterial isolate PPP13 was 0.43cms, while the radius of the

zone of hydrolysis for the bacterial isolate PPP16 was 0.52cms when grown on

Carboxy methyl cellulose media.

B. Hydrolysis of Cellulose by Yeast Isolates from Pineapple Peel.

(Fig 1b) (Plate 15)

After an incubation period of 36 hours, the following results were recorded.

The radius of the zone of hydrolysis for the positive yeast strain

Saccharomyces cerevisiae var ellipsoides was 0.20 cms when grown on Carboxy

methyl cellulose media, while the radius of the zone of hydrolysis for the yeast

isolate YP02 was 0.76cms. The radius of the zone of hydrolysis for the yeast

isolate YP06 was 0.60cms. The radius of the zone of hydrolysis for the yeast

isolate YP11 was 0.22cms when grown on Carboxy methyl cellulose media and

the radius of the zone of hydrolysis for the yeast isolate YP12 recorded was

0.35cms.

C. Hydrolysis of Cellulose by Bacterial Isolates from Jackfruit Peel.

(Fig 2a) (Plate 16)

After an incubation period of 36 hours, the following results were recorded.

The diameter of the zone of hydrolysis for the positive strain C.uda was

4.5cms when grown on Carboxy methyl cellulose media, while the diameter of

the zone of hydrolysis for the bacterial isolate CKJB01 was 4.30cms. The

diameter of the zone of hydrolysis for the bacterial isolate CKJB08 was 3.6cms

and the diameter of the zone of hydrolysis for the bacterial isolate CKJB17

recorded was 4.2cms.

D. Hydrolysis of Cellulose by Yeast Isolates from Jackfruit Peel. (Fig

2b) (Plate 17)

After an incubation period of 36 hours, the following results were recorded.

The diameter of the zone of hydrolysis for the positive yeast strain

Saccharomyces cerevisiae var ellipsoides was 0.37 cms when grown on Carboxy

methyl cellulose media, while the diameter of the zone of hydrolysis for the yeast

isolate YP02 was 4.1cms. The diameter of the zone of hydrolysis for the yeast

isolate YP06 was 3.4cms and the diameter of the zone of hydrolysis for the yeast

isolate YP11 recorded was 3.8cms.

VI. Fermentation Studies.

Before inoculating the pretreated media with the different isolates and

strains, an initial physical and biochemical estimations tests were conducted for

both pineapple and jackfruit mash. (Table 9 & 10)

1. Pineapple peel: The Initial pH was maintained at 4.2, with a Brix of 19, a sugar

concentration of 10 per cent, cellulose percent of 1700(µg/ml) and percent of

tartaric acid, acetic acid and citric acid was maintained at 0.102%, 0.081% and

0.163% respectively. (Plate 18)

2. Pineapple pulp: The Initial pH was maintained at 4.5, with a Brix of 21, a sugar

concentration of zero per cent, cellulose percent of 1040(µg/ml) and percent of

tartaric acid, acetic acid and citric acid was maintained at 0.192%, 0.153% and

0.087% respectively. (Plate 19)

3. Jackfruit NEFP: The Initial pH was maintained at 6.3, with a Brix of 22, a sugar

concentration of 10 per cent, cellulose percent of 1900(µg/ml) and percent of

tartaric acid, acetic acid and citric acid was maintained at 0.112%, 0.090% and

0.096% respectively. (Plate 20)

4. Jackfruit EFP: The Initial pH was maintained at 5.8, with a Brix of 24, a sugar

concentration of zero per cent, cellulose percent of 1400(µg/ml) and percent of

tartaric acid, acetic acid and citric acid was maintained at 0.21%, 0.168% and

0.192% respectively. (Plate 21)

VII. Estimation of Cellulose

A. Estimation of Cellulose in Pineapple Peel Wastes. (Fig 3a) (Plate 22)

The concentration of cellulose in pineapple peel wastes were estimated by the

anthrone method after the completion of fermentation by the bacterial isolates

and the result recorded are as follows.

1. The concentration of cellulose that remained after degradation of pineapple peel

wastes by the positive strain C. uda was 284.49µg/ml, 159.13µg/ml, 275.75µg/ml

and 167.86µg/ml, at 22°C, 27°C, 32°C and 35°C respectively.

2. The concentration of cellulose that remained after degradation of pineapple peel

wastes by the bacterial isolate PPP04 was 249.55µg/ml, 193.55µg/ml,

135.6µg/ml and 94.39µg/ml, at 22°C, 27°C, 32°C and 35°C respectively.

3. The concentration of cellulose that remained after degradation pineapple peel

wastes by the bacterial isolate PPP13 was 374.91µg/ml, 331.76µg/ml,

154.5µg/ml and 258.21µg/ml, at 22°C, 27°C, 32°C and 35°C respectively.

4. The concentration of cellulose that remained after degradation of pineapple peel

wastes by the bacterial isolate PPP16 was 405.35µg/ml, 275.75µg/ml,

241.33µg/ml and 150.39µg/ml, at 22°C, 27°C, 32°C and 35°C respectively.

B. Cellulose estimation in Pineapple Pulp Wastes (Fig 3a)

The concentration of cellulose in pineapple pulp wastes were estimated by the

anthrone method after the completion of fermentation by the yeast isolates and

the result recorded are as follows.

1. The concentration of cellulose that remained after degradation of pineapple pulp

wastes by the positive strain Saccharomyces cerevisiae var ellipsoides was

409.85µg/ml, 573.75µg/ml, 504.9µg/ml and 413.96µg/ml, at 22°C, 27°C, 32°C

and 35°C respectively.

2. The concentration of cellulose that remained after degradation of pineapple pulp

wastes by the yeast isolate YP02 was 227.97µg/ml, 612.49µg/ml, 469.16µg/ml

and 435.54µg/ml, at 22°C, 27°C, 32°C and 35°C respectively.

3. The concentration of cellulose that remained after degradation of pineapple pulp

wastes by the yeast isolate YP06 was 556.79µg/ml, 660.06µg/ml, 763.84µg/ml

and 396.49µg/ml, at 22°C, 27°C, 32°C and 35°C respectively.

4. The concentration of cellulose that remained after degradation pineapple pulp

wastes by the yeast isolate YP11 was 794.16µg/ml, 686µg/ml, 794.16µg/ml and

496.17µg/ml, at 22°C, 27°C, 32°C and 35°C respectively.

5. The concentration of cellulose that remained after degradation of pineapple pulp

wastes by the yeast isolate YP12 was 453.01µg/ml, 711µg/ml, 699.11µg/ml and

323.03µg/ml, at 22°C, 27°C, 32°C and 35°C respectively.

C. Cellulose Estimation in Jackfruit Peel Wastes. (Fig 4a) (Plate 23)

The concentration of cellulose in jackfruit peel wastes were estimated by the

anthrone method after the completion of fermentation by the yeast isolates and

the result recorded are as follows.

