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Excretion of volatile fatty acids by anoxic Mytilus edulis and Anodonta cygnea
Comp. Biochem. Physiol. Vol. 80B, No. 2, pp. 299-301, 1985
0305-0491/85 $3.00 + 0.00 © 1985 Pergamon Press Ltd
Printed in Great Britain
EXCRETION OF VOLATILE FATTY ACIDS BY ANOXIC M Y T I L U S EDULIS AND A N O D O N T A CYGNEA GUIDO VAN DEN THILLART a n d INEKE DE VRIES State University of Leiden, Gorlaeus Laboratories, Department of Animal Physiology, P.O. Box 9502, 2300 RA Leiden, The Netherlands (Tel: 071-148-333)
(Received 25 April 1984)
Abstract--1. A fast and reproducible extraction method for volatile fatty acids is described. 2. During anoxia, excretion of acetic, propionic, butyric, iso-butyric, valeric and iso-valeric acid was measured. The excretion rates of acetic and propionic acid were about 100 times higher than those of the other volatile fatty acids. 3. The anoxia survival time for Mytilus edulis was 5 days (10°C) and for Anodonta cygnea 22 days (16°C). 4. Aspects of diffusional efflux are discussed and estimates of the total acid production are made. 5. The ratio of propionate vs acetate production is not constant and comes close to 1: 1, which indicates that glycogen is not the only carbon source for acetate.
INTRODUCTION Volatile fatty acids are p r o d u c e d u n d e r anoxic conditions by m a n y invertebrates, such as bivalves, including Mytilus edulis a n d Anodonta cygnea ( K l u y t m a n s a n d Zandee, 1983; Giide a n d Wilps, 1975) a n d worms, like Arenicola marina (Surholt, 1977), Tubifex (Seuss et al., 1983) a n d Nereis diversicolor (Schrttler, 1978). W h e n placed in water, these animals a p p e a r to excrete volatile fatty acids, p r o b a b l y by way of diffusion, since these c o m p o u n d s have a low mol. wt and have a m p h i p a t i c properties. So, the p a t t e r n s a n d evolution o f volatile fatty acids in the s u r r o u n d i n g water m a y represent the status o f the animals' m e t a b olism, which was surveyed in a few experiments with Mytilus edulis a n d Anodonta cygnea. Volatile fatty acids are usually extracted by a biphasic extraction system, followed by steam-distillation of the water m e t h a n o l layer ( K l u y t m a n s et al., 1975; Seuss et al., 1983). A faster m e t h o d was developed a n d tested for reproducibility a n d extraction efficiency.
MATERIALS AND METHODS
Conditioning Mytilus edulis mussels were purchased at the local market and kept at 5°C for several weeks in artificial seawater prior to the experiments. Anodonta cygnea was taken from the cooling-water pond of our laboratory and kept at 16°C in mud and water from the same pond. Experiments were carried out in the winter of 1983/1984 with apparently healthy animals. To obtain anoxic conditions, mussels were placed in erlenmeyer flasks filled with water; the flasks then were closed with perforated stoppers fitted with glass tubes and flushed with N 2 gas for 30 min. The flasks were closed by 3-way valves connected to the glass pipes by butyl-rubber (air-tight) tubing. For short experiments, water was forced out of the flasks by N 2 pressure. The first 10ml was discarded to avoid contamination by dead space water (3 ml). Long term experiments were carried out with two 299
interconnected flasks, the second one without mussels, in order to keep a constant volume in the animal chamber. By the use of precise syringes throughout the experiments, volume decrease or dilution was exactly known and corrections made correspondingly.
