DEGRADATION OF AROMATIC COMPOUNDS
BY MICRO-ORGANISMS
IN DISSIMILATORY NITRATE REDUCTION
BIBLIOTHEEK
TECHNISCHE UNIVERSITFH DELFT
DEGRADATION OF AROMATIC COMPOUNDS
BY MICRO-ORGANISMS
IN DISSIMILATORY NITRATE REDUCTION
PROEFSCHRIFT
TER VERKRIJGINGVAN DE GRAAD VAN DOCTOR IN DE TECHNISCHE WETENSCHAPPEN AAN DE TECH-NISCHE HOGESCHOOL DELFT, OP GEZAG VAN DE RECTOR MAGNIFICUS PROF.IR. L. HUISMAN, VOOR EEN COMMISSIE AANGEWEZEN DOOR HET COLLEGE
VAN DEKANEN TE VERDEDIGEN OP WOENSDAG 25 MEI 1977 TE 16.00 UUR
DOOR
GERHARD BAKKER
SCHEIKUNDIG INGENIEUR GEBOREN TE ROTTERDAM
DIT PROEFSCHRIFT IS GOEDGEKEURD DOOR DE PROMOTOR PROFESSOR DR. T.O. WIKÉN
Aan Marga,
CONTENTS
page
CHAPTER 1: Introduction and survey of literature 9
/. /. Introductory remarks 9 1.2. Literature on ttie decomfjosition of aromatic compounds 10
1.2.1. Aerobic degradation 10 /. 2.2. A naerobic degrada tion 13
CHAPTER 2: The growth of micro-organisms on aromatic compounds 21 in denitrifying conditions
Summary 21 2.1. Introduction 21 2.2. Materials and methods 22
2.2.7. Enrichment cultures 22
2.22. Media 22 2.2.3. Assays 23 2.2.4. Sterile and anaerobic Erienmeyer system 24
2.3. Results 26 2.3.1. Experiments on enrichment and isolation of micro-organisms 26
2.3.2. Experiments on degradation of other aromatic compounds 36 2.3.3. Degradation of phenol in the presence of succinate 38
2.4. Discussion and conclusions 39
CHAPTER 3: Demonstration of the degradation of aromatic 41 compounds in anaerobic denitrifying conditions
Summary 41 3.1. Introduction 41 3.2. Materials and methods 42
3.2.1. Maintenance of an active culture 42 3.2.2. Assay of enzymes and cell concentration 43 3.2.3. Culture vessel for experiments with C-labelledphenol 45
14
3.2.4. Assays of C components 45
3.3. Results 46 3.3.1. Degradation of phenol in anaerobic denitrifying conditions 46
3.3.2. Influence of KNOj on phenol degradation 47 3.3.3. Degradation of ^^C-labelledphenol 48
3.3.5. Assays of nitrate reductase, dioxygenases, and catechol or 51 o-benzoquinone as intermediates
3.3.6. Transition from anaerobic to aerobic conditions 53
3.4. Discussion and conclusions 55
CHAPTER 4: Stoichiometry and optimum conditions for anaerobic 57 degradation of phenol
Summary 57 4.1. Introduction 57 4.2. Materials and methods 57
4.2.1. Warburg experiments 57 4.2.2. Cytochrome spectra 68
4.2.3. Gas analysis 58 4.2.4. Assay of the composition of cell material 59
4.3. Results 59 4.3.1. Influence of pH and temperature 59
4.3.2. Cytochrome difference spectra 62 4.3.3. Gas production during denitrification 64 4.3.4. Stoichiometry of the overall reaction 67
4.4. Discussion and conclusions 71
CHAPTER 5: Kinetics of the degradation of phenol 73
Summary 73 5. /. Introduction 73 5.2. Materials and methods 74
5.2.1. Micro-organisms 74 5.2.2. Growth curves 74 5.2.3. Kinetic experiments 74
5.3. Results 75 5. J . / . Growth of a mixed population on phenol 75
5.5.2. Determination of the maintenance and cell yield coefficient 78 5.3.3. Determination of the Monod constant and the substrate inhibition 80
constant
5.3.4. The lowest attainable phenol concentration 82
CHAPTERS: Hypotheses concerning the anaerobic metabolism of 87 aromatic compounds
Summary 87 6.1. Introduction 87 6.2. Materials and methods 88
6.2.1. Estimation of free enthalpy changes 88 6.2.2. Breakdown of C-labelled phenol 90 6.2.3. Isolation of acetic and caproic acids 91
6.2.4. ^^C analysis 91 6.3. Results 91 6.3.1. Involvement of NO ^-phenols 91
6.3.2. Hypothetical pathway for the anaerobic microbial decomposition of 92 phenol
6.3.3. Intermediates in the decomposition of C-UL-phenol 94 6.3.4. Pathway for the anaerobic microbial decomposition of benzoic acid 100
6.3.5. Hypothetical pathway for the anaerobic microbial degradation of 103 cresols
6.4. Discussion and conclusions 105
REFERENCES 107 A B B R E V I A T I O N S , S Y N O N Y M S A N D SYMBOLS 115 S U M M A R Y 117 S A M E N V A T T I N G 121 D A N K W O O R D 124 C U R R I C U L U M V I T A E 125 S T E L L I N G E N 127
1. Introduction and survey of literature
1.1. INTRODUCTORY REMARKS
Continuous industrialization and ever-growing problems with pollution of the environment have made" knowledge of the processes involved in the degradation of waste compounds of the utmost importance. Particularly the long-term effects of waste compounds on the environment call for close attention, which has become quite evident from reports on such chlorinated hydrocarbons as DDT, PCB, and freon, as well as on aromatics like
3,4-benzopyrene and phenol.
Such knowledge should be utilized exhaustively, since the
environmental problems presented are diverse and often highly complex. For instance, DDT and PCB are poody degradable and highly toxic compounds, which may accumulate in food chains. The non-toxic freon may seriously affect the upper atmospheric strata. Benzopyrene and a great many other compounds are mutagenic. Phenol causes the quality of drinking water to deteriorate if chlorination is applied. Many such instances could be added.
In the present investigation, the microbial degradation of aromatic compounds will be studied under anaerobic, denitrifying conditions, and for reasons of comparison in some cases under aerobic conditions. That the degradability of such compounds by micro-organisms under anaerobic conditions is often doubted and sometimes even completely denied has been the incentive for the present research programme. Phenol, being a major aromatic waste,for instance in coke oven effluents, has mainly been used as aromatic compound. So far the breakdown of phenol under anaerobic denitrifying conditions has not been recorded in the literature. Such degradation would facilitate the operation of waste-water treatment units incorporating a denitrification step, for instance for the nitrification and denitrification of coke oven effluents.
In the following chapters, the experiments are described in which the existence of the anaerobic decomposition of phenol and other aromatic compounds in the presence of nitrate has been established, special attention being paid to the conditions under which the process takes place and to the mechanism of the process.
1.2. LITERATURE ON THE DECOMPOSITION OF AROMATIC COMPOUNDS
1.2.1. Aerobic degradation
Although the aerobic degradation of aromatic compounds is well known, a brief review will be given here, mainly for comparison with the anaerobic process. In practice, aerobic degradation of, for instance, phenol, has been applied in many installations for biological treatment of waste water from coke ovens and other plants. In the Pasveer ditch of DSM at Geleen, Netherlands, phenol reductions of over 99 % have been achieved.
Various species of micro-organisms of different genera are capable of breaking down aromatics under aerobic conditions as can be seen in Table 1, which does not claim completeness, but is only meant to be illustrative.
