UNIVERSITI PUTRA MALAYSIA FUNGAL SCREENING AND...

UNIVERSITI PUTRA MALAYSIA

FUNGAL SCREENING AND ISOLATION OF CELLULOLYTIC, MANNAN AND PROTEIN DEGRADING ENZYME PRODUCERS IN PALM KERNEL

CAKE SOLID STATE FERMENTATION

MOHD. FAZLI BIN FARIDA ASRAS

FBSB 2009 25



By


Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in

Fulfilment of the Requirement for the Degree of Master Science

July 2009

DECLARATION

I hereby declare that the thesis is based on my original work except for the quotations and citations which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any degree at UPM or other institutions.

________________________________

MOHD. FAZLI BIN FARIDA ASRAS Date: 2 July 2009

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in Fulfilment of the Requirement of Degree of Master Science



By


July 2009

Chairman: Rosfarizan Mohamad, PhD

Faculty: Biotechnology and Biomolecular Sciences

Palm Kernel Cake (PKC), an agro-industrial by-product obtained after extraction of oil

palm from oil palm seeds is used extensively in the animal feed industry but has

limited used in poultry feed due to its high fiber and low protein contents. In this

study, PKC was used as a substrate in solid state fermentation (SSF) by locally-

isolated strains and their feasibility for cellulase and mannanase enzymes production

were investigated. The potential isolates were obtained from various sources such as

peat soil, rotten rice chaff, ‘Tanah Bakar’, rotten palm frond and raw PKC. The

isolates were screened based on the clearing zone method and on selective agar media

containing substrates such as locust bean gum (LBG), carboxymethylcellulose (CMC),

potato dextrose, mannan and PKC.

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Forty-eight fungal cultures have been screened and isolated based on the selective

agars. Only thirty-one isolates were able to grow well after multi-subculturing

techniques. The microbial activities of the isolates were accessed through clearing

zone by chromogenic substrates such as Azo-carob galactomannan and Azo-CM

cellulose. The diameter of clearing zone on the agar plate was observed every 24 h

until 120 h.

Cultivation of the strains was carried out at 50% moisture content using shake flask

and pre-germinated spores were preferred as the inoculum. The effects of cultivation

conditions such as moisture level, inoculum concentration and agitation were

investigated with the aim to achieve maximum production of cellulase and mannanase

enzymes. Shaking at 120 rpm was found as the best agitation speed in the pre-

germination process to be used as the inoculum. The samples were analyzed for neutral

detergent fiber, acid detergent fiber, crude fiber and crude protein using Near Infrared

Reflectance Spectroscopy analysis.

The best enzymes producer was fungal isolate D1 with specific enzyme exoglucanase

activity of 17.9323 U/mg, specific enzyme endoglucanase activity of 41.6008 U/mg

and specific enzyme β–glucosidase activity of 79.2626 U/mg using the pre-optimized

conditions on the fifth day of fermentation process. About 50.1036 U/mg of specific

enzyme mannanase activity was achieved on the fourth day of fermentation process

using PKC as the substrate. The fibre degradation increased significantly. Neutral and

acid detergent fibers were reduced from 85.16 to 21.72% on the sixth day and 45.18 to

17.18% at eighth day of fermentation process, respectively. The protein content

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increased from 13.31 to 31.53% on the eighth day. Lower cellulase and mannanase

enzymes activities were obtained in other isolates.

The highest cellulolytic and mannan-degrading enzymes producer was identified using

microscopic. Under the microscopic view, isolate D1 was identified as Aspergillus sp.

The identity of the isolate was further confirmed and belongs to Aspergillus sp. after

observation under Scanning Electron Microscope (SEM). As a result, isolate D1 was

identified as Aspergillus sp.

