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The biodegradation of quaternary ammonium compounds by mould fungi Zheng, Yu 1995

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T H E BIODEGRADATION OF QUATERNARY A M M O N I U M COMPOUNDS B Y M O U L D FUNGI by Yu Zheng B.Sc. (1990) and M . Sc. (1993), Fujian Normal University, P. R. China A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF T H E REQUIREMENTS FOR T H E D E G R E E OF M A S T E R OF SCIENCE in T H E F A C U L T Y OF G R A D U A T E STUDIES (Faculty of Forestry, Department of Wood Science) We accept this thesis as confinning to the required standard T H E UNIVERSITY OF BRITISH COLUMBIA November, 1995 ®Yu Zheng, 1995 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of &oA ^ C ^ ^ The University of British Columbia Vancouver, Canada Date DE-6 (2788) ABSTRACT Didecyldirhethylarrmionium chloride (DDAC) has been described as one of the most effective quaternary ammonium compound (QAC) wood preservatives, based on laboratory experiments. Subsequently, QAC-treated stakes failed rapidly in field tests. It was suggested that the degradation of QACs by mould and stain fungi had reduced the efficacy of QACs. This thesis describes a study of the role that moulds play in limiting the performance of DDAC. Fungi were isolated from DDAC-treated stakelets placed in a field test site at UBC. Other fungal strains were obtained from culture collections. A comparison was made of their tolerance and ability to degrade DDAC. Experiments were performed both in solid malt agar and Vogel liquid media with, and without, DDAC. It was found that tolerance and ability to degrade DDAC shown by the fungal strains were common properties of many of the moulds studied. The metabolites produced by the action of moulds on DDAC were extracted by methylene chloride and analyzed by high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). The results showed that DDAC had been degraded by mould fungi, either by fission of the C-N bond or co-and (3-oxidation of the decyl group. Electrophoresis was performed to try to identify additional proteins arising from the presence of DDAC. New proteins bands were obtained in the extracellular supernatant. The DDAC concentration in liquid media was decreased when inoculated and incubated with the i i extracellular supernatant recovered from Verticillium bulbillosum grown in liquid media containing 250 ppm DDAC. No new proteins were obtained in the intracellular extract, and no degradation of the DDAC was recorded when the intracellular supernatant was incubated with DDAC. This suggested that DDAC degradation took place outside the fungal cell wall. iii TABLE OF CONTENTS Page ABSTRACT ii T A B L E OF CONTENTS iv LIST OF TABLES viii LIST OF FIGURES x ACKNOWLEDGMENTS xiv LIST OF ABBREVIATIONS xv 1. INTRODUCTION 1 1.1 The limitations of currently used wood preservatives 2 1.2 Quaternary ammonium compounds 5 1.2.1 Industrial applications 5 1.2.2 Potential as wood preservatives 7 1.3 Review of laboratory performance and field performance 8 1.3.1 Laboratory trials 8 1.3.2 Field trials 8 1.3.3 Reasons for the poor field performance 9 2. OBJECTIVES 13 3. LITERATURE REVIEW OF BIODEGRADATION OF QACS 14 3.1 Definitions of biodegradation 16 3.2 Evidence of biodegradation of QACs 18 3.2.1 Akyltrimethylammonium salts CMes^T 18 3.2.2 Alkylbenzyldimethylammonium salts CnBzMeiN* 27 3.2.3 Dialkyldimethylammonium salts (C„)2Me2N+ 2 8 3.2.4 Alkylpyridinium salts (C„)py+ 31 3.2.5 Trialkylmethylammonium salts (C^MelST 31 iv 3.3 Mechanisms of biodegradation 33 3.3.1 Oxidation of the long alkyl chain 3 3 3.3.2 Carbon nitrogen fission: cleavage of the alkyl chain 36 3.4 Factors affecting the biodegradation of QACS 41 4. MATERIAL AND METHODS 44 4.1 Evaluation of a rapid colorometric screening technique for identifying 44 QAC degrading fungi 4.1.1 Chemicals 44 4.1.2 Sample preparation 44 4.1.3 Sample analysis 45 4.2 Isolation of tolerant fiingi 46 4.2.1 Wood treatment and exposure 46 4.2.2 Isolation of tolerant fungi from treated wood exposed in the 46 field area 4.3 Comparison of the tolerance of isolates from the field experiment with those 48 from culture collections 4.3.1 Tolerance of fungi in solid media 48 4.3.2 Tolerance of fungi in liquid media 49 4.3.3 The growth curve of V. bulbillosum 51 4.4 Assessment fungal degradation of DDAC 52 4.4.1 Solid media 52 4.4.2 Liquid media 53 4.5 Extraction and identification of metabolites, following fungal growth in 57 liquid media 4.4.1 Methylene chloride 57 4.5.2 Complex formation 57 4.5.3 Identification of metabolites 58 v 4.6 Attempt to fractionate the protein that degrade D D A C 60 4.6.1 Comparison of extracellular and intracellular protein in selected 60 fungi exposed to D D A C and without DDAC, using electrophoresis 4.6.2 Degradation of D D A C by supernatants from cultures of 61 V. bulbillosum containing 0 ppm D D A C and 250 ppm D D A C 4.7 The distribution of D D A C in starch, silica gel, and solution 63 4.8 Fungal growth in liquid media containing decyldimethylamine (Barlene 10S) 65 4.8.1 Fungal species 65 4.8.2 Growth conditions 65 4.8.3 Fungal biomass 65 5. RESULTS AND DISCUSSION 66 5.1 Use of a colormetric approach to monitor changes in D D A C 66 concentration during fungal growth 5.1.1 Bromophenol blue 66 5.1.2 Methyl orange indicator 68 5.2 Recovery and identification of fungal strains from field area 71 5.3 Comparison of the tolerance of isolates from the field experiment with 78 those from culture collections 5.3.1 Solid media 79 5.3.2 Liquid media 96 5.3.3 The growth curve of V. bulbillosum 99 5.4 Fungal degradation of D D A C 101 5.4.1 Solid media 101 5.4.2 Liquid media 104 5.5 Identification of D D A C metabolites and implications for 115 fungal degradation process 5.5.1 Decanoic acid 115 vi 5.5.2. Tertiary amine 119 5.5.3 Possible process in biodegradation of QACs by moulds 119 5.6 The growth of fungi in liquid media containing decyldimethylamine 121 5.7 The distribution of D D A C in starch, silica gel and aqueous solution 5.7.1 The recovery of D D A C from liquid media without starch 123 but with silica gel 5.7.2 The distribution of D D A C 125 5.7.3 Residual amount of D D A C after fungal growth in liquid media 128 with silica gel 5.8 Attempts to fractionate the protein produced by fungi growing in liquid media 129 with and without D D A C 5.8.1 Extracellular protein pattern 129 5.8.2 Intracellular protein pattern 132 5.8.3 Decreased amount of D D A C after treatment with intracellular 132 extracts and supernatants from cultures of V. bulbillosum containing 0 ppm and 250 ppm D D A C 5. CONCLUSIONS 135 6. RECOMMENDATION 136 7. REFERENCES 137 vii LIST OF TABLES Page Table 3.2.1 Biodegradation data of alkyltrimethylammonium 19 (RMeslST) type quaternary surfactants (Swisher, 1987) Table 3.2.2. Biodegradation data of alkyldimethylbenzylammonium 21 (RBzMe2lNr) type quaternary surfactants) (Swisher, 1987) Table 3.2.3 Biodegradation data of dialkyldimethylarnmonium 22 (R^M^ISH) type quaternary surfactants) (Swisher, 1987) Table 5.2.1 Fungi isolated in July from treated and untreated stakelets 72 placed in field area Table 5.2.2 Fungi isolated in October from treated and untreated stakelets 74 placed in field area Table 5.2.3 Fungi isolated in October from treated and untreated stakelets 76 placed in fungal cellar Table 5.3.1.1 Slope and intercept of linear regression of fungal growth 93 (hyphal diameter in cm) 'over the first fifteen days and incubation period (days) Table 5.3.1.2 Slope and intercept of linear regression of growth" of G. roseum 94 (hyphae diameter in cm) over the first fifteen days and the incubation period (days) Table 5.3.2.1 Fungal growth in the liquid media containing 250 ppm 97 D D A C Table 5.4.1.1 D D A C degradation in solid media 102 Table 5.4.2.1 The recovery of D D A C from liquid media and buffer using 105 different extraction procedures Table 5.4.2.2 Residual concentration of D D A C in liquid media 107 after fungal growth Table 5.6.1 Fungal growth in nominal Vogel liquid media containing 122 1000 ppm decyldimethylamine viii Table 5.6.2 Fungal growth in normal Vogel liquid media containing 100 ppm 122 decyldimethylamine Table 5.7.1.1 The recovery of D D A C from liquid media 124 Table 5.7.2.1 Distribution of D D A C in starch, silica gel, starch and silica gel, 126 and aqueous solution Table 5.7.2.2 The biomass of fungi in liquid media counting 250 ppm D D A C 127 and silica gel Table 5.8.3.1 Amount of D D A C in buffer after treatment with extracellular and 134 intracellular supernatants from cultures containing 0 ppm and 250 ppm D D A C ix LISTS OF FIGURES Page Fig. 1.2.1.1 Diagrams of main types of alkylammonium compounds 6 compared to ammnium chloride Fig. 3.3.1 Possible mechanisms of degradation of QACs by bacteria 34 Fig. 3.3.1.1 Biodegradation of alkyltrimethylammonium chloride, initiated 3 5 at the terminal methyl group of long alkyl chain, followed by P-oxidation of the chain with the formation of betaine." Subsequent cleavage of the methyl groups from the nitrogen yields sarcosine (Cain, 1976) Fig. 3.3.2.1 Proposed biodegradation mechanism for hexadecyltrimethyl- 37 ammonium chloride by Pseudomonas strain B \ (Van Ginkel et al, 1992) Fig. 3.3.2.2 Possible pathways for the biodegradation of trimethylethyl- 39 ammonium chloride by Pseudomonas (Ghisalba and Kuenzi, 1983) Fig. 5.1.1.1 Absorption spectra of 1) standard bromophenol blue 67 (2.04xl0"5 M) 2) complex formed by bromophenol blue (2.04xl0"s M) anddidecyldimethylarnmonium chloride (l.O^xlO-^atpHl.SO Fig. 5.1.1.2 Absorption spectra of 1). standard bromophenol blue 69 (2.04xl0"s M) 2) complex formed by bromophenol blue (2.04x10*5 M) and didecyldimethylammonium chloride (l^xlO^lVQatpHn.O Fig. 5.3.1.1 Fungal growth in malt agar 80 (S-l-6 is Z. molleri; S-2-11, S-l-1 and S-2-1 are F. avenacewn; F-l-1 is T. virens) (Standard deviations < 0.6 cm) Fig. 5.3.1.2 Fungal growth in malt agar (Standard deviations < 0.6 cm) 81 Fig. 5.3.1.3 Fungal growth in malt agar (Standard deviations < 0.6 cm) 82 Fig. 5.3.1.4 Growth of G. roseum on MA with 250 ppm DDAC 83 (Standard deviations < 0.6 cm) Fig. 5.3.1.5 Fungal growth on MA with 500 ppm DDAC 84 (S-l-6 is Z. moiled; S-2-11, S-l-1 and S-2-1 are F. avenacewn; F-l-1 is T. virens) (Standard deviations < 0.6 cm) Fig. 5.3.1.6 Fungal growth on M A with 500 ppm D D A C 85 (Standard deviations < 0.6 cm) Fig. 5.3.1.7 Growth of G. roseum on on M A with 500 ppm D D A C 86 (Standard deviations < 0.6 cm) Fig. 5.3.1.8 Fungal growth on M A with 750 ppm D D A C 87 (S-l-6 is Z. molleri; S-2-11, S-l-1 and S-2-1 are F. avenaceum; F-l-1 is T. virens) (Standard deviations < 0.6 cm) Fig. 5.3.1.9 Fungal growth on M A with 750 ppm D D A C 88 (Standard deviations < 0.6 cm) Fig. 5.3.1.10 Growth on of G. roseum M A with 750 ppm D D A C 89 (Standard deviations < 0.6 cm) Fig. 5.3.1.11 Fungal growth on M A with 1000 ppm D D A C 90 (Standard deviations < 0.6 cm) Fig. 5.3.1.12 Growth of G. roseum on M A with 1000 ppm D D A C 91 (Standard deviations < 0.6 cm) Fig. 5.3.1.13 G. roseum (TC-NP-16) grown on M A containing 0 ppm 95 and 750 ppm D D A C A: Face up B: Face down Fig. 5.3.2.1 Fungal growth in liquid media containing 250 ppm D D A C 98 A: G. roseum (TC-NP-16) B: F. avenaceum (S-l-1) Fig. 5.3.3.1 Growth curve of V. bulbillosum 100 Fig. 5.4.1.1 The different locations on the malt agar plate for assessment 103 of D D A C C: center E: edge P: perimeter Fig. 5.4.2.1 HPLC spectra of extract from liquid media containing 108 250 ppm D D A C after fungal growth of V. bulbillosum A: Standard D D A C (776 ppm) B: Control (concentrated extract from liquid media without DDAC) C: Concentrated extract from Vogel liquid media with D D A C D: Concentrated extract from Vogel liquid media with D D A C and silica gel xi Fig. 5.4.2.2 Fig. 5.4.2.3 Fig. 5.4.2.4 Fig. 5.4.2.5 Fig. 5.4.2.6 Fig. 5.5.1.1 Fig. 5.5.1.2 Fig. 5.5.1.3 HPLC spectra of extracts from liquid media counting 109 250 ppm D D A C after fungal growth of Acremonium sp. A: Standard D D A C (776 ppm) B: Control (concentrated extract from liquid media without DDAC) C: Concentrated extract from Vogel liquid media with D D A C D: Concentrated extract from Vogel liquid media with D D A C and silica gel HPLC spectra of extracts from liquid media counting 110 250 ppm D D A C after fungal growth of G. roseum (TC-NP-16) A: Standard D D A C (776 ppm) B: Control (concentrated extract from liquid media without DDAC) C: Concentrated extract from Vogel liquid media with D D A C HPLC spectra of extracts from liquid media counting 111 250 ppm D D A C after fungal growth of F. avenaceum (S-l-1) A: Standard D D A C (776 ppm) B: Control (concentrated extract from liquid media without DDAC) C: Concentrated extract from Vogel liquid media with D D A C HPLC spectra of extracts from liquid media counting 112 250 ppm D D A C after fungal growth of G. rosem (321C) A: Standard D D A C (776 ppm) B: Control (concentrated extract from liquid media without DDAC) C: Concentrated extract from Vogel liquid media with D D A C HPLC spectra of extract from liquid media containing 113 D D A C without any fungal growth A. Standard D D A C (776 ppm) B. Extract from Vogel liquid media with 250 ppm D D A C C. Extract from Vogel liquid media with 800 ppm D D A C and siica gel GC-MS spectra of a component ( retention time 13.584 min) 116 in the extracts from a liquid culture of V. bulbillosum GC-MS spectra of a component (retention time 12.567 min) 117 in the extracts from a liquid culture of G. roseum (TC-NP-16) GC-MS spectra of a component ( retention time 13.551 min) 118 in the extracts from a liquid culture of Acremonium sp. xii Fig.5.8.1.1 Silver stained SDS-PAGE phast gel (homogenious 12.5%) of 130 extracellular proteins from culture of V. bulbillosum and Acremonium sp. containing 250 ppm D D A C and 0 ppm D D A C lane 1: low molecular weight standard lane 2: 2 u,g protein from Acremonium sp containing 0 ppm D D A C lane 3: 2 u,g protein from Acremonium sp containing 250 ppm D D A C lane 4:2ug protein from V. bulbillosum containing 0 ppm D D A C lane 5: 2 p,g protein from V. bulbillosum containing 250 ppm D D A C Fig. 5.8.1.2 Silver staining TEF gel (pi 3-9) of extracellular protein from 131 culture supernatants of V. bulbillosum and Acremonium sp. containing 0 ppm D D A C and 250 ppm DDAC. Pharmacia TEF standards are shown in lane 5. Protein were loaded at approximately 2 u,g per lane, lane 1: supernatant from culture of. V. bulbillosum containing 0 ppm DDAC. lane 2: supernatant from culture of. V. bulbillosum 250 ppm D D A C lane 3: supernatant from culture of. Acremonium sp containing 0 ppm D D A C lane 4: supernatant from culture of Acremonium sp. containing 250 ppm D D A C Fig.5.8.2.1 Silver stained SDS-PAGE phast gel (8-25%) of intracellular 133 protein from culture of V. bulbillosum with 500 ppm D D A C and without D D A C lane 1 and 6: low molecular weight Standard lane 2: lpg protein from V. bulbillosum withDDAC lane 3: lug protein from V. bulbillosum withoutDDAC lane 4: 2ug protein from V. bulbillosum with D D A C lane 5: 2u,g protein from V. bulbillosum without D D A C xiii ACKNOWLEDGMENTS I would like to sincerely thank.my supervisor, Dr. John N. R. Ruddick, whose patience, enthusiasm and encouragement enabled me to complete this work. I wish able to thank Dr. Breuil, for her helpful advice and guidance. I also want to thank the members of my thesis committee, Dr. Simon Ellis and Dr. Leigh Walker for their help and support and my co-workers Mr. Changshi Xie and Dr. Jochen Burgel for their generous help, advice and support throughout my studies. I would like to acknowledge the assistance of Dr. Paul Morris for teaching me how to prepare selective media and isolate fungi from wood. I gratefully acknowledge financial support provided by Lonza Inc. Finally, my greatest gratitude goes to my parents who care for, support and encourage me during my educational studies. xiv LIST OF ABBREVIATIONS A A C Alkylammoniurri compound A C A Ammonium copper arsenate B S A Bovine serum albumin B T M A Benomyl-tetracycline-malt agar C C A Chromated copper arsenate D D A C Didecyldimethylammonium chloride D D A Decyldimethylamine GC-MS Gas chromatography mass spectra HPLC High performance liquid chromatography IEF Isoelectric focusing M A Malt agar P A G E Polyacrylamide gel electrophoresis PAHs Polycylic aromatic hydrocarbons PCP Pentachlorophenol QAC Quaternary ammonium compound SCNR Starch-casin-nitate-rose bengal SDS Sodium dodecyl sulphate XV 1. INTRODUCTION Wood being a naturally biodegradable product, is often vulnerable to attack by biological organisms, which can degrade it and cause loss in value. Fungi of various kinds can grow in wood, resulting in stains and decay. In general, the basic requirements of deteriorating organisms are: oxygen, a suitable temperature (15-30 °C) and pH, water, and a source of nutrients. If one or more of the basic requirements can be controlled, wood decay could be prevented. Under normal exposure conditions the oxygen and the environmental temperature can not be controlled. One of the simplest preservation approaches is to keep wood dry. However, various uses of wood can not eliminate all the decay risk situations. So to prevent colonization by sapstain, mould and wood-destroying fungi, wood can be protected by application of chemical preservatives, and this is the most common method of protecting wood used in biologically hazardous situations. Each year the industry in Canada treats approximately 2.0 million cubic meters (70.1 million ft3) of wood. In 1992, 51.5% of the treated wood went to the consumer lumber, 21.3% to pole production and 24.1% to industrial lumber. In 1992 the value of the treated lumber was $547 million. The value of the total volume of treated wood installed in Canada annually is in excess of $10 billion (Stephens et al. 1994). The wood preservation industry is therefore an important contributor to the Canadian economy. 1 1.1 The limitations of currently used wood preservatives Wood preservatives can be divided into two categories: those which are oilborne and those which are waterborne. The traditional oilborne preservatives include creosote, pentachlorophenol, bis(tributylin)oxide, copper-8-quinolate and copper naphthenate. However, of the five preservatives, only creosote and pentachlorophenol have been widely used in Canada. Creosote was the first commercially successful preservative and it is still used today. It is produced by the high temperature carbonization of bituminous coal in the absence of air. The principal product of the process is coke. One of the by-products is coal tar, which is distilled to produce creosote. Creosote is neither a pure compound nor a well-defined mixture of compounds. The composition of creosote varies from one source to another, being also greatly influenced by the coking process. Many of the minor components in creosote remain unidentified. Further, creosote contains polycyclic aromatic hydrocarbons (PAHs), some of which may cause cancer or be mutagenatic. However, creosote is still the principal preservative used for railway ties in Canada and is also widely-used to preserve marine piling and timbers. Pentachlorophenol the second generation oilborne preservative was developed in the mid 1930's. It is a pure crystalline organic compound, and is produced commercially by the chlorination of phenol, using an aluminum trichloride catalyst. However, dioxins, which are present as a minor impurity in technical grade pentachlorophenol have caused the use of this preservative to decrease, principally because one member of the dioxin family (2,3,7,8-tetrachlorodibenzo-p-dioxin) has been shown to be very toxic. This causes pentachlorophenol to have a poor image. 