Table 1
Micro-organisms capable of breaking down aromatics under aerobic conditions
Acetinobacter spec\es (Renier, 1971) Alcaligenes eutrophus (Stanier, 1971) Arthrobacter speaes (Horvath, 1970) Aspergillus niger (Kumar et al., 1972)
/4^ofo6acfer species (Renier, 1971)
Candida species (Mills et al., 1971; Neujahr et al., 1974) Fusarium oxysporum (Barz, 1971)
Hydrogenomonas facilis (de Cicco and Umbreit, 1964) Mycobacterium species (Lippelt and Bönicke, 1968) Neurospora crassa (Gross et al., 1956)
Nocardia rubra (Hartmann et al., 1973) Pseudomonas species (Stanier, 1948) Pseudomonas arvilla (Nozaki et al., 1963)
Pseudomonas putida (Hopper and Taylor, 1975; Ornston and Stanier, 1966) Rhodotorula species (Mills et al., 1971)
Saccharomycescerevisiae (Millsetal., 1971) Thiobacillus species (Taylor et al., 1969) Trichoderma lignorum (Vidal, 1969) Trichosporon cutaneum (Mills et al., 1971)
o
0 2 phenolO
OH O2r^^^^
OH COGH -COOH - ^ etc.catechol cis-cis-muconic acid
- ^ etc.
a-Hydroxymuconic semialdehyde
Fig. 1
Aerobic degradation of phenol by micro-organisms
The pathway first leads by hydroxylation to catechol. Molecular oxygen then brings about the ring rupture by ortho- or meta-cleavage, resulting in unsaturated, non-cyclic compounds (Ornston and Stanier, 1966). Cleavage of protocatechuic acid occurs similarly.
The biochemistry involved in the degradation of various aromatic compounds is well-known. Key-point in the breakdown process is the rupture of the aromatic nucleus by free molecular oxygen. Three different metabolic pathways are known, two of them involving catechol or protocatechuic acid, the third gentisic acid as intermediates. The essential steps of the first two pathways, with catechol and protocatechuic acid as substrates, are indicated in Fig. 1. The influence of molecular oxygen on the ring rupture has been studied extensively. A proposed mechanism is shown in Fig. 2. The oxygenases have been described and even crystallized (Hayaishi, 1966). Subsequent
metabolism of the breakdown-products follows a pathway resulting in
compounds of known biochemical cycli. Much research on the decomposition of various aromatics has been reviewed by van der Linden and Thysse (1965), Raymond (1969), Ornston (1971) and Dagley (1971, 1976).
-©
a
OHJ"
WA
M
/O-o
W^§H
® OH* t 0 H
"OH Metapyrocatechase" \ I I
O
^ C " / O H ^OHa
VOHX /O®
4 OH O ^ catechol ^-fc- Pe^* Pyrocatechase FIg. 2Oxidative fission of catechol; proposed mechanism for catalysis by metapyrocatechase and pyrocatechase
Catechol is degraded by molecular oxygen by
In the last few years a number of publications on the catabolism of other cyclic compounds have appeared in literature. Sparnins and Dagley (1975), for
instance, reported on the degradation of gallic acid by Pseudomonas putida, and Haider et al., (1974) on the degradation of chlorinated aromatics and cyclohexane derivatives. Chapman (1976) discussed the microbial degradation of halogenated compounds.
McKenna and Kallio (1965) reviewed the degradation of non-benzenoid cyclic hydrocarbons. De Klerk and van der Linden (1974) proved the
feasibility of aerobic breakdown of cyclohexane; they demonstrated the oxidation of cyclohexane to cyclohexanol by an n-alkane oxidizing bacterium, as well as the utilization of cyclohexanol by a strain of Pseudomonas.
Murray et al. (1974) showed that aerobic degradation of cyclohexanone by a
Nocardia species is possible, with 2-hydroxycyclohexane-1-one as an
intermediate. Other compounds, such as cyclohexanol, cyclohexane-1,2-dione and cis, trans-cyclohexane-1,2-diol, are also oxidized by microbes using free oxygen. Trudgill (1976) discussed the degradation of alicyclic and heterocyclic compounds.
A number of authors believe that most aromatic compounds can be decomposed only with free molecular oxygen via the known pathways. They base their opinion on the fact that the aromatic nucleus is stabilized by its marked resonance behaviour and that, hence, initial ring rupture can only be effected by the high energy input resulting from oxidation by direct coupling to molecular oxygen (Stanier, 1972).
1.2.2. Anaerobic degradation
As early as 1934 the degradation of aromatics during methane fermentation was reported (Tarvin and Buswell, 1934). Since 1952, a great number of publications have appeared on the breakdown of both aromatic and other cyclic compounds under anaerobic conditions; information was provided about the conditions prevailing during dissimilatory nitrate reduction, methane fermentation, photosynthesis, and dissimilatory sulphate reduction.
Using a manometric technique. Proctor and Scher (1960) proved the possibility of degradation of benzoic acid, protocatechuic acid and catechol under anaerobic conditions in the light by a Rhodopseudomonas species. An extract from cells grown anaerobically on a medium of mineral salts and benzoate could not oxidize these substrates, either in the light or in the dark; the extract could, however, catalyze the anaerobic decarboxylation of protocatechuic acid to catechol in the dark. For anaerobic conditions and
exposure to light, they therefore proposed a pathway similar to that assumed for aerobic conditions. An analogous observation was made by Hamasaki and Uchida (1969) with a cell-free extract of an Aerobacter cloacae mutant, which under anaerobic conditions decarboxylated protocatechuic acid to catechol. Leadbetter and Hawk (1965) reported on the photosynthetic growth of
Rhodopseudomonas palustris on 4-hydroxybenzoic acid under anaerobic
conditions. According to Hegeman (1967), the enzymes responsible for the aerobic breakdown of 4-hydroxybenzoic acid are not present under
photosynthetic, anaerobic conditions, which is borne out by the observations of others.
The evidence underlying the suggestions of Proctor and Scher (1960) was criticized by Dutton and Evans (1967), who stated that molecular oxygen plays no part in the anaerobic, photosynthetic breakdown of benzoic acid. Dutton and Evans (1968, 1969) and Guyer and Hegeman (1969) suggested, and presented evidence for, a new reductive pathway for the degradation of benzoic acid under anaerobic conditions (Fig. 3). Dutton and Evans (1969) demonstrated growth in the light of Rhodopseudomonas palustris on benzoic acid, 2-, 3-, and 4-hydroxybenzoic acid, as well as on protocatechuic acid. 2,3-Dihydroxybenzoic acid was slowly assimilated, whereas the other
di hydroxy benzoic acids and catechol were not assimilated at all. The cell-free extracts prepared from cells grown photosynthetically on benzoate failed to bring about any detectable reduction of benzoate, not even after various combinations of Fe2+, Mg2+, NADH2, ATP and GSH had been added. Ferrodoxin, prepared from Rhodopseudomonas pa/ustris cells did not act as electron-donor for benzoate reduction (Whittle, 1975). It was shown that benzoate is converted to benzoyl-CoA and then further metabolized (Whittle et al., 1976). The inhibition of aromatic photometabolism in
Rhodopseudomonas palustris by fatty acids was reported by Dutton and Evans
(1968, 1970). Degradation of benzoic acid and 4-hydroxybenzoic acid was inhibited by long-chain fatty acids and also by some low-molecular
monocarboxylic acids.
Van Iterson (1904) was the first to investigate denitrification in the presence of aromatic compounds. However, he could not prove the microbial degradation of aromatic compounds with nitrate as oxidant in the absence of free oxygen. Van Allen and van Niel (1952) came to the same conclusions with Pseudomonas fluorescens on a benzoate mineral medium under denitrifying conditions. Hansen and Kallio (1957) were likewise unable to detect any utilization by bacteria of nitrate as electron acceptor in
COGH COOH + 4H ^HaO benzoic acid COOH
ó
COOH COOH +H2O<3
COOHCr
COOHó
pimelic acid Fig. 3Proposed pathway of the photometabolism of benzoic acid by Rhodopseudomonas palustris under anaerobic conditions
Benzoic acid is first hydrogenated and finally converted t o pimelic acid by ring rupture (Dutton and Evans, 1969; Guyer and Hegeman, 1969). This route has been modified by Williams and Evans (1975), and Whittle et al. (1976), as discussed in Chapter 6.
COOH COOH + 3H2O benzoic acid dihydroxypimelic acid COOH Fig. 4
Degradation by soil micro-organisms of aromatic compounds in the presence of nitrate was first shown by Oshima (1965). This author stated that only a mixture of two micro-organisms, which he referred to as strains 5233 and 5207, was capable of degrading protocatechuic acid, phenylalanine, 4-hydroxybenzoic acid, benzoic acid or tyrosine anaerobically with nitrate as oxidizing agent. Strain 5233 seemed to be a Pseudomonas species, which could be replaced by other species of Pseudomonas. Strain 5207 was rod-shaped, non-motile and gram-negative. A mixture of these two organisms could not bring about the degradation of catechol, tryptophan or anthranilic acid. Free oxygen appeared to inhibit the degradation of protocatechuic acid, whereas a filtrate of the culture was found to accelerate it. In cell-free extracts only very slight degradation was obtained after the culture filtrate had been added, a small amount of beta-ketoadipic acid being detected. Addition of NADH2, NADPHo, NAD, NADP, FAD, ATP or ADP had no effect on the degradation.