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk Ijazah Sarjana Sains

PEMENCILAN DAN PENYARINGAN KULAT PENGHASIL ENZIM BERSIFAT PENGURAI SELULOSA, MANNAN DAN PROTEIN DALAM

ISIRUNG KELAPA SAWIT FASA FERMENTASI PEPEJAL

Oleh


Julai 2009

Pengerusi: Rosfarizan Mohamad, PhD

Fakulti: Bioteknologi dan Sains Biomolekul

Isirung Kelapa Sawit (IKS) adalah sisa buangan industri pertanian yang diperolehi

selepas pengekstrakan minyak daripada buah kelapa sawit telah digunakan secara

meluas dalam industri makanan ternakan tetapi penggunaannya adalah terhad dalam

makanan ternakan berpunca daripada kandungan fiber yang tinggi dan protein yang

rendah. IKS digunakan sebagai substrat dalam fermentasi fasa pepejal oleh pencilan

tempatan dan dikaji kebolehannya untuk penghasilan enzim selulase dan mannanase.

Pencilan yang berpotensi dipencilkan daripada pelbagai sumber seperti tanah gambut,

tanah bakar, sekam padi dan tandan kelapa sawit yang telah reput serta isirung kepala

sawit itu sendiri. Pencilan-pencilan itu disaring berdasarkan pada zon penjernihan dan

juga pada media agar yang bersifat pemilih mengandungi substrat seperti locust bean

gum, carboxymethylcellulose (CMC), potato dextrose, mannan dan IKS.

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Empat puluh lapan pencilan dipencilkan dan disaring berdasarkan agar bersifat

pemilih. Hanya tiga puluh satu pencilan boleh hidup dengan baik selepas teknik multi-

subculturing. Aktiviti-aktiviti mikrob kultur tersebut dinilai melalui zon penjernihan

pada substrat berwarna seperti Azo-carob galactomannan dan Azo-CM selulosa.

Diameter zon penjernihan pada piring agar diperhatikan setiap 24 jam sehingga 120

jam.

Pengkulturan pencilan dilakukan pada kelembapan 50% menggunakan kelalang

berkocak dan spora pra-percambahan diutamakan sebagai inokulum. Kesan keadaan

pengkulturan seperti aras kelembapan, kepekatan inokulum dan pengocakan

ditingkatkan dengan sasaran untuk mencapai penghasilan enzim selulase dan

mannanase yang maksimum. Kelajuan 120 rpm dipilih sebagai kelajuan pengocak

yang terbaik dalam proses pra-percambahan dan digunakan sebagai inokulum. Sampel

dianalisa untuk serat neutral detergen, serat asid detergen, serat kasar dan protein kasar

menggunakan analisis Spektroskop Pemantulan Cahayamerah Bersebelahan.

Penghasil enzim yang terbaik ialah pencilan D1 dengan aktiviti enzim exoglucanase

spesifik 17.9323 U/mg, aktiviti enzim endoglucanase spesifik 41.6008 U/mg dan

aktiviti enzim β–glucosidase spesifik 79.2626 U/mg pada hari kelima proses

fermentasi menggunakan keadaan yang optimum. Sebanyak 50.1036 U/mg aktiviti

enzim mannanase spesifik dicapai pada hari keempat proses fermentasi menggunakan

IKS sebagai substrat. Penguraian serat meningkat dengan nyata sekali. Serat neutral

dan asid detergen masing-masing menurun daripada 85.16 kepada 21.72% pada hari

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keenam dan 45.18 kepada 17.18% pada hari kelapan. Aktiviti enzim selulase dan

mannanase lebih rendah dicapai oleh pencilan yang lain.

Kulat penghasil enzim pengurai selulosa dan mannan dikenalpasti menggunakan

pemerhatian secara mikroskopik. Di bawah pemerhatian mikroskopik, pencilan D1

dikenalpasti sebagai spesies Aspergillus. Pencilan ini dipastikan sebagai spesies

Aspergillus melalui pemerhatian yang dibuat menggunakan Mikroskop Pengimbas

Elektron.

ACKNOWLEDGEMENT

Special appreciation and sincere thanks to Dr. Rosfarizan Mohamad as my main

supervisor, for all her invaluable advice, encouragements and patience throughout the

period of completing this project. I would like to thank for all the good arrangements

that she made. Words are not enough to express the depth of my gratitude for the

guidance which she gave me during the proposal presentation and thesis writing. Her

concerned is very much appreciated. I am, most indebted to Prof. Dr. Arbakariya Ariff

as my co-supervisor for showing concern in my completion of thesis from time to

time, for all his generous assistance and allowing me a lot of flexibility in writing this

thesis. Also, his kind advice, overwhelming support and willingness to share his

personal view with me are most appreciated.