1. The concentration of cellulose that remained after degradation of jackfruit peel

wastes by the positive strain C. uda was 224.38µg/ml, 206.39µg/ml, 56.88µg/ml

and 101.07µg/ml, at 22°C, 27°C, 32°C and 35°C respectively.

2. The concentration of cellulose that remained after degradation of jackfruit peel

wastes by the bacterial isolate CKJB01 was 188.93µg/ml, 87.2µg/ml, 78.46µg/ml

and 83.09µg/ml, at 22°C, 27°C, 32°C and 35°C respectively.

3. The concentration of cellulose that remained after degradation of jackfruit peel

wastes by the bacterial isolate CKJB08 was 164.78µg/ml, 188.93µg/ml,

105.69µg/ml and 140.63µg/ml, at 22°C, 27°C, 32°C and 35°C respectively.

4. The concentration of cellulose that remained after degradation of jackfruit peel

wastes by the bacterial isolate CKJB17was 254.69µg/ml, 161.18µg/ml, 24µg/ml

and 193.04µg/ml, at 22°C, 27°C, 32°C and 35°C respectively.

D. Cellulose Estimation in Jackfruit Pulp Wastes. (Fig 4b)

The concentration of cellulose in jackfruit pulp wastes were estimated by the

anthrone method after the completion of fermentation by the yeast isolates and

the result recorded are as follows.

1. The concentration of cellulose that remained after degradation of jackfruit pulp

wastes by the positive strain Saccharomyces cerevisiae var ellipsoides was

74.35µg/ml, 142.69µg/ml, 164.78µg/ml and 54.83µg/ml, at 22°C, 27°C, 32°C and

35°C respectively.

2. The concentration of cellulose that remained after degradation of jackfruit pulp

wastes by the yeast isolate CKJY02 was 101.07µg/ml, 97.07µg/ml, 81.03µg/ml

and 37.36µg/ml, at 22°C, 27°C, 32°C and 35°C respectively.

3. The concentration of cellulose that remained after degradation of jackfruit pulp

wastes by the yeast isolate CKJY06 was 118.54µg/ml, 116.48µg/ml, 3.97µg/ml

and 89.77µg/ml, at 22°C, 27°C, 32°C and 35°C respectively.

4. The concentration of cellulose that remained after degradation of jackfruit pulp

wastes by the yeast isolate CKJY11 was 131.9µg/ml, 128.49µg/ml, 101.07µg/ml

and 67.67µg/ml, at 22°C, 27°C, 32°C and 35°C respectively.

VIII. Reducing Sugars

The reducing sugars formed by the bacterial and yeast isolates at the end of

fermentation was estimated by DNS method and the results obtained are as

follows:

A. Reducing Sugars formed by Bacterial Isolates in Pineapple Peel

Wastes.

1. The concentration of glucose formed after degradation of pineapple peel wastes

by the positive strain C. uda was 1758.97µg/ml, 1482.24µg/ml, 1085.36µg/ml and

1063.8µg/ml, at 22°C, 27°C, 32°C and 35°C respectively.

2. The concentration of glucose formed after degradation of pineapple peel wastes

by the bacterial isolate PPP04 was 921.01µg/ml, 1435g/ml, 1704.29µg/ml and

1633.69µg/ml, at 22°C, 27°C, 32°C and 35°C respectively.

3. The concentration of glucose formed after degradation of pineapple peel wastes

by the bacterial isolate PPP13 was 1495.63µg/ml, 1612.13µg/ml, 700.81µg/ml

and 1521.26µg/ml, at 22°C, 27°C, 32°C and 35°C respectively.

4. The concentration of glucose formed after degradation of pineapple peel wastes

by the bacterial isolate PPP16 was 1063.8µg/ml, 1137.34µg/ml, 1702.49µg/ml

and 1590.09µg/ml, at 22°C, 27°C, 32°C and 35°C respectively. (Plate 24) (Fig

5a)

B. Reducing Sugars formed by Yeast Isolates in Pineapple Pulp Wastes.

1. The concentration of glucose formed after degradation of pineapple pulp wastes

by the positive strain Saccharomyces cerevisiae var ellipsoides was

1024.78µg/ml, 1098.2µg/ml, 1646µg/ml and 1059.38µg/ml, at 22°C, 27°C, 32°C

and 35°C respectively.

2. The concentration of glucose formed after degradation pineapple pulp wastes

by the yeast isolate YP02 was 1603.4µg/ml, 1655.26µg/ml, 1732.88µg/ml and

921.07µg/ml, at 22°C, 27°C, 32°C and 35°C respectively.

3. The concentration of glucose formed after degradation pineapple pulp wastes by

the Yeast isolate YP06 was 528.3µg/ml, 472.32µg/ml, 472.32µg/ml and

739.83µg/ml, at 22°C, 27°C, 32°C and 35°C respectively.

4. The concentration of glucose formed after degradation pineapple pulp wastes by

the yeast isolate YP11 was 1245.05µg/ml, 752.67µg/ml, 752.67µg/ml and

869.21µg/ml, at 22°C, 27°C, 32°C and 35°C respectively.

5. The concentration of glucose formed after degradation pineapple pulp wastes by

the yeast isolate YP12 was 476.42µg/ml, 1542.88µg/ml, 1197µg/ml and

523.68µg/ml, at 22°C, 27°C, 32°C and 35°C respectively. (Fig 5b)

C. Reducing Sugars formed by Bacterial Isolates in Jackfruit Peel

Wastes.

1. The concentration of glucose formed after degradation of jackfruit peel wastes by

the positive strain C. uda was 917.05µg/ml, 280.95µg/ml, 217.86µg/ml and

630.4µg/ml, at 22°C, 27°C, 32°C and 35°C respectively.

2. The concentration of glucose formed after degradation of jackfruit peel wastes by

the bacterial isolate CKJB01 was 903.41µg/ml, 440.23µg/ml, 340.74µg/ml and

552.48µg/ml, at 22°C, 27°C, 32°C and 35°C respectively.

3. The concentration of glucose formed after degradation of jackfruit peel wastes by

the bacterial isolate CKJB08 was 809.47µg/ml, 417.05µg/ml, 265.32µg/ml and

407.14µg/ml, at 22°C, 27°C, 32°C and 35°C respectively.

4. The concentration of glucose formed after degradation of jackfruit peel wastes by

the bacterial isolate CKJB17 was 875.19µg/ml, 425.69µg/ml, 230.4µg/ml and

301.89µg/ml, at 22°C, 27°C, 32°C and 35°C respectively. (Plate 25) (Fig 6a)

D. Reducing Sugars formed by Yeast Isolates in Jackfruit Pulp Wastes.

1. The concentration of glucose formed after degradation of jackfruit pulp wastes by

the positive strain Saccharomyces cerevisiae var ellipsoides was 67.56µg/ml,

169.4µg/ml, 250.58µg/ml and 769.5µg/ml, at 22°C, 27°C, 32°C and 35°C

respectively.