Extraction and analysis of volatile fatty acids To a 15 ml tube with Teflon lined screw cap 5 ml water sample, 0.1 ml internal standard (46 mM valeric acid), 2 ml diethyl ether and 1.5 ml 1 NHCI were added. Contents were mixed and spun for 10min at 1000g. From the clear ether layer 1-10pl was injected on a 2ram (i.d.) SP-1200 GLC column (Supelco). The column conditions were: carrier gas flow rate 10 ml N 2 min; temperature: initial 5 min at 9OJC, raised 5°C/min, final 140°C for 9 min. Analyses were carried out with a Packard 428 gas-chromatograph with FID detection and a Packard 603 integrator. Identification was made by comparison with Supelco standards. The retention times relative to valeric acid appeared to reproduce within 1%. Peak separation of all volatile fatty acids was complete with separation factors > 4. Extraction efficiencies were checked over a concentration range from 10 to 0.1 mM, with acetic, propionic, butyric, valeric and caproic acids and found to be over 90% over the whole range. To compensate for small changes in extraction efliciencies, mainly due to evaporation of diethyl ether, valeric acid was added to each water sample as internal standard. Extractions with CHCI3 or CS2 were tested in preliminary experiments, but did not give a relationship between peak area on chromatograms and the concentration of fatty acids. It was also found that water sample and standard solutions should have a pH > 7.5 to ensure reproducible GLC signals.
RESULTS AND DISCUSSION
Survival In a series o f experiments at 10°C with 5-10 mussels per flask, the m e d i a n lethal time (LTs0) for Mytilus edulis was f o u n d to be 5.0 days, from 50 animals, 90% dying between 3.5 a n d 7.0 days. As a criterion for d e a t h the shock sensitivity was taken:
300
G U I D O VAN DEN THILLART a n d INEKE DE VRIES
~C2
04
f
t
m
I I I I
_
l
02
OI
/
/,
tt
o
/
!
/
¢
io =L
0
2
4
6
o
g
4 Days
6
t
0
I I
I
Aie5
.......;I.[/p ic4 2
4
6
Fig. 1. Volatile fatty acid excretion by anoxic Mytilus edulis at 10'C. Three experiments with 5 mussels each are indicated by panels I, II and III; total wet wt was respectively 17.6, 19.4 and 18.5g (O: propionate, ~: acetate, O: butyrate, []: iso-butyrate, A: iso-valerate). Dashed lines indicate the occurrence of one or more dead animals. during anoxia, all mussels have their shells opened ajar and close them after a modest blow against the container until a certain moment which is taken as the lethal time. Anodonta cygnea has a much higher anoxia resistance. Although not extensively studied, 4 specimens survived 22 days of anoxia and died within 3 days thereafter.
Anodonta was much higher than by Mytilus, which was mainly due to the much higher anoxia resistance of Anodonta. The initial rates when expressed per gram wet wt, are rather similar to those of Mytilus. c3 5
I
Cz
Volatile fatty acid excretion The excretion of volatile fatty acids by anoxic
Mytilus edulis is presented in Fig. 1. Of three experiments (out of 10) the accumulation in the surrounding water of acetic and propionic acid is given in the upper panels and of butyric, iso-butyric and iso-valeric acid is given in the lower panels. Other fatty acids like formic, valeric and caproic acids, were not found. In all experiments with Mytilus acetic acid was absent until the fourth day of anoxia. Very high levels of acetic acid were found thereafter, especially when one or more dead animals were present in the flasks. Propionic acid was always found prior to acetic acid. A significant excretion of butyric, isobutyric and iso-valeric acid was apparent, starting with the second day of anoxia. The concentrations of these acids though, were about 100 times lower than of propionic acid. In order to test for microorganismal infections, propionic acid was added at the end of one experiment during which 3 out of 5 mussels died. The initial concentrations remained rather constant, although a slight decrease of all volatile fatty acids was observed, probably due to diffusional loss. Similar results were obtained with diluted standards in the same experimental set-up. The volatile fatty acid excretion by anoxic Anodonta cygnea is presented in Fig. 2. The total excretion by
I
A~AtC5
E
~'
Days
~k.~k.-..A-~k' ' ' - ' A ' A ~ A -
O~:~r---~
"°~
io
~'o
2'0
iC zo~
o
Days
Fig. 2. Volatile fatty acid excretion by anoxic Anodoma
cygnea at 16°C. Two experiments with 2 mussels each are indicated by panels [ and If; total wet wt was respectiveb 32.0 and 30.7 g (O: propionate, £x: acetate, O: butyrate, D: iso-butyrate, A: iso-valerate).