My sincere thanks to Miss Jame’ah Hamid, Mr. Abdul Rahman and Mrs. Noraini

Samat for their motivation and valuable suggestion during the difficult moments of the

project. In addition, I would also like to express my deep appreciation to Mr. Rosli

Aslim, Mrs. Renuga Panjamurthi, Mrs. Aluyah Marzuki and Mrs. Norazlina Mohamad

for their guidance, help and generous use of the fermentation laboratory facilities and

all the laboratory staffs for their co-operation, forbearance and promptness during my

work.

Also, not forgetting to all my labmates (Azman, Radin, Fadzli and Azlan) for all the

support I received, all of whom were of immense help during this revision. In addition,

I extend special thanks to Miss Maherah, Mr. Shahrul and Mr. Zarizal who have

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contributed in an indirect but essential way to the completion of this project especially

on the molecular part.

Last but definitely not least, my deepest thanks go to my beloved parents, sisters for

their love, sacrifice and encouragement that will remain in my mind forever. It is of

utmost importance that a report is accompanied by complete and accurate supplements.

I take part in mentioning that the supplements prepared for this text possess these

qualities and much more. It is my pleasure to thank all the friends and lecturers with

whom I enjoyed working during this revision.

This thesis submitted to the Senate of Universiti Putra Malaysia and has been accepted as the fulfilment of the requirement for the degree of Master of Science. The members of the Supervisory Committee were as follows: Rosfarizan Mohamad, PhD Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) Professor Arbakariya Ariff, PhD Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member)

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HASANAH MOHD GHAZALI, Ph.D Professor and Dean School of Graduate Studies Universiti Putra Malaysia Date: 16 October 2009

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I certify that a Thesis Examination Committee has met on 2 July 2009 to conduct the final examination of Mohd. Fazli Bin Farida Asras on his thesis entitled “Fungal Screening and Isolation of Cellulolytic, Mannan and Protein Degrading Enzyme Producers in Palm Kernel Cake Solid State Fermentation” in accordance with Universities and University Colleges Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15 March 1998. The committee recommends that the student be awarded the Master of Science. Members if the Examination Committee were as follows: Shuhaimi Mustafa, Ph.D. Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) Suraini Abd. Aziz, Ph.D. Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner) Mohd. Noor Abd. Wahab, Ph.D. Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner) Kopli Bujang, Ph.D. Professor Faculty of Resource Science and Technology Universiti Malaysia Sarawak (External Examiner)

_________________________

BUJANG KIM HUAT, Ph.D. Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date: 17 September 2009

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TABLE OF CONTENTS ABSTRACT ii ABSTRAK v ACKNOWLEDGEMENTS viii APPROVAL x DECLARATION xii LIST OF TABLES xvi LIST OF FIGURES xviii LIST OF ABBREVIATIONS xx CHAPTER I INTRODUCTION 1 II LITERATURE REVIEW

2.1 Palm Kernel Cake 2.1.1 Introduction 7 2.1.2 Nutritive Value of PKC 9 2.1.3 Problems in Utilization of PKC 11

2.2 Enzymatic Hydrolysis of Cellulose 2.2.1 Nature of Lignocellulosic Materials 12 2.2.2 Cellulose 13 2.2.3 Hemicellulose 15

2.3 The Cellulase and Mannanase Systems: Mode of Action, Production and Application 2.3.1 Properties of Cellulase 16 2.3.2 Mode of Action in Cellulolysis 19 2.3.3 Applications of Cellulase 21 2.3.4 Mannanase 22

2.4 Solid-state Fermentation 2.4.1 Introduction 25 2.4.2 Fungal Biomass 27 2.4.3 Environmental Parameters Controlling Solid State 31

Fermentation 2.4.4 Advantages and Disadvantages of Solid-state 38

Fermentation 2.4.5 Applications of Solid State Fermentation 39

III GENERAL MATERIALS AND METHODS

3.1 Screening and Isolation of Cellulolytic and Mannan-degrading Enzymes Producers 3.1.1 Sample Collection and Treatment 41 3.1.2 Primary Screening of Fibre-degrading Microorganisms 44