2. The concentration of glucose formed after degradation of jackfruit pulp wastes by

the yeast isolate CKJY02 was 180.19µg/ml, 140.63µg/ml, 149.36µg/ml and

624.1µg/ml, at 22°C, 27°C, 32°C and 35°C respectively.

3. The concentration of glucose formed after degradation of jackfruit pulp wastes

by the Yeast isolate CKJY06 was 318.77µg/ml, 34.79µg/ml, 105.18µg/ml and

584.53µg/ml, at 22°C, 27°C, 32°C and 35°C respectively.

4. The concentration of glucose formed after degradation of jackfruit pulp wastes by

the yeast isolate CKJY11 was 353.47µg/ml, 136.01µg/ml, 118.54µg/ml and

343.53µg/ml, at 22°C, 27°C, 32°C and 35°C respectively. (Fig 6b)

IX. Ethanol Content

At optimum pH, incubation period, Brix and temperature, the percentage of

ethanol are as follows.

A. Percentage of Ethanol formed by Bacterial Isolates in Pineapple Peel

Wastes. (Fig 7a) (Plate 26)

1. The percentage of ethanol formed after degradation of pineapple peel wastes by

the positive strain C. uda was 2%, 3.5%, 5.1% and 1.8%, at 22°C, 27°C, 32°C

and 35°C respectively.

2. The percentage of ethanol formed after degradation of pineapple peel wastes by

the bacterial isolate PPP04 was 2.4%, 3.1%, 4.8% and 1.9%, at 22°C, 27°C,

32°C and 35°C respectively.

3. The percentage of ethanol formed after degradation of pineapple peel wastes by

the bacterial isolate PPP13 was 2.5%, 3.9%, 4.9% and 1.2%, at 22°C, 27°C,

32°C and 35°C respectively.

4. The percentage of ethanol formed after degradation of pineapple peel wastes by

the bacterial isolate PPP16 was 2.6%, 4.7%, 4.3% and 2.1%, at 22°C, 27°C,

32°C and 35°C respectively.

B. Percentage of Ethanol formed by Yeast Isolates on Pineapple Pulp

Wastes. (Fig 7b) (Plate 27)

1. The percentage of ethanol formed after degradation of pineapple pulp wastes by

the positive strain Saccharomyces cerevisiae var ellipsoides was 12.8%, 12.3%,

10.9% and 2.2%, at 22°C, 27°C, 32°C and 35°C respectively.

2. The percentage of ethanol formed after degradation of pineapple pulp wastes by

the yeast isolate YP02 was 11.8%, 10.1%, 9.6% and 3.5%, at 22°C, 27°C, 32°C

and 35°C respectively.

3. The percentage of ethanol formed after degradation of pineapple pulp wastes by

the Yeast isolate YP06 was 12.1%, 11.9%, 10.3% and 4.1%, at 22°C, 27°C,

32°C and 35°C respectively.

4. The percentage of ethanol formed after degradation of pineapple pulp wastes by

the yeast isolate YP11 was 8.5%, 7.8%, 6.9% and 3.5%, at 22°C, 27°C, 32°C

and 35°C respectively.

5. The percentage of ethanol formed after degradation of pineapple pulp wastes by

the yeast isolate YP12 was 9.2%, 8.3%, 7.7% and 3.1%, at 22°C, 27°C, 32°C

and 35°C respectively.

C. Percentage of Ethanol formed by Bacterial Isolates on Jackfruit Peel

Wastes. (Fig 8a) (Plate 28)

1. The percentage of ethanol formed after degradation of jackfruit peel wastes by

the positive strain C. uda was 2.18%, 6.15%, 5.37% and 3.81%, at 22°C, 27°C,

32°C and 35°C respectively.

2. The percentage of ethanol formed after degradation of jackfruit peel wastes by

the bacterial isolate CKJB01 was 1.29%, 1.6%, 2.6% and 1.2%, at 22°C, 27°C,

32°C and 35°C respectively.

3. The percentage of ethanol formed after degradation of jackfruit peel wastes by

the bacterial isolate CKJB08 was 3.87%, 3.89%, 5.2% and 4.8%, at 22°C, 27°C,

32°C and 35°C respectively.

4. The percentage of ethanol formed after degradation of jackfruit peel wastes by

the bacterial isolate CKJB17 was 2.02%, 6.02%, 5.06% and 3.6%, at 22°C,

27°C, 32°C and 35°C respectively.

D. Percentage of Ethanol formed by Yeast Isolates in Jackfruit Pulp

Wastes. (Fig 7a) (Plate 28)

1. The percentage of ethanol formed after degradation of jackfruit pulp wastes by

the positive strain Saccharomyces cerevisiae var ellipsoides was 14.92%,

10.73%, 12.61% and 9.61%, at 22°C, 27°C, 32°C and 35°C respectively.

2. The percentage of ethanol formed after degradation of jackfruit pulp wastes by

the yeast isolate CKJY02 was 10.09%, 9.9%, 10.34% and 7.32%, at 22°C, 27°C,

32°C and 35°C respectively.

3. The percentage of ethanol formed after degradation of jackfruit pulp wastes by

the Yeast isolate CKJY06 was 10.16%, 14.57%, 12.05% and 9.67%, at 22°C,

27°C, 32°C and 35°C respectively.

4. The percentage of ethanol formed after degradation of jackfruit pulp wastes by

the yeast isolate CKJY11 was 11.12%, 9.6%, 12.12% and 8.04%, at 22°C, 27°C,

32°C and 35°C respectively.

X. Vinegar Content (Table 11 & 12) (Fig 9 & 10) (Plate 29 & 30)

The percentage of acetic acid formed in Pineapple and Jackfruit peel

wastes after inoculation with Acetobacter aceti and Acetobacter xylinum over a

period of 1-8 days recorded are as follows:

A. Pineapple fruit Peel ethanol inoculated with Acetobacter aceti yielded acetic acid

beginning from 0.95%, 1.17%, 1.93%, 2.45%, 2.98%, 3.56%, 3.92% and 3.92%,

at 28°C respectively.

B. Pineapple fruit peel ethanol inoculated with Acetobacter xylinum yielded acetic

acid beginning from 1.13%, 2.48%, 3.08%, 3.19%, 3.21%, 3.21% and 3.21%, at

28°C respectively.

C. Jackfruit ethanol inoculated with Acetobacter aceti yielded acetic acid beginning

from 0.44%, 0.61%, 0.98%, 1.29%, 1.95%, 2.12%, 2.71% and 3.39%, at 28°C

respectively.

D. Jackfruit ethanol inoculated with Acetobacter xylinum yielded acetic acid

beginning from 0.86%, 1.22%, 1.89%, 2.43%, 2.77%, 2.92% and 3.01%, at 28°C

respectively.

DISCUSSION

Many scientific studies have shown profound interests in cellulases for

the reason that they have many applications in industries like animal and food

production, starch processing, malting and brewing, textile industry, extraction

of vegetable and fruit juices, pulp and paper industry, (Kaur et al., 2007; Gao

et al., 2008). A lot of cellulolytic microbes and their enzyme systems include

extensive studies for the enzymatic conversion of cellulosic substances

(Boonmak et al., 2011, Gilkes et al., 1991; Bey et al., 2011). The cellulases

are primarily extracellular enzymes produced by thermophilic or mesophilic

microbes (Kim et al., 2005).