2 Waterborne preservatives are based upon formulations of inorganic chemicals that are solubilized in aqueous solutions. There are two fundamental types of waterborne preservatives. The first, chromated-copper formulations, is acidic in nature and relies upon the change in the oxidation state of the chromium compound to achieve fixation to the wood. This type of preservative system is widely used around the world, with chromated copper arsenate (CCA) being the most popular formation (Zabel and Morrell, 1992). The most commonly used formulation in North American, CCA type C, contains 47.5% chromium, 18.5% copper and 34% arsenate (expressed on an oxide basis). The fixation of CCA has been the object of considerable research but a complete understanding has still not been achieved. Only recently, it was discovered that a stable Cr(V) intermediate is formed during the fixation process (Ruddick, etal. 1994). CCA has been the most successful system used today because its stability in wood and effectiveness against a wide range of microorganisms. The second type of waterborne preservatives is that based upon basic copper solutions. The most widely used preservative of this type contains copper and another co-biocide dissolved in ammonium hydroxide [e.g. ammonical copper arsenate, (ACA)] (Zabel and Morrell, 1992). ACA is less appealing than CCA because of difficulty in handling ammonia and it is often considered to be more leachable than CCA. Both preservatives contain the toxic heavy metal arsenic. In Canada, these traditional wood preservatives still dominate the use of treated wood. Of the total production volume, 79.1% was treated with waterborne preservatives while 20.9% was treated with oilborne preservatives (Stephens, 1994). Increasingly, it is becoming apparent that problems are arising with the currently used preservatives, because of two principal factors. They are: environmental concerns and the cost of oil and future energy 3 source, especially in regard to use of oil based preservatives. Of these two, environmental concerns dominate. Disposal of treated wood is a critical issue facing the industry today. Environmental and economic pressures on currently used chemicals have led to a situation where considerable effort is now directed towards the development of new wood preservatives. Among the promising candidates resulting from the last two decades of research one group of compounds comprised of long chain quaternary ammonium compounds (QACs) and tertiary amine acid salts, [collectively known as alkylammonium compounds (AACs)] has received considerable attention. 4 1.2 Quaternary ammonium compounds 1.2.1 Industrial applications AACs are derived from ammonium by successive replacement of the hydrogen atoms with hydrocarbon (alkyl) chains of varying length, thus making a wide range of compounds possible. The following Figure 1.2.1.1 illustrates the structure of the main groups of alkylammonium compounds in relation to the simple structure of ammonium chloride. The figure shows only chloride compounds, although the anion can be selected from a wide range of chemicals including bromide, iodide and acetate. There are two basic types of AACs that are of interest for wood preservation, namely quaternary ammonium compounds (QACs) and tertiary amine acid salts. Among AACs evaluated for use in the protection of wood from fungi, quaternary ammonium compounds with either two alkyl groups, or one alkyl and one aryl group, have been shown to offer promise as wood preservatives (Nicholas et al, 1991 and Tsunoda and Nishimoto, 1983). The QAC [didecyldimethylammonium chloride (DDAC)] was chosen to be studied in this project. QACs have been widely used as general purpose microbiocides for some 30 to 40 years, and are currently employed as microbiocides such as household disinfectants, eye-drops, nappy sanitisers, paint fungicides, swimming pool algaecides and surface wood treatment. Because of their surface active properties, they are also used as lubricants, antistatic agents, fabric softeners, hair conditioners and dye additives (Boething, 1984). 5 H CH 3 I I H - N+-H Cr C10H21- N+-H CI" I I H CH 3 ammonium chloride tertiary amine acid salt C H 3 C H 3 I C 1 0 H 2 1 - iT-doH^ cr C L 0 H 2 1 - N * - C H Z - ^ JCY C H 3 C H 3 ~ ~ dialkyldimethylammonium chloride alkydimethylbenzylammonium chloride C H 3 I CioB^i-lSr-CHaCr I C H 3 alkyltrimethylammonium chloride Fig. 1.2.1.1 Diagrams of main types of alkylammonium compounds compared to ammonium chloride 6 1.2.2 Potential as wood preservatives Based upon the extensive application of QACs for several decades without negative effects, as well as the toxicological data developed for specific QACs as required by regulatory agencies, it can be concluded that they are relatively safe compounds, readily available and economical for use by the preservation industry. For example, D D A C has an oral LD50 of 600-700 mg/kg (mouse) and an acute dermal LD50 of 4300 mg/kg compared with the of PCP oral LD50 of 50-140 mg/kg (rat) (Drysdale and Preston, 1982, Anonymous, 1984). In general, the AACs showing the best potential as wood preservatives are of low toxicity. Also there is a long history of widespread and safe use of these compounds in commercial products. In particular, QACs appear to have the potential for use as wood preservatives, as they exhibit a broad spectrum on biocidal effectiveness. Their effectiveness is equal to, or higher than, that of currently used C C A (Butcher etal. 1977b). QACs exhibit low volatility and are stable, colorless chemicals which are water and solvent soluble. They are environmentally more acceptable than presently used materials and reasonably close to being cost-competitive with the presently used preservatives such as CCA. These cationic materials are highly attracted to anionic surfaces such as wood, and do not pose problems for people handling treated wood. 7 1.3 Review of laboratory performance and field performance 1.3.1 Laboratory trials The use of QACs as fungicides in paints may have initially suggested their potential as a wood preservative. Various QACs were screened in the laboratory in Pinus radiate against several types of wood decay fungi, such as Postia placenta, Fomes gilvus and Chaetomium globosum. Most of QACs had a toxic threshold of the order 3.2-6.4 kg/m (Butcher et. al. 1977a and b). Further work showed that the effectiveness of QACs against basidiomycete fungi is equal to, or higher than, that of CCA (Butcher and Drysale, 1977). The same level of protection was obtained against several decay fungi when the retention of QAC 1-2 kg/m was approximately half that of the CCA (2-4 kg/m ) (Butcher, 1979a; Preston and Chittenden, 1982; Preston and Nicholas, 1982; and Nishimoto, 1983; Hedley et al. 1982). DDAC was found by several researchers to be most effective AAC, with threshold values of the order 1-2 kg/nv* (Preston and Chittenden, 1982; Preston and Nicholas, 1982; Preston, 1983; Hedley et al. 1982; Tsunoda and Nishimoto, 1987). 1.3.2 Field trials Parallel field trials did not reflect the initial laboratory observations. QAC-treated stakes showed signs of surface decay soon after being placed in the field and did not perform as well as CCA in a field trial during a 30 month period ( Butcher et al, 1979). The failure of stakes treated with dialkyldimethylammonium chloride (14.6 kg/m )^ after only one year exposure in the field (Tillott and Coggins, 1981) confirmed an earlier observation of stake failure (Ruddick, 1981) in which stakes were exposed to a mixed QAC formulation at a retention of 12 kg/nA In a subsequent field trial Ruddick (1983) also found that stakes 8 treated with DDAC (3.2 kg/m ) failed after two years of field exposure. QAC-treated wood • • • 3 • • with retentions as high as 11 kg/m had noticeable decay after six years of exposure (Ruddick, 1987; Morris and Ingram, 1988). Drysdale (1983a) found that dialkyldimethylammonium chloride (6.2 kg/m3) treated and benzalkonium chloride (10.6 kg/m3) treated P. radiate stakes were attacked by brown rot, soft rot and white rot fungi after four years in the field. In 1978 because of the good performance in early above ground and laboratory studies in New Zealand. QACs were approved above ground use. This was subsequently restricted due to the failure of QACs treated fencing (Butcher and Greaves, 1982). These failures in above ground application mirrored the earlier failure of QAC-treated stakes placed in the field exposure trial (Ruddick, 1981; Tillott and Coggins, 1981). 1.3.3 Reasons for the poor field performance The effectiveness of QACs against fungi in laboratory and field tests has varied widely. Several factors could account for this wide discrepancy in performance, including inadequate retentions being used, leaching of QACs from treated wood, uneven distribution and biodegradation of QACs. However, field trials with higher retentions also showed sign of decay (Ruddick, 1983, Morris and Ingram, 1988). So the use of too low a retention can not be the reason for the field performance. Fixation of QACs in wood QACs are surfactants. In aqueous solution, surfactants can be adsorbed onto a solid substrate by various mechanisms: physical adsorption by polarization or dispersion forces, ion pairing and ion exchange (Rosen, 1975). Wood has been described as having strongly 9 charged sites. Fixation of QACs to wood is a complex process, during which the adsorption of the solute may occur followed successively by ion-exchange, ion pairing and hydrogen bonding. Ion-exchange would appear to be the main mechanism for 'fixation' of QACs to wood. This was confirmed by the influence of pH on QAC retention in wood. Butcher and Drysdale (1978b) suggested that more acidic treating solution would retard fixation and result in a more uniform distribution, providing better protection of wood. An alkaline treating solution would create more negative binding sites and speed up fixation, which would result in higher loadings especially in the peripheral regions. Other studies confirmed that an increase in treating solution pH increase retention of DDAC in the wood, particular in the lignin component (Jin and Preston, 1991, Doyle and Ruddick, 1994). The most likely functional groups on the wood to react with QACs are negatively charged sites, such as the carboxylic and phenolic groups on lignin or hemicellulose components (Brezny et al. 1990). This was confirmed by the recent research which found that DDAC adsorbed onto lignin preferentially, while a small amount were adsorbed onto the cellulose (Doyle, 1995; Jin and Preston, 1991). Distribution of QACs in wood The erratic distribution of QACs in wood could be a factor contributing to their poor field performance. The QAC retention in outer zone of treated lumber is much higher than that of the core (Butcher and Drysdale, 1978a; Ruddick and Sam, 1982; Drysdale, 1983b; Nicholas et al. 1991). Drysdale (1983b) did not detect uneven distribution in the outer 6 mm of P. radiata. Recent studies found that earlywood retained more QAC than latewood (Nicholas et al. 1991; Doyle and Ruddick, 1994). However, the variation observed in these studies is not sufficient to explain the poor field performance. 10 Leachability of QACs from the treated wood Leaching of QACs from treated wood has been proposed as one of the reasons for the failure in the field area. Initial research on QACs leachability using four softwood found that leaching was low and independent of the type of leaching solution used. The rate of depletion was dependent on the surface to volume ratio, but was not significant enough to account for the failure of QAC-treated wood in the field (Ruddick and Sam, 1982). Drysdale (1983b) found that 30% QAC leaching occurred from the outer 2 mm of wood samples during an 18 week leaching regime. The amount of QACs leaching from wood depends on the concentration of the preservatives, the sample size, wood species, the duration of the test and the type of medium used. For example, the higher the retention, the greater the leaching (Loubinoux et al. 1992). Water leaching studies showed that the AAC depletion was independent of the solution pH (Sundman, 1984, Ruddick and Lum, 1991). In addition, preleaching of QAC-treated blocks had little influence on their performance during soil block testing (Ruddick, 1986). Loss of DDAC from latewood during leaching in a soil substrates was 30-40%. In the study, the loss of DDAC from earlywood (35%) region was greater than the latewood region (20%) (Nicholas etal. 1991; Ruddick and Lum, 1991). In summary, All the experiments seemed to show that QACs do leach from the wood, but that the degree of depletion should not have led to the rapid failure of the treated wood in service. Biodegradation of QAC During the field exposure of QAC-treated wood, it was noted that it soon became heavily stained (Ruddick and Sam, 1982; Drysdale, 1983a). Ruddick (1986) carried out a study on the effect of pre-exposure of QAC-treated wood to staining fiingi, on the toxic threshold of selected the decay fungi. The results showed that the toxic threshold for two of 11 the three fungi used, increased two times. Recent research sponsored by the NSERC/Industrial Chair in Wood Preservation at UBC, has demonstrated that non-wood destroying fungi can reduce the efficacy of AACs as wood preservatives (Doyle and Ruddick, 1993; Doyle, Chow and Ruddick, 1993). These studies showed that at least two fungi were very tolerant to DDAC, based on the ready fungal growth over media amended with high concentrations of the chemical. Further, it was noted that the DDAC concentration was greatly reduced in malt agar after exposure to the test fungi. The reduction of DDAC concentration showed that the fungi can degrade or modify DDAC. 12 2. OBJECTIVES The principal objective of the current project was to determine the possible role of staining or mould fungi in degading QACs to reduce their performance as wood preservatives. This main objective was supported by three sub-objectives which were to: • isolate and identify tolerant mould or staining fungi from treated wood; • compare their tolerance and ability to degrade DDAC with similar fungi from stock cultures; • confirm possible biodegradation processes in liquid culture by identifying metabolites. When completed successfully, this QAC information can then be used to develop formulations with better all round field performance. It will also contribute to an understanding of the potential of these fungi for bioremediating QAC-contaminated waste. 13 3. LITERATURE REVIEW OF BIODEGRADATION OF QACS The widespread application of QACs, particularly in synthetic detergents which were introduced in the 1960's, has resulted in their becoming increasingly detected in waste water, treated sewage and the receiving waters, due primarily to their excellent foaming properties. Typical concentrations in sewage plant effluent ranged from 5 to 10 ppm. Since QACs are toxic to a wide variety of aquatic organisms, including algae, fish, mollusks, barnacles, and shrimp (Taft, 1946, Vallejo-Freire et al., 1954, Knauf, 1973, Kappeler, 1982, Waters, 1982, Lewis and Wee, 1983), the build up of QAC concentrations in waterways must be avoided. Because of the potential for widespread exposure and the established toxicity of QACs, it is very important to understand the behavior of these compounds and how they are degraded by microbes in the environment. Consequently, it was anticipated that a review of the literature of the biodegradation of QACs would enable a good understanding of the relative ease of fungi and bacteria to degrade these chemicals. This information could then be used to formulate a strategy for evaluating the potential for mould and stain fungi to reduce the preservative efficacy of QACs through biodegradation. Following a survey of the literature it is surprising to discover that all of the available research has been done on bacterial degradation of QACs, and that there was no information on the fungal degradation of QACs. While a few reports of fungal tolerance have appeared in the literature no investigation detailing the biodegradation of QACs was discovered. Ruddick (1986) reported that pre-exposure of QAC-treated wood to a mixture of isolates recovered from failing field test material, substantially reduced its subsequent performance against three basidiomycete 14 fungi. Doyle and Ruddick (1993) have shown reported that three moulds isolated from discolored lumber, showed extreme tolerance to DDAC, growing on 2500 ppm DDAC in solid agar medium. The three fungi were identified as Verticillium bulbillosum, Acremonium sp. and Gliocladium roseum. High performance liquid chromatographic analysis of the amount of DDAC remaining either in the agar medium, or bound to the fungal hyphae, confirmed its disappearance. Since there is no data in the literature on fungal degradation of QACs, this review will focus on the bacterial degradation of QACs which have been proposed or used as wood preservatives. It is understood that the mechanisms of fungal degradation of QACs may be the same or different from those reported to occur for bacteria. However, an important observation is that many of the QACs are degraded by bacteria, and that microbial action could well explain the significant reduction QAC in effectiveness as wood preservatives. 15 3.1 Definitions of biodegradation The following is the generally accepted classification of various forms of degradation: Biodegradation According to Swisher (1987), biodegradation may be defined as the breakdown of chemical compounds as a result of the microbes. In this review, the organisms of interest are fungi and bacteria. Primary biodegradation Primary biodegradation may be defined as the disappearance of a compound to such an extent, that either specific characteristic properties are no longer evident, or it no longer responds to a compound specific, analytical procedure. The limitation of this approach is that it makes no allowance for the disappearance of the chemical by substrate adsorption or other physical processes. Environmentally acceptable biodegradation Environmentally acceptable biodegradation is often used to define the state at which a chemical has been partially degraded, yielding products that are deemed to have no harmful effect on the environment. Since the acceptability of chemicals for release into the environment often changes with time, this is not a good criterion for considering that the degradation has reached a suitable state. It also requires that the by-products of the biological action are known. Again no allowance is made for loss by adsorption processes. 