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3.1.3 Secondary Screening of PKC-degrading Microorganisms 45 3.2 Solid-state Fermentation of PKC

3.2.1 Inoculum Development 46 3.2.2 Analytical Procedures 47

3.2.3 Biomass Estimation 47 3.2.4 Cellulase Activity 48 3.2.5 Mannanase Activity 51 3.2.6 Protease Activity 52 3.2.7 Fibre Analysis 53

3.3 Identification of Highest Producer of Cellulolytic and Mannan-

degrading Enzymes 3.3.1 Morphological Test 59 IV ISOLATION AND SCREENING OF CELLULOLYTIC

AND MANNAN-DEGRADING ENZYMES PRODUCERS 4.1 Introduction 60 4.2 Materials and Methods 62 4.3 Results and Discussion

4.3.1 Primary Screening of PKC-degrading Microorganisms 63 4.3.2 Secondary Screening of PKC-degrading Microorganisms 69

4.4 Conclusion 75 V INOCULUM DEVELOPMENT AND SOLID-STATE

FERMENTATION OF PKC BY FUNGI ISOLATES 5.1 Introduction 76 5.2 Materials and Methods 78 5.3 Results and discussion

5.3.1 Effects of Inoculum Types on SSF of PKC 80 5.3.2 Effects of Agitation Speed (120 and 150 rpm) on the

Fungal Morphology in Pre-germination Process using Aspergillus niger FTCC 5003 82

5.3.3 Solid-state fermentation of PKC 85 5.3.4 Cellulase Activity 89 5.3.5 Mannanase Activity 94 5.3.6 Protease Activity 96 5.3.7 Correlation between Submerged Fermentation (SmF) And Solid-state Fermentation (SSF) on Growth of Fungi 99 5.3.8 Fibre Analysis using Near Infrared Reflectance Spectrophotometer (NIRS) 101

5.4 Conclusion 108 VI IDENTIFICATION OF THE BEST CELLULOLYTIC AND MANNAN-

DEGRADING ENZYMES PRODUCER 6.1 Introduction 109 6.2 Materials and Methods 110

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6.3 Results and Discussion 6.3.1 Morphological View of Strain D1 111

6.4 Conclusion 113 VII CONCLUSIONS AND SUGGESTIONS FOR 114

FURTHER WORK REFERENCES 117 APPENDICES 142 BIODATA OF STUDENT 152 LIST OF PUBLICATIONS 153

ABBREVIATIONS

ADF : Acid Detergent Fibre

ADL : Acid Detergent Fibre

AOAC : Association of Official Analytical Chemists

aw : Water Activity

BLAST : Basic Local Alignment Search Tool

CF : Crude Fibre

CMC : Carboxymethylcellulose

CP : Crude Protein

CF : Crude Fibre

°C : Degree Celcius

DCP : Digestible Crude Protein

DE : Digestible Energy

DM : Dry Matter

DNS : Dinitro Salicylic Acid

DW : Dry Weight

EDTA : Ethylenediaminetetraacetic Acid

EE : Ether Extract

EFB : Empty Fruit Bunch

Fig. : Figure

LBG : Locust Bean Gum

LF : Liquid Fermentation

h : Hour

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μm : Micrometer

ml : Mililiter

NAG : N-acetyl-glucosamine

NCBI : National Center of Biotechnology Information

NDF : Neutral Detergent Fibre

NFE : Nitrogen Free Extract

NIRS : Near Infrared Reflectance Spectrophotometer

nm : Nanometer

NMR : Nuclear Magnetic Resonance

NTG : Nitrosoguanidine

OUR : Oxygen Uptake Rate

PDA : Potato Dextrose Agar

PDB : Potato Dextrose Broth

PKC : Palm Kernel Cake

PKO : Palm Kernel Oils

POME : Palm Oil Mills Effluent

pNPG : 4-nitrophenyl-β-D-glucopyranoside

pNP : 4-nitrophenol

rpm : Rotation Per Minute

rRNA : Ribosomal RNA

SACGLM : Azo-Carob Galactomannan

SACMC : Azo-Cm Cellulose

SEM : Scanning Electron Microscope

SmF : Submerged Fermentation

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SSF : Solid State Fermentation