Anoxic mussels produce acetic and propionic acids The excretion rates of propionic acid over the first 4 days were for Mytilus edulis 3.3/~mol/g/day and for Anodonta cygnea 2.5 #tool/g/day. The propionic acid excretion rate for Anodonta, however, increased after about 6 days to 10 #tool/g/day. Similarly to Mytilus, Anodonta excretes butyric, iso-butyric and iso-valeric acid at a rate of about 100 times lower than propionic acid. Volatile fatty acids in mussel tissue Since volatile fatty acids are amphipatic and soluble in water, they will diffuse readily through all body compartments into the surrounding water. This does not imply that concentrations in water and tissues are equal, which holds for the free acid only. The total concentration difference will depend on the pH of separate compartments and can be derived from the well known buffer equation: pH=pK+log
~
.
(1)
When pHw and pHt are the pH of water and tissue respectively, we find the gradient from the pH difference: pHw - pHt = log
x HAw] ~- log ~-~ . (2)
Since water pH > 7.5 and p K -~ 4.8, the free acid in the water phase will be negligible. As for the tissue, pH may fall to lower levels (pH ~ 6.5), due to production of organic acids (de Zwaan and Wijsman, 1976), which will raise the free acid concentration and so accelerate diffusion. Since at low pH, the dissociated acid has a much lower concentration and the free acids tend to equilibrate between tissue and water compartment, the total tissue concentration may then fall even below the water concentration. Due to a constant production however, tissue concentration will remain at higher concentrations, mainly determined by total exchange area, diffusion and production rates. Thus it can be expected that the tissue volatile fatty acid concentrations will be of the same order of magnitude as the water concentration. Since in these experiments the biomass/water ratio was kept at 1/10 or lower, we may assume that the observed excretion rates are almost equal to the actual production rates of the animals.
301
Metabolic pathways In this study it is clearly demonstrated that acetic acid is excreted at almost the same rate as propionic acid, especially by anoxic Anodonta. Over the first 3 days no acetic acid was excreted by both species, which mdxcates that either pyruvate kinase was inhibited, or that pyruvate was converted to propionate via Krebs' cycle activity (de Zwaan, 1977). Since acetate most probably originates from pyruvate, we assume that regulation at the so-called "PEP-Branchpoint" changes after a few days, such that a significant carbon flux goes via the pyruvate kinase pathways. Propionic formation via the PEP-CK pathway is not in redox-balance; for each propionate molecule one extra electron-pair is needed• When the substrate for acetate production is assumed to be glycogen, then for each acetate molecule 2 electronpairs are formed. So propionate and acetate production would be in redox-balance at a ratio of 2: 1. This does not corroborate the observations (Figs 1, 2), so there may be an additional source for pyruvate other than glycogen, which would enable a higher production ratio• •
,,
,
REFERENCES G~ide G. and Wilps H. (1975) Glycogen degradation and end products of anaerobic metabolism in the freshwater bivalve Anadonta cygnea. J. comp. Physiol. 104, 79-85. Kluytmans J. H., Veenhof P. R. and Zwaan A. de (1975) Anaerobic production of volatile fatty acids in the sea mussel Mytilus edulis L. J. comp. Physiol. 104, 71-78. Kluytmans J. H. and Zandee D. I. (1983) Comparative study of the formation and excretion of anaerobic penetration products in bivalves and gastropods. Comp. Biochem. Physiol. 7511, 729-732. Schtttler U. (1978) Investigations of the anaerobic metabolism of the polychaete worm Nereis diversicolor M. J. Comp. Physiol. 125, 185-189. Seuss J., Hipp E. and Hoffmann K. H. (1983) Oxygen consumption, glycogen content and the accumulation of metabolites in Tubifex during aerobic-anaerobic shift and under progressing anoxia. Comp. Biochem. Physiol. 75A, 557-562. Surholt B. (1977) Production of volatile fatty acids in the anaerobic carbohydrate catabolism of Arenicola marina. Comp. Biochem. Physiol. 58B, 147-150. Zwaan A. de (1977) Anaerobic energy metabolism in bivalve molluscs. Oceanogr. Mar. Biol. A. Rev. 15, 103-187. Zwaan A. de and Wijsman T. C. (1976) Anaerobic metabolism in Bivalvia (Mollusca). Characteristics of anaerobic metabolism. Comp. Biochem. Physiol. 54B, 313-324.