16 Ultimate biodegradation or mineralization Ultimate biodegradation is defined as the biological conversion of a chemical to the oxidized form of its basic components, i.e. carbon dioxide and water, together with the formation of mineralized salts and the biological waste products of the microbial action. It is often used interchangeably with mineralization. This is not strictly correct since in complete mineralization, even the carbon used by the organism to generate growth must be converted to carbon dioxide. Clearly this would take some time to accomplish and is a rather idealized concept. 17 3.2 Evidence of biodegradation of QACs QACs can be divided into five groups according to the number and type of alkyl chains, attached to the central nitrogen. The five groups are: alkyltrimethyl-, alkylbenzyldimethyl-, dialkyldimethyl- alkylpyridinium, and trialkylmethylammonium salts. Only the first three of these have been considered as wood preservatives. A comprehensive listing of the literature on the bacterial biodegradation of types has been compiled by Swisher (1987), and is reproduced in Tables 3.2.1 to 3.2.3 below. In general two criteria prevail. They are that resistance to biodegradation increases with a) the number of long alkyl chains bonded to the nitrogen; and b) with increasing alkyl chain length. 3.2.1 Alkyltrimethylammonium salts, CnMesN* This cationic surfactant group represents the most facile with respect to biodegradation and under appropriate conditions with a suitable organism, the QAC can be almost completely degraded (i.e. mineralized). Dean-Raymond and Alexander (1977) studied ten QACs and reported that of the ten, only decyl- and hexadecyltrimethylammonium bromide could be metabolized by the sewage microbes. Decyltrimethylammonium bromide was extensively degraded within 8-10 days at three concentrations tested (10, 25, 100 p. g/ml). By enrichment techniques, a mixed culture was developed which could live on 500 p, g/ml decyltrimethylammonium bromide as the sole carbon source. From the several species (at least five colony types), it was observed that while none of isolates could grow when 18 Table 3.2.1 Biodegradation data of alkyltrimethylammonium (RMe3N ) type quaternary surfactants (Swisher, 1987) Substrate RMe3N Extent Method Time hours (h) or days (d) Reference c 2 c r 100 In 72h;196h;215h Ghisalba and Kiienzi, 1983 Ci6 Br" 0 BOD 5d Sheets and Malaney, 1956a,b 0.1 Wa 6h Barden and Isaac, 1957 100 CAS 8h Pitterand Svitalkovd, 1961 0 BOD 5d Winter, 1962 37;44 In 39d c 8 c r 73 In lOd Masuda et. al., 1976 cio cr 91 In lOd C12 cr 100 In 2d 63;90 In 10;12d C14 cr 100 In 2d 59;82 In 10;12d ci6 cr 100 In 3d 35;57 In 10;12d cis cr 100 In lOd 0;22 In 10;12d 56 In 20d Ci6 Br" 100; 100 In 7d Pitter and Svitalkova, 1976 93;94 In 7d Et02CCi5 Br" (Septonex) 91;79 In 7d 80;73 In 7d ci6 cr *100 Sew.Acc 7;2d Baleux and Caumette, 1977 *100 RW 7-13d 100 In 65h? Van Ginkel et. al., 1992 Ci6 Br" *100 Sew;Acc 4;2d Cio Br" «100;0.62 In 3;10d Dean-Raymond and Alexander, 1977 Ci6Br" 0.58 In 17d Ci6 Br" 91-98 CAS 3h Gerike et. al, 1978 107±19 CAS 3h 15;6 In 15h? Mackrell and Walker, 1978 109±19 CAS 3h Gerike and Fischer, 1979 53 BAS 14d 0-7 In 14-40d 0 In 14-40d 0 In 14-30d 0 In 28d 19 Table 3.2.1 Biodegradation data of alkyltrimethylammonium (RMe3N ) type quaternary surfactants (contd.) Substrate^  Extent Method Time Reference RMe3N hours (h) or days (d) Tallow CI" 0 In lOd Itoh et. al., 1979 CgCl- 84 In lOd Miura et. al., 1979 cio cr 98 In lOd en cr 100;88 In 2;10d C14 cr 100;83 In 2;10d cie cr 100;46 In 3;10d cig cr 100;0 In lOd en cr 98;99 RW 3;4d Ruiz Cruz, 1979 Ruiz Cruz and Dobarganes Garcia, 1979 cie cr 99 In 4d cis cr 99 In 5d Ci6 Br" 83,107 CAS 3h Gerike et. al., 1980 91 BAS 19d Gerike and Fischer, 1981 c i6 cr 70;92 In 0.26;0.22 Larson and Perry, 1981 cn cr 98 RW 4d Ruiz Cruz, 1981 cig cr 81;<0 In 25d Games et. al., 1982 ( a - 1 4 i 9 (Me- C) 99 BAS Id 96;65 BAS 7d 98;88 BAS 7d Ci6 Br" 98 CAS 3h Gerike, 1982 cig cr 57;0 .- 1 4C)79 In 20d Itoh and Naito, 1982 *Cig Cl"(o RW 21d Larson and Wentler, 1982 Larson and Vashon, 1983 74;0 In 25d 84 1 4 C ) 70 RW 23d Larson and Vashon, 1983 Ci6 Br"(a- RW 23d ci6 cr 84 In 13d cig cr 0 In 25d Ci6 Br" 98;107±19 CAS 3h Berth et. a/.,1984 71 GW/Soil 36d Larson, 1984 20 Table 3.2.2. Biodegradation data of alkyldimethylbenzylammonium (RBzMe2N ) type quaternary surfactants (Swisher, 1987) Substrate Extent Method Time Reference RBzMe2N+ hours (h) or days (d) R=Ci2NHCOCH2-(Quartolan) 0 Wa Id Winter, 1962 CCBz cr (Benzalkonium) 0 In ? Lambin et. al., 1966 C14BZ CI" 95 SF 2d Gawel and Huddleston, 1972 C14ED CI" 95 SF 2d C14BZ CI" 63-79 CAS 3h Fenger et. al., 1973 Cl2-16*Bz CI' (Hyamine 3500) 75;33 RW;Soil 20;64d Krzeminski et. al., 1973 95;92 BAS Id C X B Z cr 100 In l i d Arpino and Ruffo, 1976 C xCl2Bz cr 75 In 7d CgBz CI" 79 In lOd Masuda et. al, 1976 CioBz CI" 95 C12BZ cr 97; 100 In 10;2d C14BZ cr 83 In lOd Ci8Bz cr 0;62 In 10;25d Masuda et. al, 1976 Ci6Bz cr 96;88 In 20d Pitter et. al, 1976 85;81 In 20d Ci6Bz cr 0 In 60d Dean-Raymond and Alexander, 1977 cnBz cr 96;54±16 CAS 3h Gerike et. al., 1978 83±7 CAS 3h Gerike and Fischer, 1979 0-95 In 14-40d 0 In 14-30d 50 In 28d CocoBz cr 0 In lOd Itoh et. al, 1979 94;0 CAS;BAnD 3h;70d Janicke and Hilge, 1979 CnBz Cl";Br" 99 In 4d Ruiz Cruz, 1979 Ruiz Cruz and Dobarganes Garcia, 1979 C14BZ CI" 99 Ci6Bz CI" 98 In 6d Ci8Bz CI" 98 In 8d br-C xBz 61 In 15d C12BZ CI" 61 In 15d C12BZ CI" 84 BAS 19d Gerike and Fischer, 1981 CnBz CI" 97 RW 5d Ruiz Cruz, 1981 96 CAS 3h Gerike, 1982 83±7 CAS;BAS 3h;40d Gerike et. al., 1984 90;75 In 30;28d 21 Table 3.2.3 Biodegradation data of dialkyldimethylammonium (R2Me2N ) type quaternary surfactants (Swisher, 1987) Substrate Extent Method Time Reference R2Me2N+ hours (h) or days (d) (C8)2 CI" 95 SF 2d Gawel and Huddleston, 1972 (Cl0)2 cr 98 (Cl8)2 cr 100 In 7d Arpino and Ruffo, 1976 (cioto cr 50 In 19d Masuda et. al, 1976 (Cl2)2Cl" 0;25-75 In 10;25d (Cl4)2 Cl- 0;54 (Cl6)2Cr 0;42 (Cl8)2Cl" 0;23 (Cl8)2Cr 91-93 CAS 3h May and Neufahrt, 1976; Neufahrt et. al, 1976 (Cl8)2 cr 0 RW 30d Baleux and Caumette, 1977 (Cl2)2Br" 0 In 60d Dean-Raymond and Alexander, 19" (Cl8)2Br" 0 (Cio)2 cr 95;0±38 CAS 3h Gerike et. al, 1978 (Cl8)2 cr 95;78 CAS 3h 108±9 CAS 3h (Tai)2 CI" 0 In lOd Itoh et. al, 1979 62-82 CAS 3h Janicke andHilge, 1979 0 BanD 70d (Cl2)2 20 In 25d Miura et. al, 1979 (Cl4)2 30 (Cl6)2 40 (Cl8)2 25 (Cl0)2 cr 98 RW 4d Ruiz Cruz and Dobarganes Garcia, 1979 (cn)2 cr 90-92 RW 7d (Cio)2 cr 99 RW 7d Ruiz Cruz, 1981 (Cio)2 cr 95; 100 CAS;TF 3h;- Gerike, 1982 94±3 TF -(Cl8)2 100;86 TF -99±12 TF -(Cl8)2 90-96 CAS 3h Topping and Waters, 1982 77-100 CAS 5h 98-99 CAS ? Vos and de Henau, 1982 (*Ci8)2(U-14 86 TF -C) 66;43 RWg;RW 62;74d; Larson 1983 and Larson and Wentler, 1983 (Cl8)2Cr 4 In 33d (Cl8)2 (*Ci8)2(oc-(U- C) >95 CAS ? Neufahrt et. a/.,1984 C) 61;>31 81;>31 BAS 39d Sullivan, 1983 (Ci8)2*Me2 77;>53 (Cl8)2 73-92 •CAS ? HP Wee, 1984 22 List of abbreviations used in Table 3.2.1-3.2.3 The Method column are as follows: Acc Special acclimation BAnD Batch anaerobic digester BAS Batch or semicontinuous activated sludge BOD Standard BOD bottle procedure CAnD Continuous anaerobic digester, usually fed in small daily or weekly increments(thus actually semicontinuous) CAS Continuous-flow activated sludge GW Groundwater In Natural or synthetic medium inoculated with acclimated or unacclimated organisms; although the shake-culture, BOD, and Warburg procedures fall in category also, they have been designated individually. RW River water dieaway Sew Sewage dieaway SF Shake-flask culture Soil Percolation through a soil medium, usually intermittently, or dieaway in soil TF Trickling filter Wa Warburg respirometer, biodegradation may be estimated from other appropriate analysis besides 0 2 uptake * Indicate field test or large-scale trials in sewage treatment equipment 23 inoculated by themselves onto decyltrimethylammonium bromide amended media, a mixture of two pure strains, a Pseudomonas and a Xanthomonas, were able to grow on the amended media using the QAC as a sole carbon source. Both of these bacteria were Gram-negative rods which gave a positive Kovac oxidase test. It was suggested that the Xanthomonas acted on the decyltrimethylammonium bromide, but since this bacterium could not grow on the amended media alone, the Pseudomonas was thought to provide one or more of the growth factors needed, while being sustained by partial degradation of the QAC by the Xanthomonas. Metabolites extracted from culture by methylene chloride and identified by GC/MS included 9-carboxynonyl- and 7-carboxyheptyltrimethylammonium compounds. Their formation is consistent with the initial oxidation of the terminal carbon, followed by 0-oxidation of the acetyl units, leading ultimately to the formation of lower molecular weight carboxylic acids. Cells from cultures of Pseudomonas and Xanthomonas were grown in the presence of decyltrimethylammonium bromide, trimethylamine, decanoic acid and other possible by-products. Both decyltrimethylammonium bromide and trimethylamine were rapidly and almost completely oxidized, while trimethylamine, methyldecylamine (40%) and decanoic acid (65%) were substantially degraded within 3 hours or less, with almost no lag phase. This was interpreted by Dean-Raymond and Alexander as suggesting the formation of trimethylamine upon completion of P-oxidation. Hampton and Zatman (1973) studied the metabolism of tetramethylammonium chloride by the bacterium 5H2. This Gram-negative, non-mobile, rod-shape organism isolated from soil, was described as being capable of growth on tetramethylammonium chloride and trimethylamine, when they were present as the sole source of carbon. The authors suggested 24 that the tetramethylammonium chloride was completely biodegraded via the intermediates of trimethylamine, trimethylamine N-oxide to methylamine. Van Ginkel et al. (1992) reported that a bacterium, Pseudomonas strain B i , isolated from activated sludge could utilize hexadecyltrimethylammonium chloride as a sole carbon source. Of the various alkylsubstituted quaternary ammonium salts (i.e. alkyltrimethylammonium, dialkyldimethylammonium etc.) tested, Pseudomonas strain could grow only on the monoalkyl compounds. It was able to utilize these compounds over a wide range of alkyl chain length (C12 to C^l)- Van Ginkel and co-workers, also demonstrated that Pseudomonas strain B \ could use a wide range of organic compounds as sole carbon sources, including acetate, ethanol, glucose, hexadecanoate, and hexadecanal. However, none of the amines tested (hexadecyldimethylamine, hexadecylamine, trimethylamine and methylamine) could serve as growth substrates for this bacterium and these amines were not oxidized by bacterial cells grown on hexadecyltrimethylammonium chloride. These observations suggested that hexadecanal and hexadecanoate were likely intermediates formed during the degradation of hexadecyltrimethylammonium chloride. However, the inability of the Pseudomonas strain Bi to metabolize any of the amines, strongly suggests that this bacterium is only capable of cleaving and oxidizing the alkyl chain, and that the amines were not formed as intermediates in the biodegradation pathway. When the Pseudomonas strain Bi was grown in liquid culture in the presence of silica gel, the stoichiometric formation of trimethylamine was confirmed by gas chromatography. These observations suggested that the ultimate biodegradation of hexadecyltrimethylammonium chloride must involve at least two species of bacteria. 25 In a very recent study, Nishiyama et al. (1995) have confirmed the de-alkylation of the alkyltrimethylammonium halide as the first step in the degradation using an inoculum of activated sewage sludge. They suggested that the tertiary amine initially formed underwent rapid demethylation. It was also proposed that the amines were present as a salt. Using enrichment techniques, Ghisalba and Kuenzi (1983) isolated a number of bacteria from the sludge of an industrial sewage treatment plant, which were capable of utilizing trimethylethylammonium salt. Nine of these bacteria, classified as Pseudomonas sp., were able to grow on a wide range of QAC and amine substrates as a sole carbon and nitrogen source, including methylamine, dimethylamine, trimethylamine, dimethylamine dimethylethylamine and tetramethylamine. Interestingly, the growth rate on the amines was more rapid than that on the alkyltrimethylammonium chloride. In shake cultures with an unbuffered medium (MV7) and 5g/l of trimethylethylammonium chloride as the sole carbon source, a decrease in the pH (from 7 to 3.5) was observed due to the formation of hydrochloric acid. It was found that the trimethylethylammonium salt was biodegraded and utilized without the accumulation of intermediate degradation products, but with the formation of biomass and C0 2 and the release of NH4CI and HC1. In studying the biodegradation of C 1 8Me 3N +Cl' using 1 4 C labeling of the carbons adjacent to the nitrogen, Games etal. (1982) observed that when placed at the first carbon of N-methyl bonding, the formation of 1 4 C 0 2 was significantly faster than when it was placed in the Cig-alkyl chain. This was interpreted as being due to the N-methyl carbon being used mainly for energy, while the Cig carbon was involved more in the synthesis of biomass. 26 3.2.2 Alkyl benzyldimethylammonium salts, C nBzMe2N + Alkylbenzyldimethylammonium salts have been thought as superior wood candidates as wood preservatives. Based upon the oxygen uptake in respirometric experiments Masuda et al. (1976) found that 80 to 100% of the linear C nBzMe2N + cationic surfactants could be degraded in 10 days, when the alkyl chain length varied from C 8 to Ci 4 . However, longer alkyl chains, Ci6 and C^, were much more resistant to degradation, with the oxygen demand falling to almost zero. The addition of an anionic surfactant enhanced the degradation of the hexadecylbenzyldimethylammonium chloride to about 45% in 15 days. Using a preformed activated sludge, in a continuous flow experiment, Fenger et al. (1973) were able to reduce the detectable amount of tetradecylbenzyldimethylammonium chloride by up to 85%. The debenzylated amine, as well as benzoic acid and acetic acid were identified by infra-red spectroscopy and gas chromatography, but products from further degradation (for example methylamine) were not detected. This would support the conclusion that the cleavage of the benzyl group occurs preferentially as the first step in the degradation. The authors also concluded that at concentrations in the range of 20 ppm, tetradecylbenzyldimethylammonium chloride could be degraded in activated sludge even when it is the sole carbon source. Examination of the adapted sludge revealed that the most resistant bacteria to the tetradecylbenzyldimethylammonium chloride were almost exclusively Pseudomonas and Comamonas bacteria. A similar observation was recorded by Gawel and Huddleson (1972) who found that tetradecylbenzyldimethylammonium chloride at a concentration of 10 mg/1 was biodegradable in a shake culture. Confirmation of the disappearance of the QAC was based upon a colorometric and UV spectrometric analysis. Substitution of an ethyl group in 27 the benzyl ring caused the rate of degradation to slow, although 95% biodegradation was recorded in 2 days. Krzeminski et al. (1973) examined the biodegradation of alkyl(Ci2.i6)*BzMe2N+ (Hyamine 3500) by monitoring the generation of 1 4 C 0 2 from the 1 4 C tag in the methylene group of the benzyl substituent. They reported that more than 90% of the 1 4 C appeared as carbon dioxide with less than 10% being found in the effluent. Gerike et al. (1978) investigated the biodegradation of dodecylbenzyldimethyl-ammonium chloride based upon the Confirmatory Test using an activated sludge developed by the Organisation for Economic Cooperation and Development (OECD). In this test, the elimination of the QAC was monitored by the disappearance of the QAC using a disulphine blue active complex formation. Such an approach is limited since even a small modification of the molecule would cause a reduction in the QAC detected. Therefore, the authors also compared the dissolved organic carbon (DOC) before and after biological treatment. Under these conditions, the dodecylbenzyldimethylammonium chloride concentration was shown to be reduced by 54%. It was concluded that this QAC was degradable at least at a primary level. 3.2.3 Dialkyldimethylammonium salts (C n )2^ e 2^ + Dialkyldimethylammonium salts are considered to have the best potential wood preservatives based upon laboratory evaluations. As anticipated, the addition of a second hydrophobic alkyl chain on the QAC decreases its aqueous solubility compared to the monoalkyl QAC. At the same time, it is rendered more bacteriotoxic. However, the same 28 strategies that were successfully used for biodegrading benzalkonium chloride, can be employed with the dialkyldimethylammonium chlorides. These include maintaining QAC concentrations below those levels that are toxic to the micro-organism as well as complexing the QAC with a suitable anionic surfactant. The usefulness of the latter strategy has been ably demonstrated by Masuda et al. (1976). Based upon the reduction in oxygen uptake in respirometric experiments, almost 50% of didecyldimethylammonium chloride was biodegraded in 10 days, while higher homologues were not degraded at all. Addition of sodium dodecyl sulphate allowed the biodegradation to reach almost 80% in 20 days. Sullivan (1983) studied the degradation of ditallowdimethylammonium chloride using a "semi-batch" aerobic activated sludge, in which labeled surfactant was added at a concentration of 2 ppm to the initial charging of the system. In three replicate experiments, samples of ditallowdimethylammonium chloride were radiolabelled [14C] at the N-methyl, the 1 position of the Cig chain, or uniformly in the chain. The approximate initial [14C] ditallowdimethylammonium chloride concentration was 2.1 mg/1 in each test reactor. The 14CO*2 formation was monitored to determined the susceptibility of the different positions to being metabolized. Although the ditallowdimethylammonium chloride rapidly disappeared (95%) from the solution, it had been adsorbed by the sludge. Subsequently, over the remaining 35 days, it was biodegraded with mineralization being demonstrated by the production of 14CO*2. Between 20 and 50% of the ditallowdimethylammonium chloride was mineralized by day 39, based upon the production of labeled carbon dioxide. This observation confirmed that each of the three major carbon positions in the molecule could be mineralized, and that ultimate biodegradation was, possible for this QAC. The highest concentration of 1 4 C 0 2 was recorded for the N-methyl tagged molecule, suggesting that it was converted more 29 readily than carbons in the alkyl chain. A statistical analysis found no difference in the rate constants for the primary biodegradation, suggesting that all three labelled molecules were removed from the solution at the same rate. Based upon radio thin layer chromatographic scans, Sullivan also reported that since the concentrations of any intermediates were very low during the biodegradation and they did not accumulate in the sludge, they must be short lived and the rate of degradation must be limited by the primary degradation process, during which the QAC is removed from the solution. This is consistent with the observations of Gerike et al. (1978) who by measuring the soluble carbon content in the effluent of the test reactors, reported the complete removal of ditallowdimethylammonium chloride and any breakdown products from the test solution. Similar experiments with didecyldimethylammonium chloride gave much less satisfactory results with almost no degradation being reported (Gerike etal. 1978). Subsequent studies on this same QAC using a trickling filter demonstrated 94% removal of didecyldimethylammonium chloride, based upon the analysis of dissolved organic carbon. However, such results should be treated with caution as adsorbed or precipitated QAC would not be separated from the biomass, leading to an overestimation of biodegradation. The influence of the sediment on the biodegradation of QACs present in solution has been demonstrated by several researchers. Larson and Vashon, (1983b) confirmed the mineralization of dioctadecyldimethylammonium chloride in river water at 0.005 and 0.05 ppm, based upon the amount of 1 4 C 0 2 produced during biodegradation of the alkyl chains which were labeled uniformly with 1 4 C . Without any sediment the 1 4 C 0 2 production was much less (10 and 20% of theoretical) than that achieved when 5000 ppm of sediment was 30 present (65%). Thus under the right conditions the dioctadecyldimethylammonium chloride was biodegradable. 