SSU : Small Subunit

TCA : Trichloroacetic Acid

TDN : Total Digestible Nutrient

THAM :Tris-hydroxymethylaminomethane

LIST OF TABLES Table Page 2.1 Chemical Composition of PKC and POM 8 2.2 Amino Acids Profile of PKC 8 2.3 Nutrient Compositions of Solvent Extracted and Expeller 10 Pressed PKC

2.4 The Contents of Cellulose, Hemicellulose and Lignin in common 13

Agricultural Residues and Waste

2.5 Cellulase Producers by Bacteria and Fungi 17 2.6 Mannanase Producers by Bacteria and Fungi 23 2.7 Some Examples of Solid-state Fermentations 26

2.8 Correlation of Chemical Composition of Fungal Cell Walls with 28

Taxonomic Group

2.9 Indirect Methods in estimating Biomass in Solid-state 30 Fermentation

2.10 Heat Generation in some Solid-state Fermentation Systems 35

2.11 Pretreatments for Improving Lignocellulose Bioconversion 37

4.1 Primary Screening of Cellulolytic and Mannanase Enzymes Producers 64

4.2 Subculturing of Cellulolytic and Mannanase Enzymes 68 Producers

4.3 Secondary Screening of Cellulolytic and Mannanase Fungi based on 71 Diameter (cm) Clearing Zone on Cellulose Substrate

4.4 Secondary Screening of Cellulolytic and Mannanase Fungi based on 72 Diameter (cm) Clearing Zone on Mannan Substrate

4.5 Clear Zone Diameter (cm) of Agar containing Cellulose as s Substrate 74 4.6 Clear Zone Diameter (cm) of Agar containing Mannan as a Substrate 74

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5.1 Percentage of Fibre Degradation (NDF) using Different Types of 81 Inoculum for SSF of PKC

5.2 Effect of Agitation Speed (120 and 150 rpm) on the Fungal Morphology 84

in Pre-germination Process 5.3 Moisture Content of A. niger and Strain D1 during SSF of PKC 98

5.4 Growth Kinetics of the Isolates in Submerged Fermentation using 101

Defined Media

5.5 The Actual Values of NDF (in percentage) of the Isolates using 103 NIRS Analysis per Day

5.6 The Reduction Values of NDF (in percentage) of the Isolates using 103 NIRS Analysis per Day

5.7 The Actual Values of ADF (in percentage) of the Isolates using 105 NIRS Analysis per Day

5.8 The Reduction Values of ADF (percentage) of the Isolates using 105 NIRS Analysis per Day

5.9 The Actual Values of Crude Protein (in percentage) of the Isolates 107 using NIRS Analysis per Day

5.10 The Increment Values of Crude Protein (in percentage) of the Isolates 107

using NIRS Analysis per Day

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LIST OF FIGURES Figure Page 2.1 Structure of Cellulose where n equals the Number of 14

Anhydroglucose Units

2.2 Schematic Representation of the Hydrolysis of Amorphous and 19 Microcrystalline Cellulose by Noncomplexed (A) and Complexed (B) Cellulase Systems

3.1 Several Types of Samples 42 3.2 Schematic Diagram of Experimental Design on Solid-state 43

Fermentation of Palm Kernel Cake

4.1 Formations of Clearing Zones by the isolates B1, C3, A15 and D8 73 Incubated at 37ºC, 48hr on Chromogenic Substrates (A. Cellulose. B. Mannan)