A range of agricultural substrates by-products and microbial cultures

have been used effectively for cellulases production (Yang et al., 2006). A

variety of bacterial species are able to hydrolyse cellulose and being

prokaryotes have different optimal growth conditions. Bacterial cellulases

have a wide range of pH and temperatures to degrade cellulose as when

compared to yeast cellulases.

In the present study, from among the bacterial and yeast isolates from

pineapple peel, the bacterial isolate PPP04 showed a maximum zone of

0.75cm radius, while the positive strain showed 0.77cm radius. From among

microbes from jackfruit peel and culture strains, the microorganism which

showed maximum zone of hydrolysis on CMC media was the bacterial isolate

CKJB17 with a zone of 4.2cm in diameter, while the positive strain C.uda

showed 4.4cm hydrolysis on CMC media.

In the current study, the bacterial isolate PPP04 from pineapple peel

hydrolysed maximum quantity of cellulose from the initial amount of 1700µg/ml

to 94.39 µg /ml at 35°C. The isolates PPP16, PPP13 and the positive strain

C.uda with about 160 µg/ml of residual cellulose between 32°C to 35°C,

indicated that the bacterial isolates and strains were active above 32°C. The

isolate PPP16 hydrolysed the least amount of cellulose at 22°C as the

residual cellulose remained at 405.35 µg /ml.

Amongst yeast isolates, the isolate YP2 had a residual cellulose

content of 227.97 µg /ml. This was followed by the isolate YP12 with 323.03

µg/ml of residual cellulose. For all the yeast isolates and culture strain

Saccharomyces the temperature of 22°C was the ideal temperature for the

breakdown of cellulose.

Cellulose degradation by bacterial isolates from Jackfruit peel showed

maximum hydrolysis by the isolate CKJB01 having residual cellulose less than

100 µg/ml. The Isolate CKJB17 had residual sugar of 24 µg/ml. For most of

the isolates and the strains the temperature of 32°C was the ideal one.

Among yeast isolates from Jackfruit peel, the isolate CKJY06 had

the least residual cellulose of 3.97µg/ml, at a temperature of 32°C. The most

active isolate was CKJY02 which had residual cellulose content of less than

100 µg/ml and exhibited activity at all four temperatures chosen. C.uda

showed good cellulose hydrolysis in jackfruit peel failed to show similar results

in the jackfruit pulp and the residual cellulose was found to be 160µg/ml.

In general all the isolates from pineapple peel and jackfruit pulp,

bacterial isolates seemed to be having better cellulose degradation ability than

the yeast isolates.

Quantity of reducing sugar utilization and formation of ethanol differs in

a variety of sugars like mono & disaccharides. This is for the reason that

monosaccharide like glucose are carried across the cell membrane by a

simple and smooth transport mechanism. Disaccharides like sucrose is

transformed to glucose and fructose at the surface of the cell using invertase

present in the cell wall of yeast (Johannes, P., et al., 1994).

The maximum residual sugar formed in the pineapple peel fermented

broth was by the bacterial isolate PPP04 was 1704.29 µg/ml at 32°C,

compared to the positive culture strain Cellulomonas uda at 1758.97 µg/ml at

22°C. For all the isolates the temperature of 32°C was the ideal one with an

inoculums size of 1.6x 107 cells /ml.

In the same way, in the pineapple pulp fermented broth, the yeast

isolate YP02 formed the maximum residual sugar at 1732.88µg/ml at both

32°C, followed by Saccharomyces strain at 1646 µg/ml at 32°C. All the yeast

isolates’ activity reduced at 35°C. The isolate YP02 was active at all the

three temperatures of 22°C, 27°C and 32°C.

The amount of residual sugar formed in the jackfruit peel fermented

broth by the bacterial isolate CKJB17 was 250 µg/ml at 32°C, followed by the

culture strain Cellulomonas uda at 350 µg/ml at 32°C. For all the isolates the

temperature of 27°C was the ideal one with an inoculums size of 1.6x 107 cells

/ml.

In the same way, in the jackfruit pulp fermented broth, the yeast isolate

CKJY06 formed the least residual sugar at 40µg/ml at 27°C, followed by

Saccharomyces strain at 70µg/ml at 22°C. All the yeast isolates activity

reduced at 35°C. The isolate CKJY02 was active at all the three

temperatures of 22°C, 27°C and 32°C.

With enhanced inoculum level, the residual sugar content diminished

significantly in all the isolates and culture strains. This could be due to the

cause that quantity of sugar uptake and the conversion rate by the yeast cells

dependent on the high affinity and low affinity systems. The affinity uptake

depends upon the subsistence of hexokinase and the active SNF3 gene, while

the low affinity uptake is kinase independent, constitutive and facilitated

diffusion process.

Percentage of ethanol formed after the fermentation of pineapple and

jackfruit pulp was more compared to the peel fermentation. Maximum ethanol

formed in pineapple pulp was 12.1% by the yeast isolate YP06 at 22°C, while

in jackfruit pulp it was 14.57% by the yeast isolate CKJY06 at 27°C when

compared to the positive strain Saccharomyces cerevisiae var ellipsoides

which yielded 12.8% in pineapple at 22°C and 14.97% at 22°C in jackfruit

respectively. Maximum ethanol content in Pineapple and jackfruit peel was

5.1% and 6.15% by the positive strain Cellulomonas uda at 32°C and 27°C

respectively. The bacterial isolate PPP13 from pineapple yielded maximum

ethanol of 4.9% at 32°C, while in jackfruit peel, the bacterial isolateCKJB17

yielded 6.02% ethanol at 27°C.

As per the studies of Ndoye et al. (2006), acetic acid bacteria are

Gram negative, strictly aerobic and frequently originate in nature on different

plants (fruits, grains, herbs etc). Acetobacter is oxidase positive.

Fermentation of vinegar by simple batch process is quite slow and needs 4 to

5 weeks for completing the fermentation process (Ethiraj and Suresh, 1990).

In modern submerged fermentation processes called acetators, the final acetic

acid concentration is upto 1.7°depending upon certain strains of bacteria

(Ndoye, 2007).

The normal acidity of vinegar of pH 2.8 is due to the acetic acid present.

The acetic acid, butyric acid and the propionic acid from the basic raw material

together form the volatile acids, which interfere with the quality, final acidity

and flavor of vinegar (Yang and Choong, 2001). As per Walter (2005), apart

from acetic acid formed certain other organic acids like tartaric acid, succinic

acid and citric acid etc., contribute to the overall acidity of the vinegar.