3.2.4 Alkylpyridinium salts (Cn)py+ Without exception, studies of alkylpyridinium salts confirm that they are some of the most difficult QACs to biodegrade. Masuda etal. (1976) measured the degradation of Ci 2Py + at only 18% based upon the 10 day oxygen uptake. However, Pitter (1976) using the "die-away method" found that the rate of primary biodegradation for the pyridinium bromide was comparable to that of the benzalkonium chloride. Wright and Cain (1972) reported that a bacterium Achromobacter D, isolated from garden soil was able to utilize N-methylisonicotinic acid as a sole carbon source. The metabolic products were identified as, methylamine from the N-methyl group, succinate and formate from the pyridine ring, and carbon dioxide derived from the carboxylic group on the heterocyclic ring. Barden and Isaac (1957) reported that 15 ppm of cetylpyridinium bromide was completely degraded by Pseudomonas pictorium. Baldwin et al. (1966) isolated a strain of Lipomyces starkeyi which was able to utilize paraquat and related pyridine compounds, as a source of nitrogen. 3.2.5 Trialkylmethylammonium salts (Cn)3MeN+ As expected these QACs are much more recalcitrant to biodegradation, although very few studies of their biodegradation have been reported. Ruiz Cruz (1979) studied the 31 biodegradation of tridecylmethylammonium chloride in river water at a concentration of 5 ppm. The time to 50% disappearance was 7.4 days, which is almost 3.5 times that of didecyldimethylammonium chloride. 32 3.3 Mechanisms of biodegradation The mechanisms by which bacteria degrade QACs are still not fully understood. Examination of the literature failed to reveal clear support for any one of the proposed models. In general, two basic biodegradation processes have been advanced (Figure 3.3.1). In the first, the micro-organism initiates attack (co-oxidation) at the end carbon of one of the long alkyl chains. Further degradation (P-oxidation) occurs at this long chain gradually removing carbon atoms. In the alternative biodegradation process, the initial step involves the breakage (fission) of one of the carbon-nitrogen bonds, leading to the formation of a tertiary amine and a residual alkan-l-al. A brief review of the supporting evidence for each of the mechanisms is presented below. 3.3.1 Oxidation of the long alkyl chain. Cain (1976) has noted that the long alkyl chains of QACs are potentially susceptible to co- oxidation at the terminal methyl position. Subsequent P-oxidation of this chain in the case of alkyltrimethylammonium chloride, results in the formation of betaine. Cain cites the fact that betaine can support the growth of selected bacteria, which demethylate it, forming first dimethylglycine and subsequently sarcosine (Figure 3.3.1.1). Sarcosine can be further demethylated to glycine or decarboxylated to form the secondary amine. The detection of the inducible dimethylglycine and sarcosine dehydrogenases by Frissell and Mackenzie (1962) was cited by Cain as providing supporting existence for this mechanism. 33 o- and P- oxidation (CHa^NCCH^CHs (CH3)3NCH2COOH betaine (CH 3 ) 2 NCH 2 COOH dimethylglycine -HCHO CH 3 NHCH 2 COOH sarcosine Fig 3.3.1.1 Biodegradation of alkyltrimethylammonium chloride, initiated at the terminal methyl group of long alkyl chain, followed by P-oxidation of the chain with the formation of betaine. Subsequent cleavage of the methyl groups from the nitrogen yields sarcosine (Cain, 1976) 35 This biodegradation mechanism in which attack is initiated at the end of the long alkyl chain was also supported by Dean-Raymond and Alexander (1977). When studying the biodegradation of decyltrimethylammonium bromide they observed the accumulation of 9-carboxynonyl- and 7-carboxyheptytrimethylammonium as intermediates, consistent with the to co-oxidation of the decyl group. Smaller amounts of lower molecular weight carboxylic acids derivatives were also possibly formed. The formation of such intermediates is consistent with an initial oxidation of the terminal carbon atom of the long chain by Xanthomonas, followed by stepwise cleavage of the acetyl units by P-oxidation. 3.3.2 Carbon nitrogen fission: cleavage of the alkyl chain This mechanism has also been proposed by Van Ginkel et al. (1992) working with bacteria (Figure 3.3.2.1). They employed a Pseudomonas strain B l to biodegrade hexadecyltrimethylammonium chloride, which was served as a carbon and energy source. However, while the bacterium could grow on hexadecyltrimethyl- ammonium chloride or related intermediates, it could not grow on amines. This observation formed the basis of the proposal by these authors, that a second bacterium was needed to achieve the ultimate biodegradation of this molecule. Confirmation for their hypothesis was obtained by the stoichiometric formation of trimethylamine when the bacterium was grown on hexadecyltrimethylammonium chloride. This proposal made by van Ginkel et al. (1992), that a consortium of micro-organisms would effectively lead to the ultimate biodegradation of QACs had not been identified earlier, but appears to be very plausible. The fact that the bacterium B l could successfully grow on hexadeconal and hexadecanoate, indicates that both are intermediates during the degradation, supporting of alkyl chain cleavage as a key step in 36 C H 3 I C H 3 ( C H 2 ) 1 4 C H 2 - t f - C H 3 BT + NAD(P)H NAD(P)+ C H 3 ( C H 2 ) u C H O NAD + IT + N A D H . H 2 0 CH 3 (CH 2 ) 1 4 COOH p- oxidation C 0 2 + H 2 0 + biomass N(CH 3 ) 3 another bacterium N H 3 + C 0 2 + H 2 0 + biomass Fig. 3.3.2.1 Proposed biodegradation mechanism for hexadecyltrimethylammonium chloride by Pseudomonas strain B \ (Van Ginkel etal, 1992) 37 the mechanism. The direct formation of trimethylamine is in agreement with the results reported by Hampton and Zatman (1973) who also observed the production of trimethylamine and methanol during biodegradation of tetramethylammonium chloride. Ghisalba and Kuenzi (1983) found that trimethylethylammonium chloride could be utilized by nine strains of bacteria, as both a carbon and nitrogen source. The pathways proposed by authors for the biodegradation of trimethylethylammonium chloride by Pseudomonas strains are depicted in Figure 3.3.2.2. They found that the Pseudomonas strains were able to grow much better on amines such as dimethylamine and dimethylethylamine than on either tetramethylammonium chloride or trimethylethylammonium chloride. This suggests that the first stage in degradation of the QAC, namely the loss of one alkyl group with the formation of a tertiary amine will be the rate limiting step in the biodegradation of the QAC. One question that remained unanswered was when the ethyl group was cleaved. Loss of the ethyl group during initial fungal attack would correspond to the left metabolic pathway in Figure 3.3.2.2. This resembles the biodegradation process for other alkyltrimethylammonium chlorides after the initial loss of the alkyl chain. The anticipated products from the first step would be trimethylamine and methan-l-al. In an alternative mechanism the ethyl group was cleaved at a later stage with the formation of methan-1 al and the corresponding amine acid salt. During fermentation studies, no significant accumulation of anticipated metabolites was detected. This would support the suggestion that the initial degradation of the QAC is the slowest step. However, it prevented any conclusion being made as to when the ethyl group was cleaved. 38 NADfP) (CH3)3NFf cr + <CH3CHO> o 2 H 2Q NAD(P)H NAD(P) CFGCOOH (CH 3 ) 2 NH 2 + Cr + <HCHO> _ 0 2 H20> NAD(P)H NAD(P) CH 3NH 3 +Cr + <HCHO> O; CH 3NH-CH 2-CH 3C: I CH 3 o2 H20 PQQ NH4+Cr+<HCHO>+-partially H a -*• Biomass «-C 0 2 •+<HCHO> NAD(P)H NAD(P) CH 3 NH 2 CH 2 CH 3 CI- + <HCHO> o2 H 20' -NAD(P)H •NAD(P) CH 3 NH 3 + CI" + <CH3CHO> CH3COOH Fig. 3.3.2.2 Possible pathways for the biodegradation of trimethylethylammonium chloride by Pseudomonas (Ghisalba and Kuenzi, 1983) 39 In a recent study Nishiyama et al. (1995) examined the biodegradation of several alkyltrimethylammonium halides by an activated sludge. Based upon analysis by ^ - N M R the formation of trimethylamine (as an acid salt) after 40 hours incubation was positively identified for the decyl-, dodecyl- and tetradecyltrimethylammonium bromides. Subsequently, after 48 hours of incubation, the acid salt of dimethylamine was identified as being formed in the medium. The third step in the degradation pathway, the further demethylation with the formation of methylamine (as an acid salt) was confirmed by the NMR examination after 76 hours. Finally, following almost five days of incubation neither the parent QAC nor any of the degradation products could be detected. These observations clearly support the cleavage of the long alkyl chain as the initiating step for alkyltrimethylammonium halides. Interestingly, these authors also confirm the formation of acid salts of the amines in the media together with their gradual disappearance as the incubation time extends. These observations may have a bearing on the failure of other researchers to identify the formation of anticipated metabolites. 40 3.4 Factors affecting the biodegradation of quaternary ammonium compounds A number of factors have been identified which can affect the biodegradability of QACs. Two of the most important are the length of the alkyl chain and the number of long alkyl chains present in the QAC. Based upon the body of research available, it is clear that the resistance of QACs containing linear hydrophobic chains to biodegradation increases in the order RMe 3 N + < RBzMe 2N + < R 2 Me 2 N + < RPy + < R 3 MeN + (Boethling, 1984). Ruiz Cruz and Dobarganes Garcia, (1979) recorded the time taken for 5 ppm of selected QACs to decrease by 50%. While this could be achieved for dodecyltrimethylammonium and decylbenzalkonium chlorides in 1 to 2 days, dialkyldimethylammonium chlorides required 2 to 3 days and trialkylmethylammonium compounds took approximately 7 days. Masuda et al. (1976) measured oxygen uptake after 10 days incubation of 20 ppm of various QACs. While the theoretical oxygen demand approached 100% for decyltrimethylammonium chloride, and decylbenzyldimethylammonium chloride, it was only 50% for didecyldimethylammonium chloride and 4% for decylpyridinium chloride. Van Ginkel and Kolvenbach (1991) have reported the influence of structure and chain length on the biodegradability of selected QACs. They observed that as the alkyl chain length increased from C i 0 to C i 8 the oxidation rates of mono- and dialkylammonium chlorides decreased. However, the changes were less dramatic than those arising from an increase in the number of hydrophobic alkyl chains. This is consistent with the views presented by Boethling, (1984). Ghisalba and Kiienzi (1983) reported that Pseudomonas strains which were tolerant to tetramethylammonium chloride were not able to utilize tetraethylammonium chloride, 41 methyltrimethylethylamninium chloride and dimethyldiethylammonium chloride. Mackrell and Walker (1978) also found that their enrichment culture grown on tetramethylammonium chloride and adapted to trimethylethylammonium chloride was not able to grow on the higher homologue tetraethylammonium bromide. They suggested that this was probably due to the close-packed tetrahedral structure which protects the central nitrogen atom against biodegradation attacks. However, Ghisalba and Kiienzi (1983) observed that ethylamine are not easily utilized by the bacteria and reasoned that the enzymes which demethylate the QAC may be specific for the methyl group. Conversely, triethylamine was reported to be a better carbon source than either the mono- or dimethylamines. A third factor influencing the biodegradability of QACs is the presence of substrates that can adsorb or complex with the QAC thereby limiting the concentration present in the aqueous phase and/or reducing the surfactant properties of the QAC. There have been several studies which clearly demonstrate that the addition of sediment can encourage the biodegradation of QACs at much higher levels than can be biodegraded without the sediment (Krzeminski etai, 1973; and Larsen and Vashon, 1983). In a similar manner, the addition of anionic complexing agents can be used to enhance the biodegradability of QACs (Gerike et al. 1978). A fourth factor to be considered is the presence of tolerant micro-organisms. It is known that there are relatively few bacterial genera that can demethylate alkylamino-compounds, although their distribution is ubiquitous. Their presence can therefore enhance the biodegradability of QACs. 42 A fifth factor that will influence the degree of biodegradation will be the presence of a suitable consortium of micro-organisms that can undertake the degradation of the QAC and the metabolites that will be produced during the degradation process. Thus, van Ginkel et al. (1992) has proposed that at least two bacteria are required to biodegrade hexadecyltrimethylammonium chloride. Also of importance is the phenomenon of acclimation. Gawel and Huddleston (1972) reported that following acclimation, a second aliquot of a QAC was degraded much more rapidly than that present originally. The germicidal properties of the cationic do not necessarily interfere with their ultimate biodegradation provided that operating concentrations are kept below the toxic threshold. Above that threshold, toxicity can be mitigated by neutralization of the cationic with an equivalent amount of anionic of ultimate biodegradation. Finally, the biodegradation rates experience in the environment will also be influenced by factors other than chemical structure. These include such factors as microbial biomass, aeration, temperature, available nutrients, solar radiation and pH (Boethling, 1984). 43 4. MATERIAL AMD METHODS 4.1 Evaluation of a rapid colormetric screening technique for identifying QAC degrading fungi 4.1.1 Chemicals The quaternary ammonium compound used in the project was commercial grade, ® Bardac 2280 (Lonza Inc.) which contained 80 % didecyl (Cio,Cio) dimethyl-ammonium chloride (DDAC) active ingredient, 10% ethyl alcohol and 10% water. All amounts of DDAC are expressed in terms of the active ingredient only. Bromophenol blue used was supplied by Fisher Scientific Company, while the methyl orange indicator was purchased from Anachemia Chemical Ltd. 4.1.2 Sample preparation DDAC solution with a wide range of concentrations (2.04X10"6 M, 2.04xl0"5 M, 4.08xl0'5 M, 2.04X10"4 M, 4.08X10"4 M, 4.08xl0'3 M and 1.02xl0'2 M ) and one standard 2.04X10"4 M solution of bromophenol blue were prepared. For determination of the spectra test solutions containing both DDAC and bromophenol blue or methyl orange were prepared. The solution was adjusted using 1M sodium hydroxide or 1M hydrochloric acid to produce values of 1.80, 3.50, 6.80 and 12.00. All pH measurements were made with an Accumet pH meter 915 (Fisher Scientific). It is generally expected that the stoichiometric relationship of a compound formed from monovalent ions of opposite charge will be 1:1. At high pH values (12.0), the phenolic 44 groups of bromophenol blue are completely dissociated, so that the possibility of two D D A C molecules combining with one mole of anionic dye exists. For experiments under acidic conditions, 10 ml of bromophenol blue were added to 10 ml of each D D A C solution. For the alkaline pH of 12.0, 10 ml of bromophenol blue were added to 20 ml of each D D A C solution. The final solution concentrations tested were: 2.04xl0"7 M , 2.04X10"6 M , 4.08x10"* M , 2.04xl0'5 M , 4.08xl0"5 M , 4.08X10"4 M and 1.02xlO"3M of D D A C and 2.04xl0"5 M bromophenol blue. For the studies using methyl orange the procedure was identical to that employed for bromophenol blue, with the exception that pH values of 1.1 and 11.5 were studied. 4.1.3 Sample analysis All the samples were analyzed by Cary UV-Vis spectrophotometer (200 900 nm) to determine the relative absorbances of the bromophenol blue-DDAC and methyl orange-D D A C compounds. Standard solutions of bromophenol blue and methyl orange were also analyzed to determine the changes due to the compound formation. 45 4.2 Isolation of tolerant fungi In order to isolate fungi which are very tolerant to QACs or tertiary amines, small wooden stakelets treated with several retentions of DDAC and decyldimethylamine (Barlene 10S) were placed in a field test site at the University of British Columbia and recovered at selected time intervals. The treated stakelets were sampled to recover the tolerant fungi after different time intervals. 