1 Q
In an experiment with '°0-labelled nitrate, Oshima established a certain enrichment of cell material with the nitrate oxygen. On the basis of these experiments, he suggested that the metabolic pathway might be similar t o the aerobic route.
Taylor et al. (1970) isolated a Pseudomonas strain, which, by nitrate respiration, can grow anaerobically on each of the following acids:
benzoic acid, 3- and 4-hydroxybenzoic acid and protocatechuic acid and, after a lag of 9 to 11 days, also on hydrocinnamic acid or on 2-hydroxybenzoic
acid. No growth occurred — whether the conditions were aerobic or anaerobic — on phenol, catechol, benzyl alcohol, 6-hydroxynicotinic acid, cyclohexane carboxylate, cyclohex-3-ene carboxylate or a mixture of cis- and trans-isomers of 1,2-cyclohexane diol. These authors found that hardly any aerobic
oxygenases were present during the anaerobic degradation of aromatics. They were able to plot a growth curve for their Pseudomonas species on
4-hydroxybenzoic acid and nitrate; moreover,they established a stoichiometric relationship between CO2 and N2 production and the growth of this species on KNO3 and benzoic acid. They criticized the suggestions of Oshima (1965) about the aerobic degradation pathway. The reductive pathway mentioned by Dutton and Evans (1969) for photosynthetic breakdown was also
considered unlikely, seeing that 'such a mechanism raises the question of the source of reducing power'. They therefore proposed a degradation pathway for benzoic acid in which the compound is hydroxylated with water to trihydroxycyclohexane carboxylic acid, which in turn is metabolized to a dihydroxycyclohexanone compound by dehydrogenation. Subsequently, the
both '^C-ring-labelled and ^C-carboxyl-labelled benzoic acid under strictly denitrifying conditions.
Williams and Evans (1973) showed that Pseudomonas stutzeri can degrade benzoic acid under denitrifying conditions. They found by
'^C-labelling that trans-2-hydroxycyclohexane carboxylate is an intermediate. This is in agreement with the anaerobic pathway of benzoate in the presence of
Rhodopseudomonas palustris proposed by Dutton and Evans (1969). Williams
and Evans (1975) demonstrated the production of adipic acid in benzoic acid decomposition by a Moraxella species under nitrate-reducing conditions. Harder (1974) showed that 4-hydroxybenzoic acid could be degraded under denitrifying conditions by a mixed population. He isolated motile rods with one polar flagellum, identifying them as a species of Pseudomonas. The degradation data were felt to be disputable, however. Harder plausibly suggested that degradation is more effective in a mixed population.
A part of the work in this thesis on the degradation of phenol under anaerobic denitrifying conditions will be published (Bakker, 1977). Some data of this degradation process formed the subject of a patent application
(Bakker, 1972).
A few publications deal with degradation of aromatics during methane fermentation. Tarvin and Buswell (1934), and Buswell and Hatfield (1938) have already reported the breakdown of aromatics like phenol, salicyclic acid, o-phthalic acid, benzoic acid and cinnamic acid in an anaerobic digestion tank of one litre. Buswell et al. stated that fatty acids might act as the intermediate compounds. They found no degradation of lignin, benzene, toluene,
benzaldehyde, bromobenzene and aniline. Clark and Fina (1952) used mixed cultures and showed that benzoic acid can be decomposed to CO2 and CH4. They further proved that the CH4 was formed not only from CO2, but also from benzoic acid and, principally, from the carboxyl group of benzoic acid, which was confirmed by Fina and Fiskin (1960) using benzoic acid-1-^C and -7- C. Clark and Fina (1952) suggested a ring rupture mechanism that
differed from the aerobic one. Using ring-labelled ^^C-benzoic acid, Nottingham and Hungate (1969) likewise found that CH4 and CO2 were formed.
Van Velsen (1976) reported on the methanogenic fermentation of phenol and p-cresol in pig manure. Keith (1972) proposed heptanoate as intermediate in anaerobic benzoate fermentation, involving a carbon - carbon single bond rupture; Balba and Evans (1977), however, proposed adipate.
Some publications deal with degradation under sulphate-reducing conditions. Novell! and Zobell (1944) and Rosenfeld (1947) claimed the breakdown of long-chain aliphatic hydrocarbons such as hexadecane by
dehydrogenase system which is capable of dehydrogenating hexadecane in the presence of methylene blue. Such a dehydrogenation mechanism under aerobic conditions has been discussed extensively in the literature but was finally disproved by Gallo et al. (1973). Updegraff and Wren (1954) doubt the possibility of degradation of oil by Desulfovibrio, and also Baars (1927) was sceptical. Shelton and Hunter (1975) showed that aromatic components from effluent crude oil found in bottom sediments were not degraded under sulphate-reducing conditions. They demonstrated that slow degradation of partly oxidized compounds (e.g. aldehydes, ketons, carboxylic acids) took place. No other reports have been found in the literature on the degradation of
aromatic compounds under anaerobic sulphate-reducing conditions. Bacteria of the genus Lactobacillus are capable of transforming aromatic and/or other cyclic compounds. In Fig. 5 it is shown that
dehydrogenation, hydrogenation, dehydration and decarboxylation take place under anaerobic conditions and that finally catechol is formed. Various articles have been written about this reaction sequence, the diagram of Fig. 5 having been published by Whiting and Coggins (1971).
Several references are found in the literature on the capacity of micro-organisms in the rumen fluid to degrade aromatic or other cyclic compounds under anaerobic conditions. Simpson et al. (1969) and
Krishnamurty et al. (1970), as well as others, reported on the degradation of flavonoid compounds, like rutin, quercitrin and naringin, by a Butyrivibrio species. The degradation was shown to proceed until two aromatic molecules are produced which are not further metabolized. The degradation of
phloroglucinol, one of these aromatic molecules, was demonstrated by Simpson et al. (1969), and confirmed by Tsai and Jones (1975), who isolated gram-positive cocci. The authors identified one strain as Streptococcus bovis and another strain was brought under a genus 'Caprococcus'. Yokohama and Carlson (1974) brought about the dissimilation of tryptophan using ruminal micro-organisms in vitro. Dehority et al. (1958) reported on the metabolism by rumen micro-organisms of proline, which is subject to a reductive ring cleavage and deamination to form valeric acid. Hungate (1966) and Scott (1964) studied the Stickland reaction under anaerobic conditions with both aromatic and non-aromatic amino acids. It appeared that two amino acids yield a carboxylic acid, ammonia and carbon dioxide by a redox reaction.
Harary (1957) effected the degradation of nicotinic acid by
anaerobic bacteria isolated from soil and suggested that 6-hydroxynicotinic acid was an intermediate. Pastan et al. (1964) isolated a Clostridium species
HO. ^COOH ÓH Quinic acid HO COOH ÓH 3-Dehydroquinic acid
COOH COOH COOH
O'" \ ^ "OH HO
OH OH 3-Dehydroshikimic acid Enol form
; 0 0 H COOH • 2 H OH HO OH Protocatechuic acid COOH -H,0 OH Catechol "OH H O ' ' " \ . ^ „ „ ^ ^ O H ÖH ÓH Shikimicacid Dehydroshikimic acid
COOH
Fig. 5
Pathways of anaerobic metabolism of quinate and shikimate by Lactobacilli
extracts appeared capable of utilizing this intermediate in thepresence of pyruvate. Finally, nicotinic acid is degraded anaerobically to propionic acid, acetic acid and carbon dioxide. The presence of a large amount of Bi2-coenzyme was reported, but additional evidence on its significance was not obtained.