In the present study, vinegar was successfully produced from both

pineapple peel and jackfruit peel alcoholic beverage. Two culture strains were

used as inoculums for vinegar production viz., Acetobacter aceti and

A.xylinum. Of the two strains, A.aceti gave a higher yield of vinegar of 3.92

percent in pineapple alcoholic beverage as against 3.39 percent vinegar in

jackfruit by the eighth day of fermentation. The strain A.xylinum comparatively

produced lesser percent of vinegar of 3.21 percent in pineapple and 3.01

percent in jackfruit alcoholic beverage. Also the percentage of vinegar formed

by the eighth day of fermentation in pineapple peel was higher in contrast to

jackfruit.

SUMMARY

Alcoholic beverages have been an important part of individual being,

spiritual and civilizing life throughout various societies of the world. In the

Middle East and European countries, indigenous alcoholic beverages are

produced chiefly from fruit. In the Asia-Pacific region alcoholic beverages are

produced from cereals and provide an important source of nutrients. Rice

wine is popular in countries like Korea, Vietnam, Japan, China, Philippines,

etc.

When compared to fruit alcoholic beverages, fruit peel alcoholic

preparation has an additional step i.e., saccharification of cellulose to simple

sugars. The present investigation was carried out to hydrolyse cellulose to

fermentable monomers, as fruit peel is rich in cellulose content, optimization of

the pre-treatment conditions with acidic and enzymatic treatment for obtaining

maximum reducing sugars for alcoholic preparation was done.

The initial cellulose content of pineapple/ jackfruit peel and pulp varied

significantly. The pineapple peel had an initial cellulose content of

1700µg/ml, while the jackfruit peel had 1900µg/ml. In the same way, the initial

cellulose content recorded in pineapple pulp was 1040µg/ml, whereas in

jackfruit pulp it was 1400µg/ml.

To know the efficiency of the pre-treatment, cellulose hydrolysis and

reducing sugars were estimated. The acidic and enzymatic pre-treatment

methods employed showed positive influence on the release of reducing

sugars. In case of acid pre-treatment, there was significant increase in the

release of reducing sugars. Acid concentration of 4 per cent coupled with 2

hours of incubation showed maximum release of reducing sugars.

It is to be concluded that using pineapple and jackfruit wastes, both the

bacterial and yeast isolates were able to adapt to the pineapple and jackfruit

raw material faster than the culture strains. The bacterial isolates were not

able to ferment sucrose but were able to ferment lactose, glucose & maltose.

The yeast isolates readily fermented sucrose. Although all the bacterial and

yeast isolates were able to hydrolyze cellulose and also expressed ethanol

fermentation, individually their capabilities differed. The bacterial isolate

CKJB01 showed high cellulose degrading ability but it could not ferment

efficiently compared to other bacterial isolates. The cellulose hydrolyzing

ability of the yeast isolate CKJY06 was less but its fermentation ability for

ethanol yield was high. Thus, there arises the need for isolating or genetically

manipulating a microbe of both high cellulose hydrolyzing and ethanol

fermenting ability. The concept of using bio-ethanol from Pineapple and

Jackfruit wastes could help to clean the surroundings from wastes and also

overcome the problems of fossil fuel exhaustion by forming renewable energy.

It can also encourage the farmers of jackfruit plantations in the development of

bio-ethanol that has higher commercial value.

Bioethanol present a possible replacement to fossil fuels, along with

the added advantage of decreased CO2 emissions in the atmospheric. Major

CO2 emissions from burning of bio-fuels are again reabsorbed by the second

generation of plant feedstock which is used to produce the fuel, thereby

effectively concluding the carbon cycle. Thus the transportation segment

could become a globally well-organized neutral in carbon.

As a result of the recognition that pineapple and jackfruit are

underutilised fruits that has a significant possibility for income generation,

alleviating malnutrition, and contributing to sustainability which would

eventually increase the cultivation of both pineapple and jackfruit. These fruit

wastes hence could be converted into usable end products for the betterment

of mankind and to ensure the utilization of wastes, thereby reducing the

accumulation of these wastes which would otherwise simply remain unnoticed

and unaltered unless deterioration by natural processes occurs only leading to

the degradation without much relevance to the utilization of the degraded

wastes. Hence, the present study reveals the utilization of these wastes to be

harnessed, converted and degraded by the process of fermentation to

produce bio-fuel, which over time may be put to effective use by commercial

establishments. Further, the vinegar produced could also be put to effective

use in areas required.

It is also to be considered that the process needs improvisation and

technical assistance along with marketing strategy and therefore there is

scope for much research and findings in this field, which would also require

expertise from multidisciplinary sciences to make this experimental work to be

put to effective use.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

    Table‐1  Isolation of  Fungi from Pineapple Peel & Pulp  

Sample   

Media   

Dilution & incubation   

No.of    

 Types of   Colony   

Pigment of Colony 

Size of  Colonies in mm 

Name of   Organism 

           Pineapple 

           MRBA 

10‐2  23  6  Black  0.6  Aspergillus. Niger 

Black with grey border 

0.3  Curvularia 

Grey cottony 

0.8  Botrytis.Sps 

Pale green   0.2  Aspergillus. Sps 

Green with white border 

0.3  Penicillium. Spe 

Blue green 0.2  Penicillium.Spe 

Pink rhizoidal edge 

  Yeast

10‐3  11 3 Pinkish white 

0.2  Yeasts

Pink with white border 

0.4  Yeasts

 

  Table‐2   Isolation of Bacteria from Pineapple Peel & Pulp Bacteria 

White with yellow centre 

0.6  Aspergillus

10‐4  2  2  Pink with white border 

0.4   Yeasts 

Pinkish white 

0.2  Yeasts 

10‐5  1  3  Pink with 

white border 

0.4   Yeasts 

Sample   

Media   

Dilution & incubation   

No.of   Colonies 

 Types of   Colony    Code 

Pigment of  Colony 

Gram staining 

Shape & motility   

 Pineapple          Pineapple 

 NA          NA 

10‐3  22 12 PPP01 Translucent G+ve  Cocci

PPP02  Translucent with white centre 

 G‐ve  Small plump rods in chains, motile 

PPP03 Milky white G+ve  Cocci

*PPP04  Translucentnt 

G+ve  Long thin rods with spores 

PPP05  Yellow  G+ve  Cocci in clusters 

PPP06  Creamy translucent 

G+ve  Cocci isolated 

PPP07  Cream  G+ve  Cocci 

PPP08  Orange  G+ve  Cocci in pairs/ tetrads 

PPP09  Pale pink  G+ve  Cocci 

PPP10  Chalky white 

G+ve  Cocci 

PPP11  Waxy white  G+ve  Bacilli with spores 

PPP12  Cream  G+ve  Cocci 

10‐4  19  07  *PPP13  Waxy cream  G+ve  Long thin 

rods motile 

   Table‐3  Isolation of Yeast from Pineapple Peel & Pulp 

 