4.2.1 Wood treatment and exposure Southern yellow pine (Pinus spp) sapwood was cut into small stakelets with approximate dimension ofl2x lOx 102 mm. The small stakelets were vacuum treated with 0%, 0.3% and 0.6% solutions of DDAC or decyldimethylamine (Barlene 10S supplied by Lonza Inc.) and randomly placed in an experimental test site located at the University of British Columbia. Additional samples were installed in the fungal cellar at Forintek Canada Corp. (Western laboratory). Three replicates for each concentration were used. Each stakelet was completely driven into the soil. 4.2.2 Isolation of tolerant fungi from treated wood exposed in the field area After intervals of six weeks and four months exposure in the field, representative stakelets were removed for observation. From those which showed visible fungal colonization representative samples were retained and sampled to isolate the early colonizing fungi. Selective media starch-casein-nitrate-rose-bengal (SCNR), benomyl-tetracycline-malt agar (BTMA) and preservative media (malt agar with DDAC or decyldimethylamine) were used to 46 isolate mould and stain fungi, basidiomycete, and fungi tolerant to either DDAC or decyldimethylamine, respectively. Preparation of media The SCNR media contained: starch 10.0 g/1, casein 0.3 g/1, KN0 3 2.0 g/1, NaCl 2.0 g/1, K 2 HP0 4 2.0 g/1, CaC0 3 0.03 g/1, FeS04 7H 20 0.01 g/1, agar 10.0 g/1, Rose Bengal 0.35 g/1, in one liter of water. The solutions of BTMA media contained: malt extract 20.0 g/1, agar 20.0 g/1, tetracycline 0.1 g/1 in one liter of distilled water. Solutions of media containing DDAC or decyldimethylamine were prepared by dissolving: malt extract 20.0 g/1, agar 20.0 g/1, and 100 ppm of either DDAC or decyldimethylamine in one liter of distilled water. Isolation from stakelets Each stakelet was wiped with cotton or paper tower soaked in 70% ethanol before splitting. Small pieces ( l x l x l . mm) were removed from the freshly exposed wood and placed on the selective media. The plates were incubated at 25 °C and the growth observed daily until no new fungi appeared on the plate (media). Any fungal growth which appeared in the selective media was subcultured onto malt agar containing 100 ppm chloramphenical to prevent bacterial growth. Once pure cultures were obtained through sub-culturing, all the isolates were grown on malt agar containing a threshold concentration of 250 ppm DDAC, to identify those which exhibited tolerance to this preservative. To further limit the fungi to be used in subsequent experiment, they were inoculated (core diameter 6 mm) onto malt agar containing 750 ppm DDAC. After twenty days, only those fungi which grew on the agar with a colony diameter of 5.0 cm were retained for further experiments. 47 4.3 Comparison of the tolerance of isolates from the field experiment with those from culture collections 4.3.1 Tolerance of fungi in solid media Fungal species Strains of fungi obtained from the culture collection of Forintek Canada Corp., Ottawa included four strains of Gliocladium roseum Bain (321A, 321B, 321C and 321D), Gliocladium viride (623B), Gliocladium virens* (25 8C and 25 8D), Verticillium lecanii (390A), Verticillium dahliae (163A). These fungal strains from the culture collections were compared with two strains of Gliocladium roseum (GRSB and GRTC), each of Verticillium bulbillosum and Acremonium sp. which were isolated by Dr. A. Doyle at the University of British Columbia and identified by the National Identification Service, Agriculture Canada. Five additional strains of fungi which were recovered from field stakelets treated with D D A C or decyldimethylamine were also studied. These isolates were coded as follows: S-l-1 and S-1-6 which were isolated in July, 1994; S-2-1 and S-2-11 which were isolated in October, 1994; and F-l-1 which was isolated from fungal cellar at Forintek Canada Corp. These fungi were identified by the National Identification Service, Agriculture Canada as Fusarium avenaceum (FT.) Sacc. (S-l-1, S-2-1 S-2-11), Zygorrhynchus moelleri Vuill (S-l-6) and Trichoderma virens (Miller, Giddens & Foster) V. Arx (F-l-1). Since the tolerant fungal strains studied by Dr. A. Doyle were isolated largely from wood exposed out of ground contact (except for GRSB), the tolerances of the fungi isolated from the field exposure * The two strains 258C and D were listed by Forintek Canada Corp. as Gliocladium virens. This fungus is now taxonomically more correcdy identified as Trichoderma virens. However, the name G. virens will be retained for use in this thesis. 48 (described above) were also compared to that of the standard strains of G. roseum, G. viride, G. virens, V. lecanii, V. dahliae. Preparations of different concentrations of DDAC Aliquots of DDAC (Bardac 2280, Lonza Corp. which contains 80 % a.i.), were weighed and added to 2% malt agar solution to obtain the required concentrations of DDAC. Each solution was autoclaved and poured into petri plates (approx. 20 ml per plate) and allowed to solidify on sterile conditions. The nominal concentrations of DDAC were 0 ppm (control), 100 ppm, 250 ppm, 500 ppm, 750 ppm and 1000 ppm. Inoculation and incubation of fungal cultures Cores, 6 mm in diameter, were removed from the edge of colony on which the different strains had been grown for 10 days and inoculated onto the center of the test plates prepared as above. All fungi were incubated at 25 °C. Three replicates for all concentrations of DDAC were prepared with each test fungus. The growth rate was measured in two perpendicular directions from the center of the colony over 21 day period. 4.3.2 Tolerance of fungi in liquid media Fungal species Based on the tolerance of fungi on solid media, the following fungal strains were selected for a more detailed study in a liquid media test: a) four strains of G. roseum (321 A, 321B, 321C and 321D), G viride (623B), G virens (258D and 258C), Verticillium lecanii (390A), all from the Forintek culture collection; b) from the previous investigation by D. Doyle (UBC) two strains of G roseum (GRSB and GRTC); V. bulbillosum and 49 Acremoniwn sp.; c) four isolates made from stakelets exposed at the U B C test site: Fusariumsp. (S-l-1, S-2-11), Zygorrhynchus sp.(S-\-6), and Trichoderma virens (F-l-1) Preparation of the liquid media According to a previous study (Doyle, 1995), it was observed that none of three fungi studied (G. roseum, V. bulbillosum and Acremonium sp) were able to grow in liquid media containing 100 ppm DDAC. In a preliminary experiment, three kinds of liquid media containing 0 ppm, 50 ppm, and 100 ppm D D A C were prepared. They were: 2% malt extract, Czapek and Vogel liquid media. The Czapek liquid media contained 30g sucrose, 2g sodium nitrate, lg potassium phosphate, 0.5 g magnesium sulfate, 0.5 potassium chloride, 0.01 g iron sulphate, 0.01 g zinc sulphate and 0.005 g copper sulphate in one liter of distilled water. The Vogel liquid medium contained: CaCl 2 0.4g, K H 2 P 0 4 1.0 g, Na 2 HP0 4 0.8 g, M g S 0 4 • 7H 2 0 0.5 g, NHjNOs 0.8 g, potassium hydrogen phthalate 3.0 g, 10 ml of 100 times concentrated micronutrient solution (Vogel 1956), 1 ml of filter-sterilized vitamin solution (Tan and Breuil 1986), 20 g of soluble starch (BDH) dissolved in one liter of distilled water. The pH of liquid media was adjusted to 5.8 ± 0.2 using 0.1M sodium hydroxide. Aliquots (50 ml) of liquid media were measured into 250 ml Erlenmeyer flasks, sterilized by autoclaving for 15 minutes at 103kPa and 12PC. In the final experiment, Vogel liquid media was used. Aliquots of D D A C concentrate were weighed and added into the liquid media to obtain the required chemical concentrations. The Vogel liquid media containing 0 ppm and 250 ppm DDAC, which was the threshold concentration on solid media, were prepared. 50 Inoculation and incubation of fungi Each flask was inoculated with a 6 diameter core of the pre-grown fungus grown on 2% malt agar plate. All the cores were from the edge of the colony. All the liquid culture were incubated for two weeks at 25 °C on a rotary shaker set at 200 rpm. Duplicate flasks were used for all fungi at all D D A C and decyldimethylamine concentrations. Fungal biomass The fungal biomass was measured after two weeks' incubation. In order to speed up the filtration, the liquid media after fungal growth was centrifuged at 3500 rpm for 20 minutes at 10 °C. The supernatant and the fungal mycelium was filtered under vacuum on and washed on preweighed glass fiber filters with hot water. Filters containing mycelia were oven dried at 103 °C to a constant weight, cooled in desiccator to room temperature, and weighed. 4.3.3 The growth curve of V. bulbillosum Vogel liquid medium (prepared as described in Section 4.3.2) was added (50 ml) to 250 ml Elementary flasks, which were then was inoculated with a core (6 mm) removed from the growing edge of Verticillium sp. pre-grown on 2% malt agar. All the cores were removed from the similar distance away from the center of the colony. The growth rate was determined by measuring the biomass at selected 2 day time intervals for the first two weeks. Duplicate flasks were sampled at every time interval.. 51 4.4 Assessment of fungal degradation of DDAC 4.4.1 Solid media Fungal species The fungal species were the same as that in section 4.3.1. Preparation of media The preparation of media was the same as that in section 4.3.1 but only one concentration of 750 ppm DDAC was employed. Inoculation and incubation of fungal culture The inoculation and incubation of fungal culture were the same as that in section 4.3.1. Analysis of DDAC After the fungal colony had covered approximately half of the plate (in area), the mycelium was carefully removed from the surface and oven-dried prior to weighing. Malt agar cores (0.2-0.5 g) were collected from beneath the center and edge of the hyphal growth of the fungal colony. Additional cores were removed from the perimeter of the plate which had not been colonized by the fungus. These additional cores were taken to provide a reference DDAC concentration. The cores at each location on a plate were collected together and weighed, in order to provide the minimum amount of agar needed to allow DDAC to be detected. Duplicate cores were removed from each position within each plate containing DDAC amended medium. In some cases duplicate plates were also sampled. Following weighing, the cores were placed in a tube to which was added either 1 or 2 ml of ethanol (pH = 5), acidified with formic acid. The amount of ethanol was adjusted for the mycelium mass to ensure good recovery of the DDAC. The solution containing the cores was sonicated in an 52 ultrasonic bath at 40 Hz for three hours. The solutions were centrifuged at 2,800 rpm at 9 °C for 25 minutes, after which the samples were filtered with 0.45 u.m PTFE microfilter, and analyzed by HPLC. The HPLC spectrometer was fitted with an indirect photometric detector (Larson and Pfeiffer, 1983). The system consisted of a Spectra Physics 1200 series HPLC pump equipped with a 8 mm x 10 cm Partisil SCX column (Waters 85753) containing 10 urn particles and a Spectra Physics variable wavelength U V detector set at 262 nanometers. A solution containing 0.025 M of benzyltrimethylammonium chloride, 1% acetic acid and watenmethanol mixture with a ratio of 5:1 was used as a mobile phase at a 2.2 ml/min flow rate. This method was also used for determining the amount of D D A C in mycelia. D D A C concentrate was used to dissolve in ethanol (pH - 5) which was acidified with formic acid for D D A C standard. All D D A C peak was measured by peak height. 4.4.2 Liquid media Fungal species The strains of fungi used in this study were selected from those that had exhibited good tolerance in D D A C amended liquid culture, based upon their production of biomass. The fungi selected were: V. bulbillosum, Acremonium sp. G. roseum (TC-NP-16 and 321C), Fusarium (S-l-1), and V. dahliae(390A). Preparation of media The Vogel liquid media was prepared as described earlier (Section 4.3.2). The inoculation and incubation of the liquid cultures were described in section of 4.3.2. Analysis of DDAC 53 Because there is no standard method available for recovering DDAC from the aqueous solution, it was necessary to develop one. Several different procedures were evaluated Recovery of DDAC from liquid media a. A known amount of DDAC (0.0098 g and 0.0092 g) was added to 50 ml of standard Vogel liquid media after which the solution was transferred to a separatory funnel. A 25 ml aliquot of methylene chloride was added to the separating funnel which was shaken for 5 minutes, after which the solution was allowed to stand for 20 minutes. The methylene chloride layer (lower) was collected. The procedure was repeated three times with 25 ml of methylene chloride, after which the four extracts were combined together, roto-evaporated to dryness, and redissolved in acidified ethanol (pH = 5) prior to analysis by HPLC. b. Vogel liquid media (50 ml) containing a 0.00382g and 0.00592g of DDAC were rotor-evaporated to dryness. Methylene chloride (25 ml) was added to the flask to dissolve DDAC. The flask was shaken and the methylene chloride transferred to another flask. This methylene chloride extraction was repeated three times and all four extracts were combined, after which they were roto-evaporated to dryness. The remaining solid was redissolved in acidified ethanol prior to analysis by HPLC. Since phosphate buffer was used during some experiment, the recovery of DDAC from pH 6.3 phosphate buffer was assessed using the same procedure. This experiment also produced some indication of the influence of inorganic salts on DDAC recovery. c. The experiment described above (Section b) was repeated using acidified methylene chloride (250 ml of methylene chloride was acidified with 1 ml of formic acid). In addition, 54 0.01048g and 0.00984g of D D A C were incorporated in the Vogel liquid medium containing the pH 6.3 phosphate buffer. d. Solutions of Vogel liquid media or pH 6.3 phosphate buffer alone containing a known amount of D D A C (0.0288g and 0.036g; 0.0294g and 0.0192g) were roto-evaporated to dryness. Acidified methylene chloride (250 ml of methylene chloride was acidified with 1 ml of formic acid) (25ml) was added to the flask which was then placed in an ultrasonic bath for 3 hours. The resulting solution was transferred to second flask. The remaining solid was then washed twice with 25 ml of acidified methylene chloride to recover any additional the DDAC. The acidified methylene chloride extracts were combined together and roto-evaporated to dryness. The resulting solid was redissolved in acidified ethanol and examined by HPLC. e. Flasks containing standard Vogel liquid media were used to incubate Verticillium sp. After incubation for two weeks, a known amount of D D A C (0.0024g, 0.0023g and 0.0028g) was added to the resulting media which was then roto-evaporated to dryness. Acidified methylene chloride (250 ml of methylene chloride was acidified with 1 ml of formic acid) (25ml) was added to the solid and the flasks were placed in an ultrasonic bath for 3 hours. The resulting solution was transferred to another flask. The remaining solid was twice washed with 25 ml of acidified methylene chloride to recover any remaining DDAC. The three acidified methylene chloride extracts were combine together and roto-evaporated to dryness. The resulting solid was redissolved in acidified ethanol and examined by HPLC. Based upon the biomass of V. bulbillosum, and the evaluation of the D D A C recovered from the different extraction regimes, the following procedure was adopted for the analysis of the residual D D A C content. The fungi were grown in liquid media (50 ml) for two 55 weeks, after which the culture was rotoevaporated to dryness. The dry sample was put in a desiccator overnight. Acidified methylene chloride (25ml) was added to the solid and the flasks were sonicated in an ultrasonic bath at 40 Hz for three hours. The resulting solution was transferred to a second flask. The remaining solid was washed three times to recover any remaining D D A C on the mycelium. All of the acidified methylene chloride extracts were combined and roto-evaporated to dryness. The resulting solid was redissolved in 5 ml of acidified ethanol (pH = 5), and filtered with a PTFE 0.45 um microfilter and analyzed by HPLC. 56 4.S. Extraction and identification of metabolites in liquid media, following fungal growth in liquid media 4.5.1 Methylene chloride Methylene chloride was chosen as the solvent for extraction. Since D D A C degrades when subjected to a GC-MS analysis, it was removed prior to analysis by passing the solution down a cationic ion exchange resin. V. bulbillosum., G. roseum (TC-NP-16) and Acremonium sp., were grown in Vogel liquid media containing 0 ppm and 250 ppm D D A C (prepared as described in Section 4.4.2). In further experiments, V. bulbillosum, G. roseum (TC-NP-16) and Acremonium were grown for two weeks in 50 ml of liquid Vogel media containing 0 ppm and 250 ppm D D A C at 25 °C, on a rotary shake with a shaking speed of 200 rpm. The cultures and Vogel liquid media containing 250 ppm D D A C without growth of fungi, were extracted three times with 25 ml of methylene chloride. The extracts were combined together, roto-evaporated to dryness and re-dissolved in 5 ml of 100% anhydrous ethyl alcohol. The extract solution was added to a cationic ion-exchange resin (Bio-Rad A G 50 W x 4) which was then washed with 50 ml of 100% anhydrous ethyl alcohol. The wash solution was collected, evaporated to dryness using a roto-evaporator at 40 °C and re-dissolved in 5 ml of methylene chloride, prior to examination by (GC-MS). 4.5.2 Complex formation Based upon the early data from the GC-MS results, the presence of the tertiary amine decyldimethylamine was not confirmed. Subsequently, it was found that the tertiary amine could not be washed off the cationic ion exchange resin under the experimental conditions used. Therefore the procedure had to be modified. The modified procedure was designed to 57 remove the DDAC, but not the tertiary amine. It was based upon the known ability of cationic surfactants to form compounds with anionic surfactants. The resulting compound is insoluble in both water and methylene chloride. Verticillium sp. and Acremonium sp. were grown at 25 °C for two weeks in 50 ml of Vogel liquid media containing 0 ppm and 250 ppm DDAC with a shaking speed of 200 rpm. The cultures and liquid media containing 250 ppm DDAC without fungal growth were extracted three times with 25 ml of methylene chloride. The extracts were combined, roto-evaporated to dryness and redissolved in 10 ml of distilled water. Aliquots of 3.46 x 10"3 M anionic surfactant (sodium dodecyl sulphate) were added to the solution until it became turbid. The turbid solution was extracted three times with 5 ml of methylene chloride. The clear extract (lower layer) were combined together, roto-evaporated to dryness, and redissolved in 10 ml of distilled water. The procedure was repeated until addition of sodium dodecyl sulphate did not cause any turbidity at that point, the last solution was rotoevaporated to dryness and redissolved in 5 ml of methylene chloride for analysis. 4.5.3 Identification of metabolites by GC-MS The methylene chloride solutions prepared as above were examined by GC-MS to identify the metabolites. The GC-MS was equipped with a V G Trio 1000 mass selective detector. The samples were analyzed on a 25 m long HP-5 capillary column (0.2mm ID, 0.32 um film). The injector system was maintained at 250 °C, and the oven temperature was held at 50 °C for 2 minutes and then programmed to increase to 300 °C at a rate of 13 °C/min. The separated components were identified where possible by comparing their mass spectrum with those stored in the library. Samples of authenticated compounds of interest such as 58 decyldimethylamine were analyzed when the relevant compound spectrum was unavailable the library. 