'COOH x ; : : ^ \ / C O O H • H j O -2H O ^ ^ N nicotinic acid H I COOH •*• C O O H - C H 2 - C H 2 - C - C O O H + N H T II ^ CH2 2 [ C 3 - compounds]
CH3COOH +CO2 + CH3CH2COOH
Fig. 6
Pathway of nicotinic acid degradation under anaerobic conditions
This involves hydration, hydrogenation, deamination, etc., resulting in formation of acetic acid propionic acid and CO2 (Tsai et al., 1966).
2.The growth of micro-organisms on aromatic
compounds in denitrifying conditions
SUMMARY
The breakdown of several aromatic compounds such as phenol by a mixed population of micro-organisms was demonstrated. The molar
consumption ratio of nitrate to aromatic compound was 3—5, from which it was concluded that nitrate was reduced dissimilatorily.
Isolation of pure strains from the mixed population was unsuccessful when the initial incubation of the agar plates was aerobic; anaerobic incubation yielded pure strains of gram-negative, slightly curved rods which were
monotrichously flagellated. These were not capable of growth on phenol as sole carbon source, but only on a phenol nutrient broth. Even in this case, however, the growth rate was only one tenth of that of the mixed population.
2.1. INTRODUCTION
Until now it was not known for certain if micro-organisms were capable of degrading phenol and other aromatic compounds under denitrifying
conditions. Oshima (1965) stated that the degradation of protocatechuic acid and 4-hydroxybenzoic acid was possible under anaerobic denitrifying
conditions. He also noticed that a single isolated strain was incapable of
growth, while a mixture of two strains could degrade the aromatics. One of the strains was identified as a Pseudomonas species.
While the present study was in progress, Taylor et al. (1970) reported experiments showing that anaerobic degradation of various aromatic compounds was possible, but stated that the Pseudomonas they had used was not capable of degrading phenol. Although the existence of an anaerobic degradation process in the absence of free oxygen had often been discussed in the
literature, there seemed to be a need for a conclusive answer on the matter. It was in view of this that the present research programme was initiated, to investigate whether microbes are capable of degrading an aromatic compound such as phenol as sole carbon source under denitrifying conditions in the
In this chapter, attention is given to the enrichment cultures and to the subsequent isolation of microbes capable of phenol degradation under
denitrifying conditions. Only a few aromatic substances are known to be degradable under anaerobic denitrifying conditions, viz. benzoic acid and several hydroxybenzoic acids (Taylor et al., 1970 and Williams and Evans,
1975). There are no reports on phenol or cresols, however, and these compounds were therefore investigated.
2.2. MATERIALS AND METHODS
2.2.1. Enrichment cultures
Enrichment cultures were obtained by inoculation of various liquid media contained in 100-ml or 1-litre stoppered, non-stirred bottles. The
bottles were incubated in the dark at 30 °C. Phenol and nitrate concentrations were measured twice a week, additional phenol and KNO3 being supplied in quantities as indicated. Because of production of N2 gas, liquid was forced out of the bottles; this loss was compensated by frequent supplies of fresh basal medium.
2.2.2. Media
Basal medium
Unless otherwise stated, experiments were conducted in a basal medium used by Woldendorp (1963), in a slightly modified form. The composition per litre distilled water was: MgS04.7 H2O, 0.4 g; K2HPO4,
1.0 g; NH4CI, 1.0 g; CaCl2.2 H2O, 0.05 g; MnS04.5 H2O, 3.0 mg;
FeS04.7 H2O, 10 mg (Fe weight); ZnS04.7 H2O, 0.25 mg; CUSO4.5 H2O, 0.25 mg; H3BO3, 0.25 mg; Na2Mo04.2 H2O, 0.05 mg; pH 7.0; 5 g KNO3 was added when required. The medium was sterilized for 15 min. at 120 °C, and phenol was added from a 25 mg ml '^ concentrated solution. Agar and other components were added as indicated in the descriptions of each experiment.
During the fourth isolation attempt 10 mg Fe as FeCl3.6 H2O was used.
Isolation media
Phenol-nutrient agar (PNA-1): nutrient broth (Oxoid), 13 g; KNO3, 5 g; agar (Difco), 12 g; distilled water, 1 I; phenol, 400 mg.
Phenol-nutrient agar (PNA-2); yeast extract (Difco), 1 g; Bacto-pepton (Difco), 2.5 g; beef extract (Difco), 0.5 g; KNO3, 5 g; agar (Difco), 12 g; basal medium, 1 I; phenol, 400 mg.
Phenol-nutrient agar (PNA-3): nutrient broth (Oxoid), 13 g; KNO3, 5 g; agar (Oxoid 3), 15 g; distilled water, 1 I; phenol, 400 mg.
Yeast-glucose-chalk agar (YGCA): yeast extract (Difco), 10 g; CaC03, Schaeffer type, 10 g; KNO3, 5 g; agar (Oxoid 3), 12 g; distilled water, 1 I; pH, 7.2; glucose, 10 g.
Phenol-yeast-glucose-chalk agar (PYGCA): as YGCA, but with 400 mg phenol added.
Phenol agar (PA): KNO3, 5 g; agar (Difco), 12 g; basal medium, 1 I; phenol, 500 mg.
Phenol-nutrient broth (PNB-1): nutrient broth (Oxoid), 13 g; KNO3, 5 g; basal medium, 1 I; phenol, 150 mg.
Phenol-nutrient broth (PNB-2): as PNB-1, but with 6.5 g nutrient broth (Oxoid).
Phenol-nutrient broth (PNB-3): as PNB-1, but with 4 g nutrient broth (Oxoid).
Phenol-basal medium (PB-1): basal medium, 1 I; KNO3, 5 g; phenol, 150 mg. The phenol was in all cases added from a concentrated solution (25 g \'') which had been stored at 4 °C and filtered through a millipore (0.22 fim) filter. Medium YGCA was sterilized for 15 min. before and after addition of glucose at 120 and 110 °C, respectively. All other media were sterilized before addition of phenol for 15 min. at 120 °C. When media were to be used in anaerobic experiments, they were stored immediately after sterilization in an atmosphere of nitrogen and carbon dioxide.
2.2.3. Assays
Assay of phenol
Phenol was assayed using a colour reaction with 4-aminoantipyrine, as described by Merck (undated brochure). Into a 100-ml volumetric flask, 1 ml sample, 1 ml 4-aminoantipyrine solution (6 g 1"^), 1 ml ammonia solution (15 ml 25 % ammonia in 85 ml water), and after thorough mixing, 1 ml K3Fe(CN)g solution (20 g \'') was introduced. The contents were brought up to 100 ml and then thoroughly mixed. The extinction of the red-coloured solution was measured at 470 nm in a glass cuvette with a path
length of 1 cm. As a blank, 4-aminoantipyrine and K3Fe(CN)g were added in reverse order, so that no red colour appeared; the extinction due to the cell mass of the samples was very low. All extinction measurements were carried out with a split-beam Pye-Unicam UV-Vis recording spectrophotometer of the type SP 1800, to which an automatic sample changer and recorder were connected.
Assay of nitrate ion
Nitrate was measured according to the sodium salicylate method described by Scheringa (1930).
Assay of nitrite ion
Nitrite was assayed according to the method described by Meerburg and Massink (1934), using the Griess-Romein reagent.
Assay of aromatic compounds other than phenol
Protocatechuic acid and 2-, 3-, and 4-hydroxybenzoic acid were determined by the Folin method described by Colowick and Kaplan (1957). L-tyrosine was assayed using the Folin-Ciocalteau reagent. Phenylalanine was measured by the method described by Colowick and Kaplan (1957). Benzoic acid and o-, m-, and p-cresol were assayed by measuring extinctions at 270 nm.
Assay of oxygen
Oxygen was measured in a 0.5 ml gas sample, which was separated on a molecular sieve (5 A) at 120 °C with argon as carrier gas. For detection a katharometer was used. The detection limit of this method is approximately 0.01 % oxygen.