Sample   

Media   

Dilution & incubation   

No.of  colony  

Types of   Colony   

Code Pigment  & Colony characteristics 

Microscopic features 

Name of   Organism

           Pineapple 

 YPD           

10‐3  93  6  YP1  Chalky with rhizoidal edge 

Circular cells  Yeast 

*YP2  Chalky circular  Uneven sized rods 

Yeast 

YP3  Transparent  circular 

Uneven cells  Yeast 

YP4  Creamy circular 

Spores uneven   

YP5  Creamy yellow  Circular cells  Yeast 

*YP6  Creamy white  Large ovoid cells  Budding yeast 

10‐4  11  3  YP7  Translucent spreading 

Round spores  Tiny yeast 

YP8  White   Wheat shaped in clusters 

Cocci 

YP9  Round cream  Branched spores 

Candida 

10‐5  3  2  YP10  Translucent  Tiny spores  Yeast 

YP11  Translucent  Ovoid budding cells 

Yeast 

10‐6  2 1 YP12 Cream Spores uneven Yeast

PPP14 Yellow G+ve  Cocci

PPP15  Translucent  G+ve  Cocci 

*PPP16  Cream  G+ve  Pale thin rods 

PPP17  Pale orange  G+ve  Cocci 

PPP18  Lemon yellow 

G+ve  Cocci 

PPP19  Translucent white 

G+ve  Cocci in clusters 

10‐5  07 03 PPP20 Off‐white G+ve  Diplococci

PPP21 Translucent G+ve  Short rods in chains 

PPP22 Translucent G+ve  Cocci

         

   

  Table‐4  Isolation of  Fungi from Jackfruit Peel & Pulp 

 

Sample   

Media   

Dilution & incubation   

No.  of   Colonies 

 Types of   Colony   

Pigment of  Colony 

Size of  Colonies in cm 

Name of   Organism 

           Jackfruit 

           MRBA 

10‐3  27  5  Deep brown  1.2  Spiral mycelium –Sporotrichium Sps. 

Pink with white concentric rings 

0.6  Trichothecium

Pale pink 0.3  Geotrichum

Deep pink  0.1  Dreschlera. Sps 

Pink centre with white border 

1.3  Geotrichum 

11 4 Pinkish white 0.8  Fusarium. Sps

Pink with white border 

0.4  Geotrchium

White with yellow centre 

0.9  Aspergillus. Sps.

Pink with white border 

0.4   Yeasts

5 3 Deep  Pink  0.4   Dreschlera. Sps

Pinkish white  0.2  Fusarium. Sps 

Pink with white border 

0.4   Yeasts 

3  1  Pink with white border 

0.4   Yeasts 

 

 

        Table‐5  Isolation of Bacteria from Jackfruit Peel & Pulp 

Sample   

Media   

Dilution & incubation   

No.of   Colonies 

 Types of   Colony    Code 

Pigment of  Colony 

Gram staining 

Shape & motility   

           Jackfruit     

           NA 

  

10‐2 

 229 

 11 

*CKJB01 

Small cream G+ve  Small rods 

CKJB02 Chalky white 

 G‐ve  Cocci in clusters 

CKJB03 Cream G+ve  Cocci

CKJB04 Off‐ white G+ve  Cocci

CKJB05  Creamy translucent 

G+ve  Cocci isolated 

CKJB06  Cream  G+ve  Cocci 

CKJB07  White  G+ve  Cocci 

*CKJB08 

Creamy white 

G+ve  Short rods 

CKJB09  Pale pink  G+ve  Tiny Cocci 

CKJB10  Yellowish white 

G+ve  Diplococci 

CKJB11  Translucent  G+ve  Cocci 

 

10‐3 

  19 

  07 

CKJB12  Lemon yellow 

G+ve  Cocci 

CKJB13  Translucent  G+ve  Cocci 

CKJB14  Deep orange 

G+ve  Cocci 

CKJB15  Cream  G+ve  Cocci in clusters 

CKJB16  White  G+ve  Cocci 

*CKJB17 

Waxy white  G+ve  Long thin rods motile 

    Table‐6  Isolation of Yeast from Jackfruit Peel & Pulp 

 

 

CKJB18 Translucent white 

G+ve  Cocci in clusters 

 

10‐4  07 

 03 

CKJB19 Off‐white G+ve  Diplococci

    CKJB20  Translucent  G+ve  Short rods in chains 

CKJB21  Translucent  G+ve  Cocci 

CKJB22  Light Cream  G ‐ve  Short rods 

CKJB23 Cream G+ve  Cocci in clusters 

Sample   

Media   

Dilution & incubation   

No.of  colony   

 Types of   Colony   

 Code  Pigment  & 

Colony characteristics 

Microscopic features 

Name of   Organism

           Jackfruit 

 SDA           

10‐3  

93  6  CKJY01  Chalky white  Branching type uneven cells 

Yeast 

CKJY02  Chalky circular  Uneven sized large rods 

Large yeast 

CKJY03  Transparent  Spreading 

Small isolated cells 

Yeast 

CKJY04  Creamy circular  Spores uneven  Yeast 

CKJY05  Yellow dots  Tiny circular cells 

Yeast 

CKJY06  Creamy white  Large ovoid cells 

Budding yeast 

11  3  CKJY07  Translucent spreading 

Round spores  Tiny yeast 

CKJY08  White   Wheat shaped cells 

Cocci 

CKJY09  Round cream  Branched spores 

Candida 

10‐4  3  2  CKJY10  Translucent  Tiny spores  Yeast 

CKJY11  Translucent  Ovoid budding cells 

Yeast 

2  1  CKJY12  Pale Pink  Cells in clusters  Yeast 

 

 

 

 

 

 

 

 