59 4.6 Attempts to fractionate the proteins that degrade DDAC 4.6.1 Comparison of extracellular and intracellular protein in selected fungi exposed to DDAC and without DDAC, using electrophoresis In the initial experiment, Verticillium sp. was grown at 25 °C in Vogel liquid media containing with 500 ppm D D A C and 0 ppm DDAC. The flasks were shaken at 200 rpm during incubation. After 15 days, the cultures were centrifuged at 3,900 rpm at 9 °C for 40 minutes to separate the supernatant from the mycelium. The mycelium was washed with 10 ml of 20 mM tris-HCl buffer at pH 8.0. The supernatant, which contained the extracellular protein, was concentrated by ultrafiltration (10,000 kDa) until the protein concentration was approximately 1 mg/ml. The hyphal cell wall was disrupted by grinding with a cell homogenizer to which was added sand, in order to recover the intracellular protein. The intracellular protein then was concentrated by ultrafiltration (10,000 kDa) until the protein concentration was approximately 1 mg/ml. Protein concentrations were measured by the Bradford microassay (Bio-rad), using bovine serum albumin (BSA) as the standard (Bradford, 1976). Gel electrophoresis was used to analyze the extracellular and intracellular proteins. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing electrophoresis (JJEF pH 3-9). SDS-PAGE and IEF gel electrophoresis were performed on the phastsystem (Pharmacia, Uppsala, Sweden). Separation of protein by SDS-PAGE was performed at a constant current of 10.0 mA for a total 70 V.h. Separation by IEF was performed at a constant current of 2.0 mA for a total of 431 V.h. The pi and molecular weight (MW) markers were provided in the Pharmacia kits for IEF and SDS-PAGE respectively. 60 Aliquots containing 0.25 u,g/ul and 0.5 u.g/p.1 of extracellular and intracellular protein were prepared, after which 4 ul of each sample was loaded onto the SDS-PAGE gel (gradient 8-25%). A silver staining method was used to show the location of the protein bands. In a final experiment, V. bulbillosum and Acremonium sp. were grown in liquid cultures containing 250 ppm and 0 ppm DDAC. After two weeks' incubation growth, the supernatant was collected and concentrated by ultrafiltration until the protein concentration was about 2 mg/ml. Duplicate samples were prepared. The protein was diluted with 20 mM tris-HCl buffer, pH 8.0 to 0.5 ug/ul. Aliquots of 4 ul of extracellular protein solution were loaded onto SDS-PAGE (homogenous 12.5%) and IEF (pH 3-9) gels. The SDS-PAGE and IEF gel were run in duplicate and were silver stained for proteins according to the manufacturer's recommendations (Pharmacia) to show the locations of the protein bands. 4.6.2 Degradation of DDAC by supernatants from the cultures of V. bulbilosum amtaining 0 ppm and 250 ppm DDAC The same source of supernatants and mycelia of V. bulbillosum as above without concentration were used in this experiment. The protein concentration was also measured. A 0.02355 g sample of 80 % D D A C was dissolved in 100 ml of pH 6.3 phosphate buffer. Exactly 15 ml of this D D A C solution was added to 30 ml of supernatant in which the protein concentration was 0.075 mg/ml. A 0.01286 g sample of 80 % D D A C was dissolved in 100 ml of pH 6.3 phosphate buffer. A 1 ml aliquot of this D D A C solution, was combined with 5 ml of pH 6.3 buffer and 0.5 ml of intracellular protein in which the protein concentration was 0.0017 mg/ml. 61 All of the flasks and tubes were incubated at 27 °C for 7 days with shaking at 200 rpm. After incubation, the samples were roto-evaporated to dryness at 45 °C. A 25 ml or 5 ml of acidified methylene chloride was added to each sample which was sonicated in an ultrasonic bath for three hours. The resulting solids were extracted three times with 25 ml or 5 ml of acidified methylene chloride. The methylene chloride extracts were combined and transferred to another flask for removal of the solvent. Acidified ethanol was added to the extracts and the solution analyzed by HPLC to determine the D D A C content. 62 4.7 The distribution of D D A C in starch, silica gel, and solution A known weight (41.2 mg) of D D A C was added into the Vogel liquid media (50 ml) without starch but with 0.8 g of silica gel. Replicate samples were used. Aliquots of 38 mg or 35 mg of D D A C were added into the flasks containing cultures of V. bulbillosum grown in Vogel liquid media for two weeks. All of the samples were roto-evaporated to dryness. A 25 ml solution of acidified methylene chloride (250 ml of methylene chloride, acidified with 1 ml of formic acid) was added to the solid and the flasks were placed in an ultrasonic bath for 3 hours. The resulting solution was transferred to another flask. The remaining solid was twice washed with 25 ml of acidified methylene chloride to recover any remaining DDAC. The three acidified methylene chloride extracts were combine together and roto-evaporated to dryness. The resulting solid was redissolved in acidified ethanol and examined by HPLC. Pre-determined amounts of D D A C (Og, 0.0163g, 0.0255g, and 0.0248g) were weighed and added to a 50 ml centrifuge tube which contained either i) 25 ml of Vogel liquid media (without starch) and 0.4 g of silica gel; ii) 25 ml of Vogel liquid media and 0.4 g of silica gel; or iii) 30 ml of standard Vogel liquid media. The samples containing starch were autoclaved at 103 kPa and 121 °C for 15 minutes. All the samples were prepared, shaken and allowed to stand overnight. The next day, the samples were centrifuged at 3,900 rpm for 40 minutes. The clear solution and the solid were both collected. The D D A C was recovered from the clear solution by the procedure described above. The D D A C was extracted from the silica gel, and any undissolved starch (where this was present) by adding 8 ml of acidified ethanol and sonicating in an ultrasonic bath at 40 Hz for 2 hours. 63 In the final experiment 0.8 g of silica gel was added into 50 ml of Vogel liquid media containing 0 ppm and 250 ppm DDAC. Fungi V. bulbillosum and Acremonium sp. were used. The preparation of Vogel liquid media, inoculation, and incubation were described earlier (section 4.3.2). Duplicate samples were prepared. After two weeks, the cultures were centrifuged at 3,900 rpm for 40 minutes. The D D A C in the clear solution was recovered by the procedure described in the Section 3.4.2 (subsection 'e') using methylene chloride. The D D A C in the solid was extracted by adding 5 ml of acidified ethanol and sonicating in an ultrasonic bath at 40 Hz for 2 hours. All the samples were filtered with a PTFE microfilter (0.45 urn) and analyzed by HPLC. 64 4.8 Fungal growth in liquid media containing decyldimethylamine (Barlene 10S) 4.8.1 Fungal species The following fungi were used in the first experiment : V. bubillosum, Coniophora puteana, and Trametes versicolor. Those in the second experiment were: V. bubillosum, Acremonium sp., Fusarium sp. (S-l-1) and G roseum (TC-NP-16). 4.8.2 Growth conditions Vogel liquid media was prepared and autoclaved as described above (Section 4.3.2). After the media were cooled to room temperature, decyldimethylamine was added to 50 ml of media in 250 ml Erlenmeyer flasks. The concentrations of the tertiary amine were: 1500 ppm and 800 ppm. To determine the ability of the fungus to use the tertiary amine as a sole N-source or sole C-source, the nitrogen or carbon sources were omitted from the medium during preparation of those specific samples. The various combinations of liquid media were inoculated with Verticillium sp. and incubated with shaking at low speed (140 rpm), at room temperature, for 12 days. Vogel liquid media containing 1000 ppm decyldimethylamine was prepared and inoculated with V. bulbillosum, C. puteana, T. versicolor and incubated at room temperature for two weeks. Vogel liquid media containing 100 ppm decyldimethylamine were prepared and inoculated with V. bubillosum, Acremonium sp. Fusarium sp. G. roseum (TC-NP-16) and incubated at room temperature for two weeks with shaking at 140 rpm. 4.8.3 Fungal biomass The measurement of fungal biomass was as described in section 4.3.2. 65 5. RESULTS AND DISCUSSION S.l Use of colorometric approach to monitor changes in DDAC concentration during fungal growth It was anticipated that a large number of fungi may have to be screened for their tolerance to DDAC. In order to screen these fungi quickly and identify those that can degrade QACs, it would be helpful to develop a rapid technique that did not require the chemical analysis of QAC each time. Cationic surfactants such as DDAC, readily form stable colored complexes with anionic dyes such as bromophenol blue and methyl orange (Ballard et. al., 1954; Zografi et. al., 1964; Few and Ottewill, 1956; Waters, 1976). A screening trial was designed to investigate whether the colored complex formed was changed when the QAC was degraded. If the results were positive, this would confirm that the technique could be more generally used to identify those fungi which degrade QACs. 5.1.1 Bromophenol blue At an acidic pH of 1.80 when the molar concentrations of D D A C were lower than the molar concentrations of bromophenol blue, the spectra of bromophenol-DDAC were the same as that of standard bromophenol blue. Increasing the molar concentration of D D A C so that it was equal to or higher than, that of bromophenol blue, caused an absorption peak observed around 610 nm due to the bromophenol blue-DDAC complex formed (Figure 5.1.1.1). However, no linear relationship between the intensity of the absorption and the concentrations of D D A C could be determined. At higher pH values of 3.50 and 6.80, there was no difference between the spectra of bromophenol blue-DDAC complex and standard 66 L9 o „ 4> O " I g s: a. TO g-2 o i § 1 : ° ° £ 21 © O " f i " O •— o bo 3 o *o 5* 3> a. cr cr o S cr cP cs cs cs K) CS CS CS ' CO ' 4*. CS cs cs CO cs cs cs KJ cs cs cs cs 4i. cs cs. CD CS CD CS csH cs cs ca cs cs-cs cs V. + J l { bromophenol blue, no matter whether the molar concentration of D D A C was equal to, higher than, or lower than, that of standard bromophenol blue. Increasing the solution pH to 12.00, failed to produce any difference between the UV-Visible spectra of the D D A C and the complex, when the molar concentration of D D A C was lower than that of bromophenol blue. If the molar concentration of D D A C was equal to, or higher than, that of bromophenol blue, the spectra were different (Figure 5.1.1.2), but there was no specific peak which could be assigned to the bromophenol blue-DDAC complex. 5.1.2 Methyl orange indicator At the acidic pH of 1.1, there was no difference between the spectra of the methyl orange-DDAC complex and the spectra of standard methyl orange, when the D D A C molar concentrations were equal to, lower than, or higher than, that of methyl orange. Increasing the solution pH to 11.5, produced spectra of the methyl orange-DDAC complex and standard methyl orange that showed no difference, when molar concentration of D D A C was lower than that of standard methyl orange. However, when the D D A C molar concentration was higher than, or equal to, that of methyl orange, the spectrum was changed. However, it was impossible to establish a relationship between the absorption intensity of the peaks in the spectra of the complex and the D D A C concentration. The D D A C concentrations were lower than, or similar to the critical micelle concentration (cmc). Since fungal growth in liquid media containing D D A C will produce more complex solutions, the use of UV-Visible absorption spectroscopy of complexed D D A C to monitor its degradation does not appear fruitful. Potential difficulties may be identified as a) the range of D D A C concentration 68 69 O co 4^ O S CO & o 8* ^ g 3 I cu cr 3 3 o T3 c r § cr o I-3 o o 3. C u . . ^ | S i 0 . , S g ?o Cu ffi O i—i ° 9 cu cr cr o o c r a CD CD CD N C S CD CS CD E*0 CD CD CO CO. CD CD CO £S3 CD" CD CD N CO CS CD CD 4*. CD CD CD cn co CD CD CD CD CD S i J L__J * It which may be studied by this method (i.e. below the cmc point); b) possible interference of fungal metabolites reacting with the dye; and c) lack of relationship between concentration and peak intensity. 70 5.2 Isolation and identification of tolerant fungal strains from field area The survival and colonization of organisms in wood is dependent on (a) the moisture content; (b) aeration; (c) temperature; and (d) interactions between micro-organisms. All these factors and the constantly changing nutrient status of the wood will control the microbial successions in wood (Eaton and Hale, 1993). The general succession of fungal colonization during the decay of wood in soil contact is first mould and staining fungi during stage 1, soft rot and brown rot fungi during stage 2, and white rot fungi during stage 3. Table 5.2.1 shows all fungi which were isolated in July, 1995 from pine sapwood stakelets installed at the U B C test site. A second set of stakelets were removed in October, 1995 and Table 5.2.2 describes all fungi which were isolated from the field area at that time. Table 5.2.3 shows all fungi which were isolated in October, 1995 from stakelets placed in fungal cellar. Five strains of fungi chosen for further experiments based on their growth rate on 2% solid malt agar containing 750 ppm DDAC, were identified as: Trichoderma virens (Miller, Giddens and Foster) V. Arx (F-l-1). Zygorrhynchus moelleri Vuill (S-l-6), and Fusarium avenaceum (Fx) Sacc (S-l-1, S-2-11, S-2-2). During the first four to six months, the progressive deterioration of wood will be in stage 1. Therefore it is anticipated that primarily mould and staining fungi will be isolated. This was confirmed by the low frequency of isolation of decay fungi. 71 Table 5.2.1 Fungi isolated in July from treated and untreated staklets placed in field test Treated wood Selective media Reference No. Pure culture Tolerance Code No. 250 ppm 750 ppm 0.3% DDAC* SCNR* 1 + + -2 + -3a + + -3b + + -4 -BTMA* 5 -6 -7 -8 -Preservative media (DDAC) 9 + + -10 -11 -12 -0.6% DDAC SCNR 5 + + -6 -7 + + -8a + -8b + + -BTMA 9 -10 -11 + -12 -Preservative media (DDAC) r i 1 -2 -3 + + + S-l-1 4 -0.3% DD A* SCNR 5 + + -6 + + -7 - -8 + -BTMA 9 -10 -11 + + -12 -Preservative media (DDAC) 1 -2 -3a + -3b + + -4 -*DDAC, DDA, BTMA and SCNR see LIST of ABBREVIATIONS on page xv. 72 Table 5.2.1 (contd) Fungi isolated in July from treated and untreated staklets placed in field test 0.6% DDA SCNR 1 + + -2 -3 + + -4 + + -BTMA 9 -10 + + + S-l-6 11 -12 -Preservative media (DDAC) 5 + + -6 -7 + -8 + -Control SCNR 1 -2 -3 -4 -BTMA 9 + + -10 -11 -12 -Preservative media (DDAC) 5 -6 7 -8 -*DDAC, DDA, BTMA and SCNR see LIST of ABBREVIATIONS on page xv. 73 Table 5.2.2 Fungi isolated in October from treated and untreated stakelets placed in field test site Treated wood Selective media Reference No. Pure culture Tolerance Code No. 250 ppm 750 ppm 0.3% DDAC SCNR t + - -2 + + + S-2-1 3a + + -3b +• + -4 + -BTMA 13 -14 -15 -16 Preservative media (DDAC) 9 10 -11 + + -12 + + -Preservative media (DDA) 5 - -6 + + -7a + -7b + + -8 - -0.6% DDAC SCNR 1 + -2 + + + S-2-11 3 + -4 + -BTMA 5 -6 -7 -8 -Preservative media (DDAC) 9 + + -10 -11 - + -12 + -Preservative media (DDA) 13 + -14 + -15 + -16 + + -0.3% DDA SCNR 9 + -10 + -11 - -12 + -BTMA 13 -14 -15 + " + -16 -Preservative media (DDAC) 5 + 6 + -7 + -8 + -Preservative media (DDA) 1 + + -2 + -3 + + -4 + -•DDAC, DDA, BTMA and SCNR see LIST of ABBREVIATIONS on page xv. 74 Table 5.2.2 (Contd) Fungi isolated in October from treated and untreated stakelets placed in field test site Treated wood Selective media Reference No. Pure culture Tolerance Code No. 250 ppm 750 ppm 0.6% DDA SCNR 1 + -2 + -3 + -4 + -BTMA 5 -6 + + -7 -8 -Preservative media (DDAC) 13 + -14 + -15 + -16 + -Preservative media (DDA) 9 + -10 + -11 + -12 + -Control SCNR 1 + + -1 + -2 + -3 + -4 + -BTMA 5 - -6 -7 -8 -Preservative media (DDAC) 9 + 10 -11 + -12 + -•DDAC, DDA, BTMA and SCNR see LIST of ABBREVIATIONS on page xv. 75 Table 5.2.3 Fungi isolated in October from treated and untreated stakelets placed in fungal cellar Treated wood Selective media Reference No. Pure culture Tolerance Code No. 250 ppm 750 ppm 0.3% DDAC* SCNR* 1 + + -2 + + -3 + + + F-l-1 4a + + -4b + + -BTMA* 5 + + -6 + + -7 -8 -Preservative media (DDAC) 9 + + -10 -11 -12 -0.6% DDAC SCNR 5 + + -6 + -7 + + -8 + + -BTMA 9 + -10 + -11 + + -12 -Preservative media (DDAC) 1 + -2 -3 + - -4 -0.3% DDA* SCNR 5 -6 + -6 + + -7 + + -8a + -8b + -BTMA 9 10 -11 + + -12 -Preservative media (DDAC) 1 -2 -3 + -4 -*DDAC, DDA, BTMA and SCNR see LIST of ABBREVIATIONS on page xv. 76 Table 5.2.3 (contd) Fungi isolated in October from treated and untreated stakelets placed in fungal cellar 0.6% DDA SCNR 1 + -2 + + -3 + + -4 + + -BTMA 9 -10 + -11 -12 -Preservative media (DDAC) 5 -6 -7 + -8 + -Control SCNR 1 + + -2 + -3 + -4 + -BTMA 9 + + -10 -11 -12 -Preservative media (DDAC) 5 + -6 -7 -8 -•DDAC, DDA, BTMA and SCNR see LIST of ABBREVIATIONS on page xv. 77 5.3 Comparison of the tolerance of the isolates from field experiment with those from culture collections It is also now well established that different fungal and bacterial species have significant variation in the chemical composition of the cell wall, which no doubt influences their ability to degrade QACs (Lawrence, 1970). The tolerance of these organisms to QACs has been postulated to be due to the presence of a lipid or lipid-protein material on the cell surface, which resists the disruptive surfactant forces of the disinfectant. Salton (1951) noted that bacteria treated with a cationic surfactant or other surface-active agents, could release nitrogen and phosphate compounds into the surrounding medium as a result of leakage of cellular constituents. The effect of D D A C on fungi is almost certainly similar. When considering the action of QACs, two phenomena must be considered. The first involves chemical interaction of the QAC with the fungal hyphae either by i) adsorption of the QAC from the surrounding medium by the cell wall or ii) the penetration of the QAC through the membrane into the cell cytoplasm. The second phenomenon involves the reaction of the QAC with the cell protein. The QAC can disrupt bacterial or fungal activity in many ways, ranging from reducing the surface tension at the cell membrane causing leakage of the cell contents, to replacement of protons by ion exchange. Since the complex metabolic processes are finely balanced, even small changes can cause significant effects in the physiology of the organism, jeopardizing its viability. The tolerance and degrading ability of fungi to chemicals can be an inherent characteristic or may result from the development of resistant strain through adaptive behavior. The objective of these experiments was to examine whether the tolerance and D D A C degrading ability of the isolated fungi were common properties for 78 these fungi or whether they were particularly tolerant strains. This was done by comparing their tolerance to D D A C with that of strains obtained from culture collections. 