2.2.4. Sterile and anaerobic Erienmeyer system
The anaerobic denitrification experiments were conducted in a sterilizable 500-ml Erienmeyer flask, as shown in Fig. 7. To obtain
anaerobic conditions, nitrogen which had been freed from contaminant oxygen by passage over a deoxo catalyst was admitted through the stopper. For this purpose a small amount of hydrogen had been admixed to the nitrogen supply. In the nitrogen thus obtained oxygen could not be detected (O2 < 0.01 %). The deoxo catalyst used was model D, Engelhard, Brussel. Sterile samples could be drawn without air being introduced. Evolved gases could escape
cottonwool nitrogen gas inlet liquid inlet
gas outlet
mercury lock
culture liquid
Fig. 7
Sterile and anaerobic Erienmeyer system
This Erienmeyer system was used for anaerobic experiments; with it, samples can be taken and liquids added anaerobically.
2.3. RESULTS
2.3.1. Experiments on enrichment and isolation of micro-organisms
Enrichment culture series 1, 2, 3 and 4
Thirty one-litre bottles were completely filled with liquid media consisting of various combinations of basal medium, yeast extract, a
glucose/glycerine mixture ( 1 : 1 w/w), phenol and potassium nitrate (Table 2). These media were inoculated (10 % v/v) with a mixture of garden soil, manure, loam, and soil from a phenol plant site, with or without added sludge from a phenol- and nitrate-degrading Pasveer ditch. After three days' incubation at 30 °C in the dark, changes were observed in the concentrations of phenol and nitrate. In all bottles phenol degradation had occurred, up to a maximum of 50 per cent, and nitrate reductions were all above 70 per cent. In bottles Nos. 7A — 16 B large quantities of nitrite had been produced, while in Nos. 17 — 26 virtually none was detected.
On day 8 phenol, KNO3 and compounds as indicated in Table 2 were added anew to those bottles in which phenol was no longer present. After 15 and 21 days of incubation, phenol concentrations were low in all cultures, the total percentages of phenol degraded ranging from 78 to 98 per cent (based on single observations). The average degradation rates varied from 4 to 19 ^mol 1"^ h" , depending on the phenol loading of the culture.
Table 2
Phenol degradation in first culture series
On days 0 and 8, yeast extract and a glucose and glycerine mixture were added to the
i
cultures, as indicated; 5 g I KNOo was added to all cultures.
Cultures 7A — 16B were inoculated with 100 g of a mixture of garden soil, loam, manure, and soil from a phenol plant site (MIX). Cultures 1 7 - 2 6 were inoculated with 100 g MIX to which 100 ml sludge from a phenol- and nitrate-degrading Pasveer ditch (SL) had been added. All the data are based on single values.
Table 2
Day O Day 3 Day 8 Day 10 Day 15 Day 15 Day 17 Day 21
Bottle Inoculation Yeast Glucose + Phenol Phenol Phenol Phenol Phenol Phenol Phenol Phenol % of Rate number material extract glycerine added cone. added cone. cone. added cone. cone. total * *
( m g l ' " ( m g l - l ) (mg r l ) ( m g l l ' (mg r ^ ) ( m g r l ) (mg r l ) ( m g r ^ l (mgV^) (mg l"!) * (^mol V''h^'') 7A 7B 8A 8B 9A 98 IDA 108 11A 11B 12A 12B 13A 13B 14A 148 15A 158 16A 168 17 18 19 20 21 22 23 MIX MIX MIX MIX MIX MIX MIX MIX MIX MIX MIX MIX MIX MIX MIX MIX MIX MIX MIX MIX MIX + SL MIX + SL MIX + SL MIX + SL MIX + SL MIX + SL MIX + SL 0 0 0 0 0 0 0 0 0 0 100 100 100 100 100 100
:oo
100 100 100 0 0 0 0 0 100 100 0 0 0 0 10 10 500 500 500 500 0 0 0 0 10 10 500 500 500 500 0 0 10 500 500 0 0 10 10 100 100 10 10 100 100 10 10 10 10 100 100 10 10 100 100 10 10 10 100 10 100 10 10 100 5 6 55 49 5 8 83 79 11 9 6 6 78 66 6 7 84 70 13 10 11 58 7 56 6 7 56 100 300 100 300 100 300 100 300 100 300 100 300 100 300 100 300 100 300 100 300 100 100 100 100 100 100 100 87 287 87 292 110 300 58 232 73 280 64 288 62 224 86 280 72 220 80 300 92 40 66 25 96 62 24 11 9 12 5 11 11 8 8 8 8 16 10 13 13 14 11 8 4 10 10 ND 15 ND ND 16 ND 18 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 300 300 300 300 300 300 300 107 480 97 460 102 490 39 470 67 480 111 430 43 435 33 420 40 440 57 320 310 310 240 290 310 290 270 10 85 8 20 7 88 10 36 10 23 21 52 14 23 13 32 14 20 14 160 49 54 35 32 67 19 20 95 89 97 98 97 89 97 96 95 97 90 93 95 97 94 96 95 98 93 98 88 89 91 94 84 95 4 15 6 19 4 15 6 18 4 17 4 16 6 19 4 17 6 19 4 17 8 9 8 10 7 8 10After a further 15 days of incubation phenol concentrations were increased, being brought up to 2000 mg I"' in some cultures, and the degradation rates measured. For the cultures in which the initial phenol concentration was 1000 mg r \ rates of up t o 6 5 A i m o i r ^ h"' were found, while rates for those with a concentration of 2000 mg 1'^ were significantly lower (see 5.3.3.).
From these results it could be stated that an enrichment culture for phenol degradation in an anaerobic denitrifying environment can start with 100 mg I ( — 1.1 mmol 1"^) phenol. Addition of a glucose/glycerine mixture (10 or 500 mg 1"^) or yeast extract (100 mg 1"^) gives no significant change in the degradation process. Also, the additional inoculation with sludge produces no significantly different behaviour of the culture.
In addition to these enrichment experiments in liquid cultures, mixtures of various soils and manure were supplemented with phenol and nitrate and incubated. It appeared that the average degradation rate after 21 days was much lower than that for the liquid cultures. It seems that higher dosage rates, of 100 mg 1'^ each time, allow for better adaptation of the microbial flora than low rates. As the liquid cultures showed a better degradation percentage and rate after 21 days, these soil cultures were abandoned.
In an experiment in which phenol and nitrate were added to basal medium without inoculation, no degradation was observed.
From these results and those of the preceding enrichment experiments it can be concluded that micro-organisms are responsible for the degradation process.
The liquid enrichment culture described was maintained for some weeks by fresh additions of phenol and nitrate as required. A second culture series was started in 50-ml bottles on a substrate consisting of basal medium, phenol and nitrate (PB-1) by adding a 10 % (v/v) inoculum from the first enrichment culture. The initial phenol concentrations were 100 or 300 mg 1'^ (1.1, 3.2 mmol I ). After two weeks the degradation in the second culture had become rapid, and a third series was started using a 10 % (v/v) inoculum from the second. The phenol concentration was brought up to 500 mg 1'^ (5.3 mmol I'"").
In the same way a fourth culture series was made in a 35-ml bottle, using a platinum loop, and degradation was proceding well after several weeks. With this culture an attempt was made to isolate individual micro-organisms. These were plated out on phenol agar (PA) and incubated aerobically at 30 °C.
These were tested in 20 groups of 4 mixed isolates in tubes containing PB-1. With the exception of one mixture, which was very slightly active, all proved to
be incapable of degrading phenol and nitrate anaerobically in culture tubes. Transfer of the slightly active mixture to a tube with fresh medium caused the activity to discontinue. Using the method of Oshima (1965), an attempt was made to achieve degradation with 40 pairs of 2 isolates using large inocula. It appeared, however, that nitrate reduction and phenol degradation were minimal, and so no further investigations were carried out on these 80 strains.
Enrichment culture series 5, 6 and 7
It is quite possible that the level of micro-organism enrichment in the preceding cultures was not sufficiently high for successful isolation. The bottled culture liquids of the second series were examined microscopically and a great variety of microbes found to be still present. A fifth enrichment culture was therefore prepared by platinum-loop inoculation into 100-ml bottles. After a week,a sixth enrichment culture was prepared by making a 1 % (v/v)
inoculation from the fifth, and a week later this procedure was repeated to obtain a seventh subculture. In this way it was hoped to enhance the survival chances of the phenol- and nitrate-degrading organisms at the cost of other species. The seventh culture was incubated for a week and an attempt was then made to isolate those organisms responsible for degradation by plating out on phenol-nutrient agar (PNA-2) at 30 °C. Although after a long period of incubation an active culture could successfully be started by transferring cell material from all the colonies on one agar plate, it was not possible to obtain a single active isolate in this way. This attempt was therefore also abandoned.