0305-0491/85 $3.00 + 0.00 © 1985 Pergamon Press Ltd
Printed in Great Britain
EXCRETION OF VOLATILE FATTY ACIDS BY ANOXIC M Y T I L U S EDULIS AND A N O D O N T A CYGNEA GUIDO VAN DEN THILLART a n d INEKE DE VRIES State University of Leiden, Gorlaeus Laboratories, Department of Animal Physiology, P.O. Box 9502, 2300 RA Leiden, The Netherlands (Tel: 071-148-333)
(Received 25 April 1984)
Abstract--1. A fast and reproducible extraction method for volatile fatty acids is described. 2. During anoxia, excretion of acetic, propionic, butyric, iso-butyric, valeric and iso-valeric acid was measured. The excretion rates of acetic and propionic acid were about 100 times higher than those of the other volatile fatty acids. 3. The anoxia survival time for Mytilus edulis was 5 days (10°C) and for Anodonta cygnea 22 days (16°C). 4. Aspects of diffusional efflux are discussed and estimates of the total acid production are made. 5. The ratio of propionate vs acetate production is not constant and comes close to 1: 1, which indicates that glycogen is not the only carbon source for acetate.
INTRODUCTION Volatile fatty acids are p r o d u c e d u n d e r anoxic conditions by m a n y invertebrates, such as bivalves, including Mytilus edulis a n d Anodonta cygnea ( K l u y t m a n s a n d Zandee, 1983; Giide a n d Wilps, 1975) a n d worms, like Arenicola marina (Surholt, 1977), Tubifex (Seuss et al., 1983) a n d Nereis diversicolor (Schrttler, 1978). W h e n placed in water, these animals a p p e a r to excrete volatile fatty acids, p r o b a b l y by way of diffusion, since these c o m p o u n d s have a low mol. wt and have a m p h i p a t i c properties. So, the p a t t e r n s a n d evolution o f volatile fatty acids in the s u r r o u n d i n g water m a y represent the status o f the animals' m e t a b olism, which was surveyed in a few experiments with Mytilus edulis a n d Anodonta cygnea. Volatile fatty acids are usually extracted by a biphasic extraction system, followed by steam-distillation of the water m e t h a n o l layer ( K l u y t m a n s et al., 1975; Seuss et al., 1983). A faster m e t h o d was developed a n d tested for reproducibility a n d extraction efficiency.
MATERIALS AND METHODS
Conditioning Mytilus edulis mussels were purchased at the local market and kept at 5°C for several weeks in artificial seawater prior to the experiments. Anodonta cygnea was taken from the cooling-water pond of our laboratory and kept at 16°C in mud and water from the same pond. Experiments were carried out in the winter of 1983/1984 with apparently healthy animals. To obtain anoxic conditions, mussels were placed in erlenmeyer flasks filled with water; the flasks then were closed with perforated stoppers fitted with glass tubes and flushed with N 2 gas for 30 min. The flasks were closed by 3-way valves connected to the glass pipes by butyl-rubber (air-tight) tubing. For short experiments, water was forced out of the flasks by N 2 pressure. The first 10ml was discarded to avoid contamination by dead space water (3 ml). Long term experiments were carried out with two 299
interconnected flasks, the second one without mussels, in order to keep a constant volume in the animal chamber. By the use of precise syringes throughout the experiments, volume decrease or dilution was exactly known and corrections made correspondingly.