5.3.1 Solid media The growth rate of fungi on malt agar and malt agar containing D D A C was measured by two perpendicular directions through the center of the colony. This linear measurement is the simplest method of assessing fungal growth arid is considered to be valuable as a rough estimate of growth. It is non-destructive and allows repeated observations of the same fungus. It fails to account for differences in aerial mycelium or mycelium submerged in the agar (Moore-Landecher, 1990), and does not distinguish between variations in hyphal thickness. Thus it may not relate well to mycelium spread over the medium and the total biomass production. Another potential problem is the production of non-uniform colonies. However, in the current strategy, the measurement of the diameter of the mycelial growth allowed a rapid comparison of the relative tolerance of strains of the same fungus. All strains of fungi including isolates from treated and untreated stakelets exposed in the test site, culture collections and those stored at U B C following isolation by A. Doyle grew well on malt agar without D D A C (Figures 5.3.1.1 to 5.3.1.3). Figures 5.3.1.4 to 5.3.1.12 show the relationship between the growth rate of selected fungi on malt agar containing D D A C and incubation time. These results confirmed that, except for V. dahliae (163 A) on malt agar with 1000 ppm of DDAC, this chemical did not inhibit the growth of the moulds over the concentration range studied. Increasing the D D A C concentration caused the fungal growth rate to decrease. 79 80 o CM LO to CTj Q •A O CO CD CM (UJO) J819LUBIP AUO|OQ E a o E <D i _ O < CO v J CD 0 0 w CM (D CO CO < o 8 c > o Q co CO if) CO CM w c £ d fjQ E co • • > O 81 82 CD CM CO O CO CD CM o T— (lUO) J9)9LUBip AUO|OQ 83 o CM LO A o to CO Q LO I U -I CM i C O C O I CM C O + CD ; I I C O r 3 Q Q Oh o o • S 1 O o +-< 13 6 o I CN i 00 I C/0 £ CN S 9 IT) CO bi V 9 CO i CO CO CM (UJO) J9;8LUB!P AU0|0Q 84 E (UJO) J919LUBIP AU0|0Q 85 86 o CM LO A o CO Q LO I i GO <! PH P I Oh Oh O 8 •Si I «1 bo k; •B cu I 8 3 +-» i—i a i O CN « a 0 13 V S T 1 0 w VI o • .2 ° ° . tso 3 ro •n" ^ § • t—t (0 bO i co CD CM (OIO) J9)9UJBip AU0|0Q 87 3 o (UJO) J9;9LUB!P AUOjOQ 88 89 90 16 Colony diameter (cm) During the first fifteen day of incubation, there was a linear relationship between the diameter (cm) of the mycelial mat and the incubation period (days). The slope of this plot can represent the initial growth of these fungi. The intercept can be used to show whether or not the fungal growth is subject to a lag phase. From the data which are summarized in Tables 5.3.1.1 and 5.3.1.2, it was clear that there was no relationship between the fungal growth rate for any particular fungus on malt agar alone, and the growth rate on malt agar containing DDAC. For example, of all G. roseum strains, the highest growth rate on malt agar was exhibited by strain 321C. However, this strain did not have the highest growth rate in malt agar containing DDAC. G. viride (623B) had the highest growth rate of all strains of fungi tested on malt agar alone, but it did not grow fastest on malt agar containing DDAC. Comparing the growth rate of various strains of G. roseum, it was interesting to note that strain 321A grew rapidly at 250 ppm of DDAC, but as the concentration increased it was clear that strain 32 IB was more tolerant. From the data of intercepts, it seems that none of the fungi have a lag phase during growth on malt agar or malt agar containing DDAC. With increasing concentrations of DDAC, the appearance and growth characteristics of some fungi changed. Particularly noticeable was the production of increasing amounts of aerial hyphae. Some fungi released pigment into the malt agar causing strong yellow or red colorations (Figure 5.3.1.13). All the changes are considered to arise due to a reaction to the D D A C toxicity and its surfactant properties. 92 £6 © g i Ul o •a ui © o 3 zn -4 Ul O 3 o o 3 O 0 1 R i I: O > 1 § Ul 00 a 0\ Ul 00 o en • to t'6 ro i—* ro o 3 0 3 ,—, Os SO ON O 4^  O -O w> O •o >0 3 SO -0 4^  -0 o Os p SO o SO CD *o o o >o *o 3 to 4*. O it © o to o ON o o to o t» •o 3 o -J o 4^ O i o 00 o to 00 o bs 4^  OS 00 SO o o to 00 o ro i—• ro o 3 ° © to <J1 to o p to o o p to o to o •o >o 3 o SO o to 4*. © 4^  O to o to o >o CD o o I •o *o 3 to o to SO o to © SO o to 00 o to to © I •o •o 3 Os to to SO © o to a 4^ o 4^  4^  SO o 4^  o 4^ to to 3 to o to s Cd Os En' N o 3 Fig. 5.3.1.13 G. roseum (TC-NP-16) grown on malt agar containing 0 ppm and 750 ppm D D A C A: Face up B: Face down 95 5.3.2 Liquid media The fungi V. bulbillosum and G roseum, were inoculated in all three liquid media with and without DDAC. After two weeks, both fungi grew well in Vogel and Czapek liquid media containing no D D A C but they did grow well in 2% malt extract. When the three liquid media contained DDAC, fungi grew only in the Vogel liquid media. Consequently this was chosen for the liquid culture experiment. The tolerant fungi were chosen for growth in liquid media with D D A C , based on the growth rate of fungi on solid media containing DDAC. The biomass of fungi in liquid media without D D A C and with 250 ppm D D A C is shown in Table 5.3.2.1. The fungal strains of G. roseum (TC-NP-16), G. roseum (321C), V. bulbillosum, V. lecanii (390A), Acremonium sp., and F. avenaceum (F-l-1) grew very well in liquid media containing 250 ppm DDAC. Figure 5.3.2.1 shows the growth of G. roseum (TC-NP-16) and F. avenaceum (S-l-1) in liquid media containing 250 ppm DDAC. Since a threshold of 250 ppm D D A C in solid agar media was employed to select fungi for further work, the same concentration was also used for the determination of fungal tolerance in liquid media. Examination of the results of fungal growth in solid media and liquid media (Fig. 5.3.1.4 to 5.3.1.12 and Table 5.3.2.1), however, revealed that there was no relationship between the fungal tolerance in solid media and liquid media. Some fungi T. virens (F-l-1), Z. molleri (S-l-6), G. roseum (321A and 321D) grew very well in solid media containing 750 ppm or 1000 ppm DDAC, but failed to grow in liquid media containing 250 ppm DDAC. 96 Table 5.3.2.1 Fungal growth in the liquid media containing 250 ppm D D A C Biomass (mg/ml) Fungi 0 ppm 250 ppm G. roseum (3 21 A) G. roseum ( 3 2 1 B ) G roseum (321C) G. roseum (32 ID) G. roseum (GRSB) G. roseum (TC-NP-16) 6.46 ±0.30 2.84 ± 0.49 7.15 ±0.08 2.21 ±0.01 6.26 ±2.39 5.86 ±0.01 0 1.81 ±0.22 6.17 ±0.65 0 2.99 ±0.05 6.83 ±0.34 V. bulbillosum V. lecanii 8.17 ±0.13 7.08 ± 1.12 6.25 ±0.15 6.34 ±0.05 F. avenaceum (S-2-11) F. avenaceum (S-l-1) 9.17 ±0.71 6.64 ± 0.73 1.85 ±0.20 7.07 ±0.56 G. viride (623B) G. virens (25SD) Acremonium sp. T. virens (F-l-1). Z. moelleri (S-l-6) 5.78 ±0.18 7.65 ±0.30 6.83 ± 0.01 7.46 ±0.25 6.38 ±0.01 3.24 ±0.49 0 6.40 ± 0.26 0 0 97 98 5.3.3 The growth curve of V. bulbillosum. The production of biomass by V. bulbillosum in Vogel liquid media without D D A C at different time intervals is shown in figure 5.3.3.1. Growth curves of fungal cultures normally show three stages of growth. According to the growth curve of V. bulbillosum, there was only a very short lag phase where the organism adjusted to the growing medium. The lack of a lag phase probably resulted from the transfer of cores containing highly active, growing mycelium, to a liquid media. Between days three to seven growth was linear. The fungal biomass reached a maximum during day seven and eight. After this, between the ninth day and eighteenth days, the biomass reduced in magnitude. During this period, degradation of D D A C may occur as the fungus seeks alternative source of carbon. However, according to the fungal growth on malt agar containing DDAC, the amount of D D A C under the edge of the colony was substantially reduced, which could indicated that degradation of the chemical must also take place during the early stages of fungal growth. 99 100 5.4 Fungal degradation of D D A C Previous research with Gliocladium roseum and Acremonium sp demonstrated that fungi could modify the structure of QACs in solid agar and wood, based upon the disappearance of the peak due to D D A C in the HPLC chromatogram. In the present study, the ability of the fungi from standard cultures to degrade D D A C was compared with strains of G. roseum, V. bulbillosum and Acremonium sp. isolated from lumber by Dr. Andress Doyle. The D D A C degrading capacity of the recent isolation of Fusarium sp., Zygorrhynchus sp. and Trichoderma sp. were also assessed in solid and liquid media. 5.4.1 Solid media Table 5.4.1.1 shows the concentration of D D A C in malt agar before fungal growth and the residual D D A C concentration in selected positions on the malt agar plates following fungal overgrowth. The positions on the malt agar are shown in Figure 5.4.1.1 and correspond to: P) the perimeter of the plate where there has been no fungal growth; E) beneath the growing limit of the hyphal mat; and C) close to the center of the plate. The percent reduction of D D A C was calculated from the baseline D D A C assay determined for each plate at the perimeter. The decrease in D D A C concentration may be considered as evidence of primary degradation. Although the concentration decreased, there was no evidence to suggest that ultimate degradation (or mineralization) had occurred. Nor was there any information on the toxicity of the products formed (environmentally acceptable degradation). V. bulbillosum, G. roseum 321C and 32ID seemed to have the best potential to degrade DDAC, based upon their ability to cause a major reduction in the D D A C peak when analyzed by HPLC. There did not appear to be any correlation between the decrease in 101 Table 5.4.1.1 D D A C degradation in solid media Fungal species DDAC content Percentage of DDAC reduction (ppm) (%) No growth Growth Growth Edge Center Edge Center G. roseum (321A) 826 710 477 16 42 G. roseum (321B) 833 755 659 9 21 G. roseum (321C) 1119 11 11 99 99 G. roseum (321D) 966 • 170 75 82 98 GRSB 765 551 145 28 81 TC-NP-16 821 520 164 37 80 V. bulbillosum 801 94 8 88 99 Acremonium 898 707 380 21 58 F. avenacewn (S-l-1) 688 101 173 85 75 F. avenacewn (S-2-11) 793 719 295 9 63 F. avenacewn (S-2-1) 1051 955 620 9 41 Z. moelleri(S-l-6) 915 731 514 20 44 T. virens (F-l-1) 953 590 564 38 41 G. virens (258D) 859 481 383 44 55 G. virens (258C) 944 506 443 46 53 G. white (623B) 760 190 154 75 80 Note: Standard deviations are between 2-50 ppm. 102 Fig. 5.4.1.1 The different locations on the malt agar plate for assessment of D D A C C: center E: edge P: perimeter 103 D D A C concentration and the fungal growth rate. For example, T. virens (F-l-1) and Z. molleri (S-l-6) grew much faster on malt agar containing D D A C than V. bulbillosum, but the residual D D A C concentration was much higher than that after growth of V. bulbillosum. 5.4.2 Liquid media Recovery ofDDAC from the liquid media D D A C is soluble in water and a wide range of solvents. D D A C contains two long alkyl chains, which are responsible for its hydrophobic character. However, the positive charged nitrogen atom enhances the hydrophilic property of DDAC. This combination of hydrophobic and hydrophilic character, its surfactant property and the common ion effect, makes the quantitative recovery of D D A C from solution or substrates challenging. Table 5.4.2.1 contains the percent of D D A C recovered from liquid media, using different extraction procedures outlined in Section 4.4.2. It was found that the recovery of D D A C using partitioning in methylene chloride was not good enough. If instead, the liquid media containing D D A C was roto-evaporated and extracted in acidified methylene chloride, with an ultrasonic bath, the best recovery of D D A C (>90%) from buffer and fungal cultures was obtained. Because D D A C contains cationic ions according to the following equation [DDAC] <-> [DDA] + + [CI]' ([DDA] + here is the didecyldimethylamonium cation), the use of an acidified solvent can increase the recovery of D D A C solubility in water or polar solvents, because the protonic solvent can prevent exchange of the D D A onto solids such as glassware etc, as well as enhence its release from wood at solid media. 104 Table 5.4.2.1 The recovery of D D A C from liquid media and buffer using different extraction procedures Extraction procedure Original DDAC (mg) Recovered DDAC (mg) Recovery (%) a Vogel liquid media 9.76 9.2 4 4 46 43 b Buffer 3.8 3.8 1.9 1.6 50.6 41.5 Vogel liquid media 5.9 5.9 3 3 5.9 5.9 c Buffer 10 10 9 8 90 80 Vogel liquid media 10 10 1.5 0.5 14 4.8 d Buffer 29 19 27 18 93 96 Vogel liquid media 29 36 16 33 57 90 e Fungal culture 24 28 23 24 25 21 100 89 91 a = Liquid media extracted with methylene chloride b = Liquid media roto-evaporated to dryness, extracted with methylene chloride c = Liquid media roto-evaporated to dryness, extracted with acidified methylene chloride d = Liquid media roto-evaporated to dryness, extracted ultrasonically with acidified methylene chloride e = Fungal culture roto-evaporated to dryness, extracted ultrasonically with acidified methylene chloride 105 Residual amount of DDAC in liquid media The fungi which showed very good growth in liquid media containing 250 ppm D D A C were chosen for these experiments to determine the amount of D D A C degraded in liquid media. Table 5.4.2.2 shows the residual D D A C after two week's fungal growth. It was found that D D A C concentrations were reduced to below the detectable limit of approximately 10 ppm in all but one case (G. roseum 321C). One explanation for this observation is that the D D A C had been modified or degraded into chemicals which can not be detected by the HPLC method, which was specific for cationic QACs. Figures 5.4.2.1 to 5.4.2.6 show the reduction or disappearance of the D D A C peaks at ca 9.85 minute retention time and the formation of other peaks at longer retention times. Since all the peaks are negative (ie. give a response similar to the DDAC, but with a different retention time), this suggests that these metabolites are cationic in nature. These new peaks are thought to arise from the fungal action on DDAC. They were not observed in the HPLC spectra of the control samples (Vogel liquid media containing 0 ppm D D A C after fungal growth or Vogel liquid media containing 250 ppm D D A C without any fungal growth). It was interesting to note that all the retention times of the negative peaks from the different cultures were similar, except for those in the chromatogram of G. roseum 321C. This suggests that the degradation of D D A C by different moulds follows the same mechanism. The longer retention time is consistent with the formation of QAC type compounds with shorter alkyl chain length. According to their retention times (Cg/Cg: 11.15 min; Cg/Cio: 10.05min; Cio/Cio: 10.49 min), two of the negative peaks could possibly correspond to C 8 / C 8 and Cs/Cio dialkyldimethylammonium salts. It may also be formed by QACs containing an oxidized 106 Table 5.4.2.2 Residual concentration of D D A C in liquid media after fungal growth Fungi * Original concentration (ppm) Residual concentration (ppm) V. bulbillosum 296 ND Acremonium sp. 296 N D G. roseum (TC-NP-16) 296 ND G. rosewn (321C) 296 8.6 F. avenacewn (S-l-1) 296 ND ND: not detected (Detection limit is 10 ppm) * Concentrations of D D A C based on HPLC analysis 107 Fig. 5.4.2.1 H P L C spectra of extract from liquid media containing 250 ppm D D A C after fungal growth of V. bulbillosum A: Standard D D A C (776 ppm) B: Control (concentrated extract from liquid media without DDAC) C: Concentrated extract from Vogel liquid media with D D A C D: Concentrated extract from Vogel liquid media with D D A C and silica gel 108 Fig. 5.4.2.2 H P L C spectra of extracts from liquid media containing 250 ppm D D A C after fungal growth of Acremonium sp. A: Standard D D A C (776 ppm) B: Control (concentrated extract from liquid media without DDAC) C: Concentrated extract from Vogel liquid media with D D A C D: Concentrated extract from Vogel liquid media with D D A C and silica gel 109 Fig. 5.4.2.3 KCPLC spectra of extracts from liquid media containing 250 ppm D D A C after fungal growth of G. roseum (TC-NP-16) A: Standard D D A C (776 ppm) B: Control (concentrated extract from liquid media without DDAC) C: Concentrated extract from Vogel liquid media with DDAC 110 SI ro 8. > 01 Retention time (min) A ro 8. -2 1 Retention time (min) B Retention time (min) 2 -3 ro o . a Fig. 5.4.2.4 FfPLC spectra of extracts from liquid media containing 250 ppm D D A C after fungal growth of F. avenacewn (S-l-1) A: Standard D D A C (776 ppm) B: Control (concentrated extract from liquid media without DDAC) C: Concentrated extract from Vogelliquid media with D D A C 111 -5 0-, Retention time (min) -1 -2\ B Fig. 5.4.2.5 HPLC spectra of extracts from liquid media containing 250 ppm D D A C after fungal growth of G. roseum (321C) A: Standard DDAC (776 ppm) B : Control (concentrated extract from liquid media without D D A C ) C: Concentrated extract from Vogel liquid media with DDAC 112 -5 Retention time (min) • -1H Fig. 5.4.2.6 HPLC spectra of extract from liquid media containing D D A C without any fungal growth A. Standard D D A C (776 ppm) B. Extract from Vogel liquid media with 250 ppm D D A C C. Extract from Vogel liquid media with 800 ppm D D A C and siica gel 113 terminal carbon atom. During the extraction, the chloride of D D A C may be replaced by the formate anion which would affect the retention time of HPLC peak. However, this should not pose any the problem since the standards and reference chromatograms of D D A C were all prepared using the same procedure. Based on the study of the tolerance and D D A C degrading ability of all of the fungi from the culture collection and those isolated from treated stakelets exposed in the field test, it did not seem that the fungi isolated from the field area were specially tolerant to DDAC. Nor did they have a better ability to degrade DDAC. Instead it seemed that the tolerance and the ability to degrade D D A C were common properties of these moulds. 114 5.5 Identification of DDAC metabolites and implications for fungal degradation process This research and the former study by Doyle (1995) have both shown that the concentrations of D D A C were reduced in solid and liquid media by fungal growth. But where did the D D A C go? The final products of biodegradation were not established in the previous study. In these experiments, an attempt was made to identify the major metabolites present in liquid media and thereby provide some evidence for the biodegadation pathway and the degree to which this degradation takes place. 