Table‐7 

Results of Biochemical Tests for Bacteria Isolated from Pineapple peel / pulp 

Bacteria 

Indole 

Methyl Red 

Vogesp rausker 

Simon Citrate 

Glucose  Sucrose  Lactose  Maltose Gr A Gas Gr A Gas Gr  A  Ga

s Gr A Gas

PPP01  +  +  +  +++ + + + + ‐ ‐ +  ‐  ‐  + + +

PPP02  +  +  +  +++   + ‐ ++ ‐ ‐ +  ‐  ‐  + + ‐

PPP03  +  ‐  +  + + + ‐ ++ ‐ ‐ +  ‐  ‐  + + ‐

PPP04  ‐‐  +  ‐  +++ + + + + ‐ ‐ +  ‐  ‐  + + +

PPP05  ‐  ‐‐‐  ‐  + + + ‐ + ‐ ‐ ‐  ‐  ‐  + + ‐

PPP06  +  ‐  +  ‐‐‐ + + ‐ + ‐ ‐ +  ‐  ‐  + + ‐

PPP07  ‐  ‐  ‐  ‐‐‐ ‐ ‐ ‐ + ‐ ‐ +  ‐  ‐  + ‐ ‐

PPP08  ‐‐  +  ‐  + + + + + ‐ ‐ +  ‐  ‐  + + +

PPP09  ++  ‐‐  ‐  ‐‐‐ + + ‐ ++ ‐ ‐ +  ‐  ‐  + + ‐

PPP10  +  +  ‐  ‐‐‐ + + ‐ ++ ‐ ‐ ++  ‐  ‐  + + ‐

PPP11  ++  ‐  +  ‐‐‐ + + ‐ ++ ‐ ‐ ++  ‐  ‐  + + ‐

PPP12  +  ‐‐  +  ‐‐ + + ‐ ++ ‐ ‐ +  ‐  ‐  + + ‐

PPP13  ‐  ‐  +  ‐‐‐ + ‐ ‐ + ‐ ‐ +  ‐  ‐  + ‐ ‐

PPP14  +  +  ‐  ‐‐‐ + + ‐ ++ ‐ ‐ ++  ‐  ‐  + + ‐

PPP15  +  ‐  ‐  + + + ‐ + ‐ ‐ +  ‐  ‐  + + +

PPP16  ‐  ‐  +  + + + + + ‐ ‐ +  ‐  ‐  + + ‐

PPP17  ‐  ‐  ‐  + + ‐ ‐ ‐ ‐ ‐ +  ‐  ‐  + ‐ ‐

PPP18  +  +  ‐  + + + ‐ + ‐ ‐ +  ‐  ‐  + + +

PPP19  +  ‐  ‐  + + ‐ + + ‐ ‐ +  ‐  ‐  +  + +

PPP20  +  ‐  +  + + + ‐ ++ ‐ ‐ +  ‐  ‐  + + ‐

PPP21  +  +  ‐  + + + ‐ ++ ‐ ‐ +  ‐  ‐  + ‐ ‐

PPP22  ++  +  ‐  ‐ + + + + ‐ ‐ +  ‐  ‐  + + ‐

 *Gr = Growth *A = Acid production *G = Gas production 

 

 

Table‐8 

Results of Biochemical Tests for Bacteria Isolated from Jack fruit peel / pulp 

Bacteria  Indole 

Methyl Red 

Vogesp rausker 

Simon Citrate 

Glucose  Sucrose  Lactose  Maltose Gr A Gas Gr A Ga Gr  A  G  Gr A G

CKJB01  +  ‐  ‐‐  ‐‐‐ + + ‐ ++ ‐ ‐ +  ‐  ‐  + + ‐

CKJB02  +  ‐‐  ‐  ‐‐‐ + + ‐ + ‐ ‐ +  ‐  ‐  + ‐ ‐

CKJB03   +  +  +  ‐‐ + + ‐ ++ ‐ ‐ ‐  ‐  ‐  + ‐ ‐

CKJB04  +  ‐  +  ‐‐ + + ‐ ‐ ‐ ‐ ++  ‐  ‐  + + ‐

CKJB05  +  +  ‐  ++ ‐ ‐ + ‐ ‐ +  ‐  ‐  + ‐ ‐

CKJB06  ‐  ‐  +  ++ ++ ++ ++ + ‐ ‐ +  ‐  ‐  + + +

CKJB07  +  +  +  + ++ ++ ‐ + + ‐ +  ‐  ‐  + ‐ ‐

CKJB08  ‐  ‐  ‐  + + + ‐ + ++ ‐ +  ‐  ‐  + + ‐

CKJB09  ‐  +  +  ‐ + ‐ ‐ + ‐ ‐ +  ‐  ‐  + ‐ ‐

CKJB01  ‐  ‐  +  ‐ ++ + ‐ + ‐ ‐ +  ‐  ‐  + ‐ ‐

CKJB11  ‐  +    ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐  ‐  ‐  + + ‐

CKJB12  ‐  ‐  ‐  + ++ ++ ‐ ++ ++ ‐ +  ‐  ‐  + ‐ ‐

CKJB13  ‐  ‐  +  ++ ++ ++ ++ ‐ ‐ ++  ‐  ‐  ++ ++ ++

CKJB14  +  ‐  ‐  ‐ ++ ‐ ‐ + + ‐ +  ‐  ‐  + + ‐

CKJB15  ‐  +  ‐  ‐ + ‐ ‐ + ‐ ‐ +  ‐  ‐  + ‐ ‐

CKJB16  ‐  ‐  +  ‐ + ‐ ‐ + ‐ ‐ +  ‐  ‐  + ‐ ‐

CKJB17  +  +  +  ‐ ++ ++ ‐‐ + + ‐ +  ‐  ‐  ++ + ‐

CKJB18  ‐  ‐  +  + + + ‐ + ‐ ‐ +  ‐  ‐  ++ ‐ ‐

CKJB19  ‐  +  +  ‐ + + ‐ + ‐ ‐ +  ‐  ‐  + ‐ ‐

CKJB20  +  ‐  ‐  ‐ + ‐ ‐ ++ ‐ ‐ +  ‐  ‐  + ‐ ‐

CKJB21  ‐  +  ‐  ‐ ++ ‐ ‐ + + ‐ +  ‐  ‐  + ‐ ‐

CKJB22  ‐  ‐  +  + + ‐ ++ + ‐ ‐ +  ‐  ‐  + + ‐

CKJB23  ‐  ‐  ‐  ‐ + + ‐ + ‐ ‐ +  ‐  ‐  + ‐ ‐

 *Gr = Growth *A = Acid production *G = Gas production 

Table - 9 Initial Parameters for Pineapple Fermentation S # Parameters Peel Pulp 1. pH 4.2 4.5 2. Sugar concentration % (w/v) 10 03. Brix0 19 21 4 Cellulose concentration in

(µg/ml) 1700 1040

5 Alcohol content (%) 0 06. Tartaric aid 0.102 0.1927. Acetic acid 0.0816 0.1538. Citric acid 0.163 0.087

Table -10 Initial Parameters for Jackfruit Fermentation S # Parameters EFP NEFP 1. pH 5.8 6.32. Sugar concentration % (w/v) 00 103. Brix0 24 224 Cellulose concentration in

(µg/ml) 1400 1900

5 Alcohol content (%) 0 0

6. Tartaric aid 0.21 0.1127. Acetic acid 0.168 0.09 8. Citric acid 0.192 0.096

*EFP – Edible Fleshy Part; * NEFP- Non Edible Fleshy Part

 

Table 11

Production of Acetic acid in Pineapple Peel Ethanol

Days A. aceti A.xylinum

1 0.95 1.13

2 1.17 2.48

3 1.93 3.08

4 2.45 3.19

5 2.98 3.21

6 3.56 2.21

7 3.92 3.21

8 3.92 3.21

SEM/-+0.28

CD 5% 1.33  

 

 

 

 

 

 

 

 

Table 12

Production of Acetic acid in Jackfruit Peel Ethanol

Days

A. aceti

A.xylinum

1 0.44 0.86

2 0.61 1.22

3 0.98 1.89

4 1.29 2.43

5 1.95 2.77

6 2.12 2.92

7 2.71 3.01

8 3.39 3.01

SEM/-+0.28

CD 5% 1.33  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure-1a. Hydrolysis of cellulose by bacteria from pineapple by CMC method

 

 

 

 

 

 

 

 

 

 

 

 