Third isolation attempt
The third enrichment culture series was still very active. After the two substrates phenol and nitrate had been completely consumed, 1000 mg T^ (10.6 mmol 1"^) phenol and 3 g 1"^ (30 mmol 1"^) KNO3 were added. As the growth rate was low, more cell material was obtained by adding to the cultures an amount of fresh basal medium equal to that initially used. From two of these cultures a number of strains were isolated on phenol-nutrient agar (PNA-1) which had been supplemented with 5 g 1"^ yeast extract. All isolated strains on this agar plate were incubated together and tested for phenol- and nitrate-degrading properties in an anaerobic Erienmeyer system (see 2.2.4. and Fig. 7) containing PNB-3 as medium. From the positive
Erienmeyer experiments new plates were inoculated, and the bacteria tested again in the same way. In this way isolates were obtained, and these were plated out. Finally, 65 strains were obtained, but after testing on PNB-3 only two
active strains remained: Nos. 19 and 31 (later, DSM Collection Nos. 988 and 989). The phenol-degrading capacity of these two isolates was low (approximately 6Mnnol r^ h ); they were gram-negative, motile short rods, slightly curved
under conditions of phenol degradation, and capable of reducing nitrate with phenol as sole electron donor. These strains were also capable of degrading phenol under aerobic conditions in the absence of nitrate.
Fourth isolation attempt
As described above, it appeared to be quite easy to obtain a mixed culture capable of degrading phenol under anaerobic conditions with nitrate as electron acceptor. The isolation of pure strains with the same degrading properties failed almost completely, however. In order to have a continuous, homogeneous source of active bacteria, a continuous culture was started, as will be described in 3.2.1.
After some time it was noticed that a spirillum-shaped organism was predominant in this phenol-degrading and nitrate-reducing culture (see Fig. 8).
The culture was plated out onto PNA-2 and incubated both
aerobically and anaerobically (in a glove box). After 8 days of incubation at 30 °C, the aerobic plates showed colonies of cocci, spore-formers, and motile curved rods. No spirilla were present. After 13 days the anaerobic plates had produced far more colonies than their aerobic parallels; most colonies
consisted of motile rods of various sizes. Attempts were made to isolate pure cultures from these colonies on agar plates with PNA-2, PNA-3 and PYGCA, and also on the same media without phenol. The plates were incubated aerobically and anaerobically. All cultures isolated from these plates were tested for phenol degradation in culture tubes containing 10 ml PB-1. All isolated bacteria capable of phenol degradation were plated out again anaerobically on PNA-3 and PYGCA at 20 °C in an atmosphere of nitrogen and carbon dioxide. The isolates were again tested for phenol degradation, the positive strains being subsequently plated out aerobically on PYGCA at 20 °C and tested anew.
The whole procedure, from the continuous culture to the last phenol degradation test, was then repeated, with the difference that the second and subsequent incubations were all aerobic.
In total, 17 strains were isolated, 16 from the first procedure and one from the second (see Table 3).
Table 3
Incubation conditions during the fourth isolation attempt
Initial agar plate Following agar plate Number of isolated bacteria* aerobic aerobic 0 aerobic anaerobic 2 anaerobic aerobic 10 anaerobic anaerobic 5 Initial incubation under anaerobic conditions gave the best results.
*capable of degrading phenol and reducing nitrate
As can be seen from Table 3 an initial incubation under anaerobic conditions gave the best results. Seven of these seventeen strains were selected for further examination. All were found to be capable of growth on phenol and nitrate in PB-1 under anaerobic conditions, phenol being present at a concentration of 150 mg I' . More than six months later, in the same medium and with 400 mg 1"^ phenol, growth was no longer observed, but under
anaerobic conditions in PNB-1 with 400 mg I ' ' phenol and 3 g I ' ' KNO3 the same seven strains were still active.
In a subsequent experiment, the bacteria were grown on both PYGCA and PNA-3. The strains grew about equally well in both media, and after 24 hours sufficient cell material had been obtained. This was suspended in Ringer's solution and 1-ml portions were diluted with PNB-1 in culture tubes until the extinction at 660 nm was 0.02 (9 ml of PNB-1 added). The tubes were then sealed under anaerobic conditions with rubber stoppers and incubated anaerobically under N2 and CO2 at 30 °C. Growth occurred first on the nutrient broth with nitrate as electron acceptor, at an average specific growth rate of 0.1 h"^. This period of growth lasted for 48 hours, and when the extinction was measured it was found to have increased only slightly. Subsequent growth was slower and involved the simultaneous degradation of phenol and nitrate; after a period varying from 2 to 7 days, all phenol had been degraded. Growth and other data are summarized in Tables 4 and 5; a typical growth curve is shown in Fig. 9.
Table 4
Isolation data for phenol-degrading and nitrate-reducing bacteria
DSM Collection No. 981 982 983 984 985 986 987 988 989 Incubation initial aerobic anaerobic anaerobic anaerobic anaerobic anaerobic anaerobic aerobic aerobic conditions subsequent anaerobic aerobic aerobic anaerobic aerobic aerobic aerobic aerobic aerobic Type of agar used after isolation PNA-2 PNA-3 PNA-2 PNA-3 YGCA PNA-3 PNA-2 PNA-1 PNA-1
All strains consisted of motile gram-negative, slightly curved rods. The cells of strains DSM Nos. 982 and 985 showed no flaggella; the other strains were monotrichously flagellated. The length of all cells lay between approximately 1.0 and 2.3 jum and the diameter between 0.4 and 0.9 jum.
Table 5
Growth characteristics of cultures on PNB-1 under anaerobic conditions
DSM Collection No. 981 982 983 984 985 986 987 988 989 mixed culture During Specific growth (h-1) 0.008 0.002 0.004 0.006 0.001 0.003 0.004 ND ND 0.11 1
phenol degradation period ; Ratio of rate ANOö/Aphenol (mol mol"' 2.9 1.7 2.7 2.0 2.0 4.0 3.0 ND ND ) 2.8-3.9 2 and 4.3 ' Average phenol degradation rate (Mmol 1-1 h-I) 35 35 30 25 10 30 30 6 6 _ 6 0 - 1 2 0 ^
All cultures were able to grow on PNB-1 under aerobic conditions with a high specific growth rate. Strains DSM Nos. 982 and 985 are the slowest, Nos. 981 and 984 are the fastest growers on phenol under anaerobic conditions.
1)
in continuous culture (see 4.3.4. and 5.3.1.) ' in stoppered bottles
^' PNB-2 as medium, with 5.3 mmol \'^ (500 mg \'^) phenol ND = not determined
It can be concluded (Tables 3 and 4) that the first incubation period should preferably be anaerobic and that the most suitable media are PNA-2 or PNA-3. After microscopic observations and plating-out procedures on agars, all strains seemed to be pure cultures, although the purity may be a matter for further experimentation. All observed bacteria appeared to be gram-negative, slightly curved, motile rods. All strains except DSM Nos. 982 and 985 were monotrichously flagellated. Electron-micrographs of several strains are shown in Fig. 10.
phenol (mmol I extinction at 660 nm 24 48 72 120 144 168 ^ — Time (hrs) 0.400 0.300 0.200 0.100 ^<_L0.OOO Fig. 9
Anaerobic growth of strain DSM No. 984 on PNB-1
The first part of the growth curve (from 0—24 hrs) seems to be related to growth on nutrient components accompanied by nitrate reduction. In the second part of the growth curve (after 96 hours) phenol degradation takes place, also accompanied by nitrate reduction.