5.5.1 Decanoic acid Based on the literature review, one plausible biodegradation route for D D A C would involve the initial removal of one decyl chain. The resulting metabolites formed would be a ten carbon chain organic chemical and a tertiary amine. Both of these are likely to be soluble in methylene chloride and should be detectable with GC-MS. When methylene chloride extract of the V. bulbillosum and G. roseum (TC-NP-16) cultures containing 250 ppm D D A C were subjected to GC-MS analysis, decanoic acid was identified as one of the compounds present (Figures 5.5.1.1 to 5.5.1.2). Analysis of liquid cultures of Acremonium sp., resulted in a closely related product, decanediol, being identified (Figure 5.5.1.3). The GC-MS analysis of the extracts of corresponding cultures containing 0 ppm D D A C or Vogel liquid media containing 250 ppm D D A C without fungal growth, did not identify the presence of either decanoic acid or decanediol. The D D A C metabolites produced by different fungi after the same incubation period may differ, because their growth rate may be different. Two metabolites contain a ten carbon chain (decanoic acid and decanediol). This would suggest that during degradation of DDAC, breakage of the ten 115 116 CO CM CO 0 CM CO Z * CM 10. TP IO r> LO CTi ID r-i CO Cv] si H ON CO -4 co^ g ov= N H H <X o © z <x o u ft in H H CO C0_ co; N H cs H 00 *co J » CS SO v£ .xf CO 0> 10 CS CO CS CS CO CS CS CM CS hio H CS CS H tcs 10 o cs <A csi: CS V) CSN =3 CS X CS o ,o o CM X CM X o CM X CM X o CM X o CM X 0 1 CM X O CM X o co x o 117 <H | CM O • * LO 0) cn E ~ & 3 +> •"5 t/i I $= CM 1 ® s co vO +> £ 0 31 CO AS +> «J A CO x: 1 cj C5 rH I O K H W CO <r m i m <n CO O • • 0) CO LO LO CO Si lOl If) CO in VO' * 4 H 09 00 m_ IT) co CO sO CO o IO If) N 53. co 00 co _ CO lf)_ IO WSJ CO CO' - 'so IO C9 Cy) (SJJ C9 cy> C9N cs u. S o u . \ H X H X £ I o CM X o CM X cj CM X a CM X o CM X o CM X o CM X cj CM X CJ CM X CM X CJ o X s 0 1 S O o 3 a o .2. 118 carbon alkyl chain from the nitrogen is the initial step. It has been reported (Cain, 1976) that during the bacterial degradation of QACs, initial rupture of the long alkyl chain occured yielding a tertiary amine and an alkan-l-al. The alkan-l-al was then further oxidized and degraded by P-oxidation. If this mechnism is correct for moulds, a tertiary amine should be formed. Attempts to detect a tertiary amine (eg. decyldimethylamine) were unsuccessful. Studies conducted using an authenticated sample of decyldimethylamine, found that it was removed during the pre-treatment with the cationic ion-exchange resin column designed to remove the QACs present. An alternative strategy was therefore attempted to separate the tertiary amine and the residual DDAC. Although the computer fitting of the GCMS spectra suggested the presence of decanoic acid and decanediol, some characteristic peaks were missing from the mass spectra, notably those at approximately 29 m/c and while others were observed at higher mass than the molecular weight. This might suggested that impurities were present, or that recombination of ion fragments has taken place. Since the spectra are dependent to some extent on the GCMS conditions of operation, the spectra of authenticated samples of decanoic acid and decanediol should be run. 5.5.2 Tertiary amine Cationic surfactants can form insoluble compounds with anionic surfactants. Advantage was taken of this to form an insoluble ionic compound between the anionic surfactant sodium dodecyl sulphate and DDAC. This compound was insoluble in water and methylene chloride. Sodium dodecyl sulphate was also insoluble in methylene chloride. Based on this procedure, the D D A C would complex with the sodium dodecyl sulphate leaving the tertiary amine in solution. It could then be separated and analyzed by GC-MS. 119 GC-MS analysis of the extract recovered from the liquid of culture of V. bulbillosum. and Acremonium sp. which contained 250 ppm DDAC, showed that after two weeks' growth, decyldimethylamine could be detected. This spectrum was identified by comparing it with the authenticated decyldimethylamine run on GC-MS. Although this may have originated from the degradation process, it was also found in the extract of liquid media containing 250 ppm D D A C but without fungal growth. Since decyldimethylamine is not a major inpurity in DDAC, either the amount is small as it must have arisen frim another source (such as the GCMS analysis of D D A C which had not been removed during the complex reaction with the anionic surfactant, or which had dissolved in the methylene chloride). 5.5.3 Possible process in biodegradation of QACs by moulds Based on the available information on biodegradation mechanism of QACs, it is possible to consider two possible pathways for degradation of D D A C by moulds. In the first mechanism, the fungus initiates attack (©-oxidation) of the decyl chains, after which further degradation (P-oxidation) of this chain gradually removes additional atoms. The presence of such a process is supported by the HPLC analysis (Fig. 5.4.2.1 to Fig. 5.4.2.5) which showed additional peaks at a longer retention time. Such peaks could well indicate QACs with shorter alkyl chains. However, in the liquid culture experiments, in two cases decanoic acid was identified by GCMS. This would support the second pathway which involves the initial breaking of the long decyl group. After cleavage of the decyl group, it is possbly further biodegraded by P-oxidation leading ultimately to mineralization. 120 5.6 The growth of fungi in liquid media containing decyldimethylamine One proposed mechanism for the degradation of QACs by bacteria and fungi involves carbon-nitrogen fission as the first step, which would result in the formation of decyldimethylamine. This experiment was carried out to confirm whether or not, decyldimethylamine could support fungal growth. V. bulbillosum failed to grow in normal Vogel liquid media containing 1000 ppm of decyldimethylamine as either the sole nitrogen- or sole carbon-source. In a second experiment, the mould V. bulbillosum, the brown rot fungus C. puteana, and the white rot fungus T. versicolor all failed to grow in normal Vogel liquid media containing 1000 ppm decyldimethylamine (Table 5.6.1). When the concentration of decyldimethylamine was decreased to 100 ppm, four moulds V. bulbillosum, Acremonium sp. G. roseum (TC-NP-16), and F. avenaceum (S-l-1) were able to grow in normal Vogel liquid media. The resulting biomass is recorded in Table 5.6.2. This experiment was carried out at room temperature (20 °C), and the shaker speed was approximate by 143 rpm. Besides these incubation conditions, the source of inoculum may be responsible for the low biomass produced by these fungi compared to that measured in the previous experiments (cf Table 5.6.1). While the low concentration of decyldimethylamine did not inhibit the growth of the moulds, it did not function as carbon source to increase the biomass produced. 121 Table 5.6.1 Fungal growth in the normal Vogel liquid media containing 1000 ppm decyldimethylamine Biomass (mg/ml) decyldimethylamine 0 ppm 1000 ppm V. bulbillosum C. puteana T. versicolor 5.68 ± 0 . 0 5 0 5.68 ± 0 . 0 1 0 8.72 ± 0 . 0 1 0 Table 5.6.2 Fungal growth in the normal Vogel liquid media containing 100 ppm decyldimethylamine Biomass (mg/ml) decyldimethylamine 0 ppm 100 ppm Acremonium sp. 2.20 ± 0.03 1.95 ± 0.00 V. bulbillosum 2.91 ± 0.25 2.72 ± 0.12 G. roseum (TC-NP-16) 2.22 ± 0 . 0 4 1.97 ± 0.26 F. avenaceum (S-l-1) 1.76 ± 0.01 2.31 ± 0.28 122 S.7 The distribution of DDAC in starch, silica gel and aqueous solution During the latter stages of research, difficulties were encountered when repeating some of the earlier experiments. Analysis of the experimental procedure revealed the observation that the presence of a small amount of suspended starch in the liquid media, had a profound impact on the amount of D D A C dissolved in the solution. To better understanding this problem, the relative amounts of D D A C adsorbed onto the suspended starch and dissolved in the liquid media, were determined. To further investigate the phenomenon, reference experiments were made using silica gel, which is used as an absorbing surface in liquid culture studies involving micro-organisms. Liquid media containing 50 ppm of DDAC, made using soluble starch purchased from two sources did not support good growth of Acremoniwn sp., Verticillium sp. G. roseum, orFusarium avenacewn (Fr.) Sacc. Previous experiments with a two year old bottle of soluble starch from a third source supported fungal growth when amended with 500 ppm DDAC. 5.7.1 The recovery of DDAC from liquid media without starch but with silica gel In the initial experiment, the recovery of D D A C was tested by adding known amount to: a) Vogel liquid media without starch but with silica gel, and b) liquid culture containing silica gel in which the fungus V. bulbillosum had grown for two weeks. The recoveries (shown in Table 5.7.1.1) were high. The acidified methylene chloride allowed the recovery of 85% - 96% of the DDAC. 123 Table 5.7.1.1 The recovery of D D A C from liquid media Original D D A C Recovered D D A C Recovery (mg) (mg) (%) Vogel liquid media without starch but with silica gel 41.2 38 92 41.2 35 85 Vogel liquid media after growth of V. bulbillosum 41.2 38 92 41.8 40 96 124 5.7.2 The distribution of D D A C The second experiment focused on the distribution of D D A C when added to media containing silica gel. Table 5.7.2.1 shows the percentage distribution of D D A C distributed in solutions containing starch, silica gel, or starch and silica gel. The results confirmed that when particles of starch remained in the Vogel liquid media, they absorbed a very high proportion (88.5%) of the DDAC, leaving less than 11.5% in the solution. This phenomenon can be explained by the highly sorptive nature of QACs. They are very strongly bound on to surfaces such as glass, soil, sewage, humus and proteins (Lawrence, 1970, Swisher, 1987, MacQuillen, 1950, Barbaro and Hunter, 1965). The adsorption of organic cationic surfactants such as QACs to starch and silica gel, occurs mainly by electrostatic attraction, although Van der Waals forces also exert an influence for QACs that have long alkyl chains (Theng, 1974). With the removal of most of the QAC from the media by the starch particles, the fungi are able to metabolize the small amount of D D A C remaining in the aqueous solution. Consequently the fungi could grow in liquid media containing large amounts (e.g. 250 ppm) of DDAC. Different sources of starch may have quite different solubility in water. This will lead to wide variations in the amount of D D A C present in the solution. When the solubility of starch is high, (as in the latter experiment), the D D A C concentration available to fungi was also high. This may inhibit fungal growth. The simulation of this effect by the addition of silica gel (which has large surface area) allowed V. bulbillosum and Acremonium sp. to grow very well. The resulting biomass is shown in Table 5.7.2.2. 125 9ZI < a. < < o O o ca ge OQ OQ OQ ca ge cp__ sr CD ca ge •S* Cf* liqui C liqui B. liqui g o-3 3 3 22. CD ft a. &. & ia wit ica gi ia wit 13* CP noi B* P ' 1+ 1+ 1+ to ON 1+ to 00 00 1+ p 1+ © o s 1 o' p OQ CD 5? • S-I o p OQ CD I ' CD. o C 09 o o S3 CD < CD o CD s o •-•> O -—- o O S3 X/i o. &| 00 p B.i CO O O S3 00 - J Ul 1+ © u i © 1+ U l © - J U l 1+ © U i 41 a? T 3 CD 0 ^ ? * CD i - | 03 CD 1 8 9 < C CD P cr1 Q . P CD 2 ca. o-| Table 5.7.2.2 The biomass of fungi grown in liquid media containing 250 ppm D D A C and silica gel Biomass (mg/ml) Fungi Control 250 ppm V. bulbillosum 7.6 ±0 .2 6.4 ±0 .2 Acremonium sp. 7.3 ± 0.2 7.0 ±0 .9 127 5.7.3 Residual amount of DDAC after fungal growth in liquid media with silica gel Figures 5.4.3.1 and 5.4.3.2 show that D D A C disappeared after two week growth of V. bulbillosum and Acremonium sp. based on the HPLC analysis. From the chromatographs, the metabolites of D D A C appeared to be similar to those formed in the liquid media containing D D A C without silica gel. It can be concluded that the silica gel did not change the mechanism of fungal degradation of D D A C in liquid media. The results were consistent with the degradation of the D D A C with the formation of QACs having shorter alkyl chain QACs. It is also possible that oxidation of the terminal carbon occurred producing either alcohol, aldehyde or carboxylic acid. 128 5.8 Attempts to fractionate the protein produced by fungi growing in liquid media with and without DDAC Experiments involving fungal growth on/in amended media have demonstrated a loss of D D A C based upon the reduction in the peak intensity in the HPLC spectra. The presence of several metabolites has also been suggested from the number of peaks in the chromatogram. To better understand the mechanism of fungal action, an attempt was made to fractionate the protein responsible for the degradation process. 5.8.1 Extracellular protein pattern Fig. 5.8.1.1 shows on SDS-PAGE profile of the extracellular protein produced by V. bulbillosum and Acremonium sp. derived from liquid media containing 0 ppm and 250 ppm DDAC. The protein pattern for the control differed from that of the liquid media containing DDAC. The latter contained two extra protein bands. The molecular weight of one band is at approximately 20-30 kDa and while that of the other band is slightly higher than 94 kDa. The corresponding IEF gel for the extracellular protein from media containing D D A C (Fig. 5.8.1.2) exhibited a few extra protein bands. The pi (isoelectric point) of the extra bands was between 4.55 to 5.20. It is possible that these proteins are the enzymes responsible for the degradation of DDAC. Another possibility is that they may come from the fungal cell wall or cell contents. It has been reported that the surfactant nature of D D A C leads to leakage of the cell contents due to alteration of the cell membrane structure. 129 Fig.5.8.1.1 Silver stained SDS-PAGE phast gel (homogenious 12.5%) of extracellular proteins from culture of V. bulbillosum and Acremonium sp. containing 250 ppm D D A C and 0 ppm D D A C lane 1: low molecular weight standard lane 2: 2 p,g protein from Acremonium sp containing 250 ppm D D A C lane 3: 2 p,g protein from Acremonium sp containing 0 ppm D D A C lane 4: 2 ug protein from V. bulbillosum containing 250 ppm D D A C lane 5: 2 p.g protein from V. bulbillosum containing 0 ppm D D A C 130 Fig. 5.8.1.2 Silver staining IEF gel (pi 3-9) of extracellular protein from culture supernatants of V. bulbillosum and Acremonium sp. containing 0 ppm D D A C and 250 ppm D D A C . Pharmacia IEF standards are shown in lane 5. Protein were loaded at approximately 2 p.g per lane. lane 1: protein from culture of. V. bulbillosum containing 0 ppm D D A C lane 2: protein from culture of. V. bulbillosum containing 250 ppm D D A C lane 3: protein from culture of. Acremonium sp containing 0 ppm D D A C lane 4: protein from culture Acremonium sp. containing 250 ppm D D A C 131 5.8.2 Intracellular protein pattern From the SDS-PAGE of the intracellular protein shown in figure 5.8.2.1, there did not appear to be any difference in the protein patterns derived from the control media and the liquid media containing DDAC. This would suggest that the D D A C degradation possibly took place outside the fungal cell wall. 5.8.3 Decreased amount of DDAC after treatment with intracellular extracts and supernatants from cultures of V. bulbillosum containing 0 ppm and 250 ppm DDAC The data in Table 5.8.3.1 shows that the amount of D D A C decreased after treatment with the supernatant from a liquid culture of V. bulbillosum containing 250 ppm DDAC, while the amount of D D A C was only slightly decreased after treatment with supernatant from culture of V. bulbillosum containing 0 ppm DDAC. Intracellular extracts from mycelia of V. bulbillosum which were grown in liquid media containing 0 ppm and 250 ppm D D A C also failed to reduce the D D A C content during incubation. This confirmed that D D A C degradation took place outside the fungal cell wall. However, it was not certain that the degradation of D D A C results from additional proteins shown on the SDS and IEF gels. 132 Fig.5.8.2.1 Silver stained SDS-PAGE phast gel (8-25%) of intracellular protein from culture of V. bulbillosum with 500 ppm D D A C and without D D A C lane 1 and 6: low molecular weight standard lane 2: 2u,g protein from V. bulbillosum with D D A C lane 3: 2u,g protein from V. bulbillosum without D D A C lane 4: lp.g protein from V. bulbillosum with D D A C lane 5: lp.g protein from V. bulbillosum without D D A C 133 O O P * O O fcH a a 3-B. B. 3 3. 3. g 5 3 e M O rq g 3 d d d d fe © © 00 00 © © © to to to 00 to VO to -o. 1+ 1+ 1+ © o © © © o © OV 00 © O O P» a a g1 B. B. 5 3. 3. 3 13 to o >T3 ro 3 133 d D fe o d fe to to 00 00 C/3 d CD B 3 £ 3 " i c CD 3 CD c I B. I CD OQ CD i CD 3 B- d W d £ > CD O I 3 g TO g CD 5. CONCLUSIONS Based upon all of the observation in this study, the following conclusions may be made. (1) It was not feasible to use bromophenol blue or methylene orange colored complexes to monitor the changes in the D D A C concentration in liquid media. (2) The tolerance and degrading capacity of the moulds examined in this study did not appear to indicate a special resistance in those strains isolated from D D A C treated wood. Further, it appeared to be a general property of this group of fungi. (3) There was no relationship between the fungal growth on solid malt agar and corresponding growth on malt agar containing DDAC. (4) There was no relationship between the tolerance of fungi to D D A C in solid media and that observed in liquid media. (5) Only the fungi which degraded D D A C on solid media were able to grow in liquid media containing DDAC. (6) Fungi produced additional extracellular protein when grown in liquid culture containing DDAC. Based on the examination of the extracellular and intracellular supernatant solutions, the D D A C degradation occurred outside the cell wall. GC-MS analysis of the D D A C containing liquid media following fungal growth, provided evidence for both C-N fission, and ©- and B- oxidation of a long alkyl chain, during fungal degradation. This may have arisen because of the presence of D D A C both adsorbed onto starch particles and dissolved in the liquid media. 135 6 RECOMMENDATIONS (1) The role of other fungi such as Zygomycetes and Actinomycetes in limiting the performance of QACs as wood preservatives should be studied. (3) It was found that the turbidity of the liquid media which caused by the variable solubility of the starch, affected the D D A C content in the solution. This may lead to the different mechanisms of the D D A C degradation. Further work should employ a carbon source which is totally soluble in water. (4) Additional research is necessary to find out whether the tertiary amine exists in the media after fungal growth, to confirm the pathway of degradation of DDAC. (5) It appears that the degradation of D D A C in solid media may be different from that in liquid media. Since the study focused on how the fungi reduced the ability of QACs to protect wood, more research need to be conducted on identifying the metabolites produced in D D A C amended solid media. (6) A more intensive examination of the intracellular protein and extracellular protein is recommended. The current experiments suggested that intracellular protein can not degrade DDAC. The new proteins shown on the SDS and IEF gels should be purified and their ability to degrade D D A C should be confirmed. 136 7 REFERENCES Anonymous. 1984. Alkyl ammonium compounds as wood preservatives. Proc. Amer. Wood Preserv. Assoc. 80:191-204. Arpino, A. and Ruffo, C. 1976. Contribution to the microdetermination of cationic surfactants. 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