Figure-1b. Hydrolysis of cellulose by yeasts from pineapple by CMC method

 

 

 

 

 

 

 

 

 

 

 

 

Figure-2a. Hydrolysis of cellulose by bacteria from jackfruit by CMC method

 

 

 

 

 

 

 

 

 

 

0

1

2

3

4

5

CKJB01CKJB08

CKJB17C.uda

4.3

3.64.2 4.5

Zone of Hydrolysis for Cellulose

Diameter in cmsCmss

 

Figure-2b. Hydrolysis of cellulose by yeast from pineapple by CMC method

 

 

 

 

 

 

 

 

 

 

 

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

CKJY02CKJY06

CKJY11Scve

4.1

3.43.8

3.7

Zone of Hydrolysis for Cellulose

Diameter in cms

Cms

 

Figure-3a. Estimation of cellulose by anthrone method for Pineapple peel wastes

 

 

 

 

 

 

 

 

 

 

 

 

0

50

100

150

200

250

300

350

400

450

C.uda Scve PPP16 PPP04 PPP13

Estimation of Cellulose in Pineapple Peel

22° C

27° C

32° C

35° C

OD

in n

m

 

Figure-3b. Estimation of cellulose by anthrone method for Pineapple pulp wastes

 

 

 

 

 

 

 

 

 

 

 

0

100

200

300

400

500

600

700

800

900

YP02 YP06 Scve YP11 YP12

22° C27° C32° C35° C

 

Figure-4a. Estimation of Cellulose by Anthrone Method using Bacterial Isolates on Jackfruit Peel Wastes. 

 

 

 

 

 

 

 

 

 

 

 

 

0

50

100

150

200

250

300

C.uda CKJB01 CKJB08 CKJB17

22⁰ C

27⁰ C

32⁰ C

35⁰ C

Co

nce

ntr

atio

n in

µg

/ml

 

Figure-4b. Estimation of Cellulose by Anthrone Method using Yeast Isolates on Jackfruit Pulp Wastes. 

 

 

 

 

 

 

 

 

 

 

 

 

0

20

40

60

80

100

120

140

160

180

SCVe CKJY02 CKJY06 CKJY11

22⁰ C

27⁰ C

32⁰ C

35⁰ C

Series5

Residual  Cellulose  in Jackfruit EFP after fermentation by Yeast Isolates

 

Figure-5a. Estimation of Glucose by DNS Method using Bacterial Isolates on Pineapple Peel Wastes.

 

 

 

 

 

 

 

 

 

 

 

 

0

200

400

600

800

1000

1200

1400

1600

1800

2000

C.uda Scve PPP16 PPP04 PPP13

22° C

27° C

32° C

35° C

ODin nm

Estimation of Glucose in Pineapple Peel

22° C

27° C

32° C

35° C

ODin nm

 

Figure-5b. Estimation of Glucose by DNS Method using Yeast Isolates on Pineapple Pulp Wastes.

 

 

 

 

 

 

 

 

 

 

 

 

0

200

400

600

800

1000

1200

1400

1600

1800

2000

YP2 YP6 Scve YP11 YP12

Estimation of Sugar in Pineapple Pulp

22° C

27° C

32° C

35° C

ODin nm

 

Figure-6a. Estimation of Glucose by DNS Method using Bacterial Isolates on Jackfruit Peel Wastes.

 

 

 

 

 

 

 

 

 

 

 

 

0

100

200

300

400

500

600

700

800

900

1000

22⁰ C 27⁰ C 32⁰ C 35⁰ C

Concentration of sugar in µg/ml

C.uda

CKJB01

CKJB08

CKJB17

Estimation of Glucose in Jackfruit Peel Wastes

 

Figure-6b. Estimation of Glucose by DNS Method using Yeast Isolates on Jackfruit Pulp Wastes.

 

 

 

 

 

 

 

 

 

 

 

 

0

100

200

300

400

500

600

700

800

900

22⁰ C 27⁰ C 32⁰ C 35⁰ C

SCVe

CKJY02

CKJY06

CKJY11

Estimation of Glucose in Jackfruit Pulp Wastes

 

Figure-7a. Estimation of Ethanol by Dichromate Method using Bacterial Isolates on Pineapple Peel Wastes.

 

 

 

 

 

 

 

 

 

 

 

 

0

1

2

3

4

5

6

C.uda PPP04 PPP13 PPP16

22⁰ C

27⁰ C

32⁰ C

35⁰ C

 

Figure-7b. Estimation of Ethanol by Dichromate Method using Yeast Isolates on Pineapple Pulp Wastes.

 

 

 

 

 

 

 

 

 

 

 

 

0

2

4

6

8

10

12

14

SCVe YP2 YP6 YP11 YP12

22⁰ C

27⁰ C

32⁰ C

35⁰ C

 

Figure-8a. Estimation of Ethanol by Dichromate Method using Bacterial Isolates on Jackfruit Peel Wastes.

 

 

 

 

 

 

 

 

 

 

 

 

0

1

2

3

4

5

6

7

C.uda CKJB01 CKJB08 CKJB17

22⁰ C

27⁰ C

32⁰ C

35⁰ C

 

Figure -8b. Estimation of Ethanol by Dichromate Method using Yeast Isolates on Jackfruit Pulp Wastes.

 

 

 

 

 

 

 

 

 

 

 

 

0

2

4

6

8

10

12

14

16

SCVe CKJY02 CKJY06 CKJY11

22⁰ C

27⁰ C

32⁰ C

35⁰ C

 

Figure-9a. Production of acetic acid in secondary fermentation of pineapple peel ethanol

 

 

 

 

 

 

 

 

 

 

 

0 1 2 3 4 5

1

2

3

4

5

6

7

8

A.xylinum

A.aceti

No. of days

Percentage of acetic  acid

Production of Acetic acid in Pineapple Peel waste ethanol

 

      Figure-9b. Production of acetic acid in secondary fermentation of jackfruit peel ethanol

 

 

 

 

 

 

 

 

 

 

 

0 0.5 1 1.5 2 2.5 3 3.5 4

1

2

3

4

5

6

7

8

A.xylinum

A.aceti

No. of days

Percentage of acetic  acid

Production of Acetic acid in Jackfruit Peel waste ethanol

 

Plate no.1a Plate no.1b

Plate no.1c

Plate no.1d

Plate no.2

Plate no.3

Plate no.4

Plate no.5 Plate no.6a

Plate no.8a

Plate no.6b

Plate no.7b

Plateno.8b

Plateno.9a

Plate.10a

Plate no.11a

Plate no.9b

Plate no.10b

Plate no.11b

Plate no. 12a

Plate no.12b Plate no.12b

Plate no12c

Plate no.12d

Plate no.12e

Plate no.12f

Plate no. 13

Plate no. 14

Plate no.15

Plate no.16

Plate no.17 Plate no.18

Plate no.19 Plate no.20

Plate no.21

Plate no.22

Plate no.23 Plate no.24

Plate no.25 Plate no.26

Plate no.27

Plate no.28 Plateno.29