5.1 Fermented PKC using Filamentous Type as Inoculum for 80

Solid-state Fermentation by Aspergillus niger FTCC 5003 5.2 Filamentous Form of Pre-germinated Fungal Strains to be used as 82

an Inoculum for SSF of PKC

5.3 The Growth Profile of all Isolates during SSF of PKC 86

5.4 The Profile of Reducing Sugars Concentration of all Isolates 88 during Solid-state Fermentation of PKC

5.5 The Profile of Specific Enzyme Exoglucanase Activity of all Isolates 90 during SSF of PKC

5.6 The Profile of Specific Enzyme Endoglucanase Activity of all Isolates 91 during SSF of PKC

5.7 The Profile of Specific Enzyme β-D-glucosidase Activity of all Isolates 93 during SSF of PKC

5.8 The Profile of Specific Enzyme Mannanase Activity of all Isolates 95 during SSF of PKC

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5.9 The Profile of Specific Enzyme Protease Activity of all Isolates 97 during SSF of PKC

5.10 Submerged Fermentation of Control and C12 after 72h of Incubation 100

in 500 ml Shake Flask 6.1 Morphological View of Isolate D1 (A, B and C) under Fluorescence 111

Microscope (100 X Magnification)

6.2 Morphology of Isolate D1 (A, B and C) under SEM in Different 112 Magnifications

6.3 Morphology of Fermented PKC by Isolate D1 (A and B) under SEM 113

in Different Magnifications 7.1 Illustration of Molecular Identification of Strain D1 115

CHAPTER I

INTRODUCTION

The palm oil industry produces a considerable amount of solid waste by-products.

These are in the form of fibers, shells and empty bunches discharged from the mills.

Currently, shell and fiber are used extensively as fuel for the production of steam in the

palm oil mills, combining waste disposal and energy recovery. After combustion,

about 5% ash by weight is produced and must be disposed of by other means. Palm

kernel cake (PKC), the major agro-industrial by-product of the palm oil industry in

Malaysia, Thailand and Indonesia is another good source of energy and protein for

ruminants (Setthapukdee et al., 1991). In 2006, Malaysia produced 2.20 million tonnes

of PKC and exported 2.12 million tonnes (Malaysian Palm Oil Board, 2007).

Palm kernel cake is one of the oilseed by-products widely used in ruminant feeds. PKC

being derived from an oil crop is expected to have relatively high oil content. In

practice, however, the oil content of PKC depends on the efficiency of oil extraction

from the kernel. The nitrogen free extract (NFE) contains variable quantities of

sucrose, reducing sugars and starch. PKC is used to supply both energy and protein to

animal. However, it is only sparingly used in poultry feeds because of high fiber

content and low digestibility. Because of the high fiber content, the metabolizable

energy content of PKC is very low. Furthermore, PKC protein has a poor amino acid

balance, with lysine being a major limiting amino acid (Onwudike, 1996). Palm kernel

cake being derived from an oil crop is expected to have relatively high oil content. In

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practice, however, the oil content of PKC depends on the efficiency of oil extraction

from the kernel.

The PKC comprises mainly of cell wall. This cell wall consists largely of

polysaccharides such as mannan which is responsible for the low digestibility of PKC

by poultry. In the total cell wall, mannose is the principle neutral sugar (56.4 %),

followed by glucose (11.6 %), xylose (3.7 %) and galactose (1.4 %) (Anon. 2002).

Many microorganisms are capable of decomposing celluloses and mannans; however,

enzymes from fungi such as Aspergillus niger (Ademark et al., 1998), Trichoderma

reesei (Arisan-Atac et al., 1993) and Sclerotium rolfsii (Gubitz et al., 1996) deserve

the most attention. The main components of lignocelluloses are cellulose,

hemicelluloses and lignin (Sjöströrn, 1981). In nature, lignocellulolytic microbes

interact in mixed culture to degrade lignocellulose e.g., wood decay (Bayer and

Lamed, 1992). Resembling the natural habitat of filamentous fungi growing on solid

lignocellulosic particles; solid substrate fermentation (SSF) involves the growth of

microorganisms on moist solid substrates in the absence of free water (Murthy et al.,

1993; Tengerdy, 1996).

Cellulose is known to consist of relatively easily accessible amorphous regions with

few lateral interactions between the cellulose chains as well as of crystalline domains

that are much harder to hydrolyze. Cellulosic materials can be decomposed into

fermentable sugars, which can be converted into other valuable products such as

ethanol, single-cell proteins, and hydrogen. Acid treatment and enzymatic hydrolysis

are the most common ways to break down cellulose into glucose. Compared with acid

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