981
983
987 984
Fig. 10
Electron micrograph of Strains DSM Nos. 981, 983, 984 and 987
The strains were grown on PNB-1 and shadowed with Pt.; 18,000x enlarged. (Batenburg, W. and Nieuwdorp, P.J. Techn. Univers. Delft)
From Table 5 it can be seen that strains DSM Nos. 981 and 984 showed the most rapid growth, which was accompanied by nitrate reduction and phenol degradation. The strains were grown on PNB-1. The specific growth rates of the isolates were lower than those for a mixed culture, but the AN03/Aphenol ratios were more or less the same. The low phenol degradation rates of strains DSM Nos, 988 and 989 (formerly Nos. 19 and 31) are also recorded in Table 5. Strains DSM Nos. 982 and 985 were the slowest growers, did not show flagella, and are probably different from the other strains,
2.3.2. Experiments on degradation of other aromatic compounds
A phenol-degrading and nitrate-reducing culture was transferred from one-litre to 200-ml stoppered bottles. Instead of phenol, other aromatic
compounds were added to these cultures at initial concentrations of 250 mg I' . When this amount had been consumed, another 250 mg I' was added. This procedure was repeated several times, with increasing quantities of aromatics being added; two additions of 500 mg l'^ were made, several of approximately 750 mg I , and eventually quantities of as much as approximately 1000 mg I" . As the aromatic compound and nitrate were consumed their concentrations were regularly measured and supplemented accordingly. Nitrate concentrations varied between 0 and 80 mmol T .
All the aromatic compounds tested showed similar degradation courses; as an example, that for 3-hydroxybenzoic acid is shown in Fig. 11. The
variation in nitrate concentration follows a similar path. From these results the average degradation rates were calculated, and, hence, the ratios of reduced nitrate to degraded aromatic compound. The results are given in Table 6. Because of loss of culture liquid from the stoppered bottles by gas production, the results of this experiment have an accuracy of only 20 per cent.
From Fig. 11 it can be concluded that the adaptation time for 3-hydroxybenzoic acid was less than one day. This was the case with all the tested substances except o- and m-cresol; with these two compounds, the low degradation rate made it impossible to measure the adaptation time.
After these experiments, the cultures received phenol as sole carbon source; after two to three weeks the phenol degradation rate returned to its original value, which suggests that adaptation to phenol degradation proceeds rather quickly, provided that the phenol concentration is not too high.
concentration (mmol
a
7- 6-«i /\ 7 2 1 n1
1
1
1
1
1 \ \ ', " 1 • 1 0 5 \ \1
I 1
1 1
1 \
.1
,1
1"
1, \
l l / i
1
10 15 20 2 1r r
" y r r 1
' 1 '
-4 44
1 1'
1 !
1
1 1
•1
11
1
1
11
; 11
1 tr
11
1
1
1 .1
1
1
11
1
1
i i
1
1
1
1 • 1 . 1 > 1 . 1 1 1 1 1 1 1 1 1 • 1 5 30 35 40 45 50 55 60 65 70 — Ti me (davs)
Fig. 11Degradation of 3-hydroxybenzoic acid in dissimilatory nitrate reduction by a mixed population
The concentrations of 3-hydroxybenzoic acid and nitrate were regularly measured and, when low, supplemented with quantities as indicated.
Table 6
Degradation of various aromatic compounds under denitrifying conditions by a mixed population in stoppered bottles
Compound benzoic acid 3-hydroxybenzoic acid 3,4-dihydroxybenzoic acid 4-hydroxybenzoic acid phenol o-cresol m-cresol p-cresol Average rate (/imol r 70 130 110 110 120 10 10 60 degradation 1h-1) Ratio of A N O J / A aromatic compound (mol mol'l) 5 4 3 5 4 ND ND ND
It was noticed that in the range of 30—80 mmol N O , I' the nitrate/aromatic ratio was not affected by the level of nitrate.
ND = not determined
2.3.3. Degradation of phenol in the presence of succinate
In an attempt to account for the difficulty experienced in maintaining cultures and starting new cultures, succinate was tested as an additional carbon source. Such a carbon source may possibly stimulate initial breakdown of phenol because of its favourable influence on the first hydrogenation step, discussed in Chapter 6. Succinate was added in four different concentrations to an only slowly phenol-degrading culture under denitrifying conditions in 250-ml bottles. The initial phenol concentration was 320 mg I'l (3.4 mmol \''), and KNO3 was present at a concentration of 5 g |-1 (50 mmol I'l). Succinate was added in amounts of 0, 1.1, 3.4 and 10.2 mmol \'^. Twelve cultures, three at each concentration, were tested. Degradation started in all cultures after a week. When phenol concentrations became low, supplements were made and succinate was likewise regularly added. The results after three weeks are summarized in Table 7. It is evident that phenol degradation was virtually
Table 7
Degradation of phenol* under denitrifying conditions in the presence of succinate
Succinate Average amount of Average amount of Phenol concentration phenol degraded phenol degraded in degradation
after 3 weeks % of amount added rate (mmol |-1) (mmol) (,^mol I'l h'l;
0 7.55 83 15 1.1 7.55 89 15 3.4 6.60 82 13 10.2 7.02 77 14
1
initial phenol concentration = 3.4 mmol I"
2.4. DISCUSSION AND CONCLUSIONS
In experiments with enrichment cultures inoculated with various soils, manure and sludge, it was clearly demonstrated that phenol can be degraded under denitrifying conditions.
The mixed population was also capable of degradation of several other aromatic compounds: benzoic acid, 3- and 4-hydroxybenzoic acid,
3,4-dihydroxybenzoic acid, p-cresol and phenol could all be degraded by a mixed population of micro-organisms in dissimilatory nitrate reduction
1 1
at a rate of 60—130 umol I h ; the rate for o- and m-cresol was considerably 1 1
lower, 10^lmo\ I h '. The ratio of degraded nitrate to degraded aromatic
•1
substance was on average between 3 and 5 mol mol" , proving that nitrate is used as a terminal electron acceptor, i.e. is dissimilatorily reduced.
It was suspected that succinate, as an additional carbon source, would stimulate initial breakdown of phenol owing to its favourable influence on the first hydrogenation step, discussed in Chapter 6. Results, however, showed that succinate has no influence at all on the breakdown of phenol under the
experimental conditions applied.
Further enrichment and maintenance of the mixed culture was only possible when phenol concentrations were kept low. Isolation under aerobic conditions of pure strains from the mixed population was extremely difficult. The isolated strains hardly grew at all on media with phenol as sole carbon and energy source and nitrate as electron acceptor. After several virtually unsuccessful isolation attempts it was decided to use for the experiments a
mixed population growing on phenol as sole carbon source in a continuous culture.
After some time spirillum-shaped organisms appeared in this culture. Isolation from this culture under anaerobic and, later, aerobic conditions was successful, yielding several isolated strains. Two of the most active strains were small, motile rods, monotrichously flagellated, gram-negative, slightly curved, and facultative anaerobic. They grew well on phenol-nutrient broth media with nitrate as final electron acceptor. The specific growth rates of these isolated strains were much lower than that of the mixed population. Anaerobic growth of the isolated strains on phenol as sole carbon source was also minimal.
These phenomena suggest that the micro-organisms are deficient in a factor essential for growth and that they can find this in nutrient broth or, even better, in the mixed population. The type of interrelationship is as yet, however, rather unclear, and must form the subject of further study.
3. Demonstration of the degradation of aromatic
compounds in anaerobic denitrifying conditions
SUMMARY
It was demonstrated that phenol and several other aromatic compounds can be degraded under strictly anaerobic conditions during dissimilatory nitrate reduction by a mixed population of micro-organisms. These compounds act as sole carbon and energy source in the degradation process, which experiments with labelled phenol showed to yield cell material and carbon dioxide. It was proved that the aromatic ring rupture mechanism for the anaerobic
process is different from that for the aerobic process, which involves dioxygenases and molecular oxygen. It was also shown that anaerobic degradation by the mixed population requires the presence of nitrate.
3.1. INTRODUCTION
In Chapter 2 it was shown that a mixed population of micro-organisms is capable of degrading several aromatic compounds in anaerobic conditions. In the literature, however, there is considerable discussion as to whether such a degradation process is possible or not. Stanier (1972) suggested the necessity of molecular oxygen for aromatic ring rupture, and thus denied the
possibility of anaerobic breakdown. Oshima (1965) suggested an anaerobic degradation route for aromatic compounds in the dark identical to the
aerobic one. The involvement of molecular oxygen under anaerobic conditions is however rather unlikely. The electrons from the electron donor are
transferred to the nitrogen atom in the nitrate ion, and the negatively-charged oxygen atoms in the nitrate ion combine with hydrogen ions to form water; it is generally accepted that no molecular oxygen is involved in this process. Nevertheless, in growth experiments using 1°0-labelled nitrate in addition to succinate, 4-hydroxybenzoate or protocatechuate as sole carbon source Oshima established a certain 1^0 enrichment of the cell mass. He found that growth on the aromatics gave 2—4 times more enrichment than that on succinate, and this led him to suggest that nitrate oxygen is involved in the
ring rupture process. The amount of '°0 in the cell mass fraction was only 0.01 % of the total quantity involved. The difference in 1^0 enrichment with succinate or aromatic compounds as substrate might be due to the operation of different catabolic routes leading to intermediates used for cell synthesis. For this reason the data presented by Oshima cannot be considered convincing proof of the involvement of nitrate oxygen in aromatic ring rupture.
In various other publications on the anaerobic decomposition of aromatics it was not always conclusively demonstrated that free oxygen was completely absent during the experiments, which means that the presence of a minute amount of molecular oxygen during the 'anaerobic' breakdown may have brought about a normal aerobic breakdown of the aromatic nucleus involving an oxygenase with a high oxygen affinity. After this essential ring rupture had taken place, it is indeed possible that further degradation proceeded with nitrate as terminal electron acceptor.
In this chapter the existence of a strictly anaerobic degradation process for aromatic compounds is indicated. Special measures were taken to prevent molecular oxygen entering the experimental system, especially as degradation rates and substrate concentrations were very low. The extent to which the nitrate ion influences the degradation process and the possible presence of nitrate reductase were investigated. So too was the existence of aerobic dioxygenases in the mixed population. The incorporation of I^C from a labelled substrate into cell material and CO2 was also examined.
3.2. MATERIALS AND METHODS
3.2.1. Maintenance of an active culture
Stoppered bottles
For this series of experiments the enrichment cultures described in Chapter 2 with the highest degrading activities were used, and inoculations made into stoppered bottles, which were stored in the dark at 30 °C. This mixed population of phenol-degrading and nitrate-reducing bacteria was maintained by adding supplements of 400 mg \'' (4.3 mmol I'l) phenol and 3000 mg r1 (29.7 mmol I'l) KNO3 twice a week; concentrations were also measured twice a week. To compensate for liquid forced out of the bottles by the production of nitrogen during decomposition, frequent additions of fresh basal medium were made. About once a month the cell material was
medium, phenol and KNO3, in order to reduce excessive KHCO3 salt concentrations caused by the repeated breakdown of KNO3 and phenol.
Resuspension was also applied to overcome the problem of interruptions in the degradation activities of the cultures. When more cellular material was needed, a new culture was obtained by making a 30—50 % v/v inoculation of an existing culture, an arrangement which proved quite satisfactory.
Continuous culture
In order to obtain a homogeneous culture of bacteria which could be used for several experiments, a continuous culture was started in a 1.5-litre stirred fermenter (see Fig. 12). The fermenter was placed in a water bath kept at 30 °C. The pH was regulated by titration with 1 M HNO3, the pH-values being controlled with an Ingold electrode. An anaerobic atmosphere was maintained by blowing nitrogen, containing 1 % CO2 and saturated with H2O, over the liquid at a rate of 20 I h ' l . The presence of CO2 was necessary for maintenance of a stable pH of 7.6, and its flow was therefore carefully regulated by a set of pressure control valves in order that the gas mixture
(N2 + CO2) leaving the mixing coil be of constant composition. The gas mixture was rendered oxygen-free by passage over a deoxo catalyst
(see 2.2.4.). The feed solution consisted of basal medium with a high
concentration of phenol (10—40 g I ' , pH = 7.65); the dosage rates varied up to a maximum of 44 mg |-1 h-1. The volume of liquid in the fermenter was kept constant by means of an overflow pipe.
3.2.2. Assay of enzymes and cell concentration
Nitrate reductase was assayed by the method described by Lowe and Evans (1964), using benzyl viologen as a redox indicator and sodium
dithionite as an electron donor.
Metapyrocatechase and pyrocatechase were assayed by the method of Gibson (1971), changes in extinction at 375 and 260 nm on addition of catechol being taken as a measure of activity. The cell-free extract was prepared by disintegrating washed cells in a phosphate buffer (0.03 mol I ' , pH 7.5) for 2 minutes in an MSE 100W Ultrasonic disintegrator.
The extinction at 660 nm of a 10 % dilution of cell material in water was used as a measure of the cell concentration. Extinction and cell dry weight values in ranges between 0.0—0.6 and 0—0.3 g T , respectively,were plotted on a graph. The straight line obtained had a gradient of 5.6, with an accuracy of about 16 %.
Motor cotton wool CO,
X
O
HNO,n---pH control Thermostatic bath cotton wool
A
® è>
Feed solution Wash Deoxo water catalyst flask — Effluent Fermenter Fig. 12Continuous Culture Equipment
This set-up was used for most of the continuous culture experiments. The feed solution contained 10—40 g \'' (0.1—0.4 mol \'') phenol and basal medium; pH = 7.6. The dosage
1 1 - 1 - 1
rate was varied up to a maximum of 44 mg r h"' (0.5 mmol I h ') phenol. The nitrate was added via a pH titrator as HNO3. The gases were allowed to escape with the effluent through an overflow pipe.
3.2.3. Culture vessel for experiments with ^^C-labelled phenol
In Fig. 13 the 50-ml vessel used for the ^^C experiments is shown. It features a mercury lock through which any gases produced can escape, and a side arm containing potassium hydroxide solution to collect the evolved C02-To ensure anaerobic conditions in the vessel, nitrogen which had been rendered oxygen-free by passage over a deoxo catalyst (see 2.2.4.) was flushed
through the side arm at the start of the experiment. A Hirsch cell was used to prove the anaerobicity of the gas mixture (detection limit: 1 ppm 02). A t the end of the experiment, sulphuric acid was introduced via the side arm into the main vessel, and the gaseous CO2 evolved from the culture liquid was collected in the potassium hydroxide; this solution was subsequently collected and analyzed.
cotton wool
mercury lock
alkali acid culture liquid
Fig. 13
Culture vessel for experiments with ^C-labelled phenol
In this vessel experiments can be carried out anaerobically. The CO2 produced is trapped in alkali. At the termination of the experiment, acid can be introduced to expel the
C02-3.2.4. Assays of ^ C components
Thin layer chromatography for amino acids
The cell material was centrifuged, washed twice, dried, and then
was then concentrated to 0.5 ml, and this solution used for unidimensional thin layer chromatography on silica gel (Merck HF 254) and for liquid scintillation counting. Two chromatograms were prepared, one with butanol, acetic acid and water ( 3 : 1 : 1 ) , and one with ethanol, ammonia and water (25 : 4 : 3) as solvent system. After the plates had been dried, the amino acid spots were developed with ninhydrin solution, and the phenol and tyrosine spots with diazotized p-nitroaniline solution. A spray of a-naphthol was used t o detect the presence of sugars.
Liquid scintillation counting
In the 1^C-experiments all counts were made using a Philips PW 4305 liquid scintillation counter. The scintillation liquid contained 300 ml methanol, 300 ml toluene, 3.6 g PPO, 0.045 g POPOP and 90 g naphthalene, and was stored in the dark. Counts were made by introducing a small amount of sample in aqueous solution directly into 8 ml of scintillation liquid in the scintillation vessel. For each type of sample the maximum volume that could be added without the liquid becoming cloudy was determined; all measurements could adequately be made with volumes below these maxima. By measuring the ratio of the integral to differential counts on a gauge curve, the count efficiency was estimated ('internal ratio method'). In this way a correction could be made for the quenching of the light emission caused by the |3-radiation of l ^ c (Parmentier and ten Haaf, 1969).
Autoradiography
After drying, both sprayed and unsprayed chromatograms were covered with X-ray film (Agfa Osray T4), and left in the dark for some time. The films were then developed and dried, and compared with the chromatograms.
3.3. RESULTS
3.3.1. Degradation of phenol in anaerobic denitrifying conditions
A one-litre stoppered bottle containing an active mixed population of phenol-degrading and nitrate-reducing micro-organisms was placed in an desiccator which was evacuated three times and refilled with nitrogen gas. This gas had been passed over a deoxo catalyst to remove all traces of oxygen