Extraction and analysis of volatile fatty acids To a 15 ml tube with Teflon lined screw cap 5 ml water sample, 0.1 ml internal standard (46 mM valeric acid), 2 ml diethyl ether and 1.5 ml 1 NHCI were added. Contents were mixed and spun for 10min at 1000g. From the clear ether layer 1-10pl was injected on a 2ram (i.d.) SP-1200 GLC column (Supelco). The column conditions were: carrier gas flow rate 10 ml N 2 min; temperature: initial 5 min at 9OJC, raised 5°C/min, final 140°C for 9 min. Analyses were carried out with a Packard 428 gas-chromatograph with FID detection and a Packard 603 integrator. Identification was made by comparison with Supelco standards. The retention times relative to valeric acid appeared to reproduce within 1%. Peak separation of all volatile fatty acids was complete with separation factors > 4. Extraction efficiencies were checked over a concentration range from 10 to 0.1 mM, with acetic, propionic, butyric, valeric and caproic acids and found to be over 90% over the whole range. To compensate for small changes in extraction efliciencies, mainly due to evaporation of diethyl ether, valeric acid was added to each water sample as internal standard. Extractions with CHCI3 or CS2 were tested in preliminary experiments, but did not give a relationship between peak area on chromatograms and the concentration of fatty acids. It was also found that water sample and standard solutions should have a pH > 7.5 to ensure reproducible GLC signals.
RESULTS AND DISCUSSION
Survival In a series o f experiments at 10°C with 5-10 mussels per flask, the m e d i a n lethal time (LTs0) for Mytilus edulis was f o u n d to be 5.0 days, from 50 animals, 90% dying between 3.5 a n d 7.0 days. As a criterion for d e a t h the shock sensitivity was taken:
300
G U I D O VAN DEN THILLART a n d INEKE DE VRIES
~C2
04
f
t
m
I I I I
_
l
02
OI
/
/,
tt
o
/
!
/
¢
io =L
0
2
4
6
o
g
4 Days
6
t
0
I I
I
Aie5
.......;I.[/p ic4 2
4
6
Fig. 1. Volatile fatty acid excretion by anoxic Mytilus edulis at 10'C. Three experiments with 5 mussels each are indicated by panels I, II and III; total wet wt was respectively 17.6, 19.4 and 18.5g (O: propionate, ~: acetate, O: butyrate, []: iso-butyrate, A: iso-valerate). Dashed lines indicate the occurrence of one or more dead animals. during anoxia, all mussels have their shells opened ajar and close them after a modest blow against the container until a certain moment which is taken as the lethal time. Anodonta cygnea has a much higher anoxia resistance. Although not extensively studied, 4 specimens survived 22 days of anoxia and died within 3 days thereafter.
Anodonta was much higher than by Mytilus, which was mainly due to the much higher anoxia resistance of Anodonta. The initial rates when expressed per gram wet wt, are rather similar to those of Mytilus. c3 5
I
Cz
Volatile fatty acid excretion The excretion of volatile fatty acids by anoxic
Mytilus edulis is presented in Fig. 1. Of three experiments (out of 10) the accumulation in the surrounding water of acetic and propionic acid is given in the upper panels and of butyric, iso-butyric and iso-valeric acid is given in the lower panels. Other fatty acids like formic, valeric and caproic acids, were not found. In all experiments with Mytilus acetic acid was absent until the fourth day of anoxia. Very high levels of acetic acid were found thereafter, especially when one or more dead animals were present in the flasks. Propionic acid was always found prior to acetic acid. A significant excretion of butyric, isobutyric and iso-valeric acid was apparent, starting with the second day of anoxia. The concentrations of these acids though, were about 100 times lower than of propionic acid. In order to test for microorganismal infections, propionic acid was added at the end of one experiment during which 3 out of 5 mussels died. The initial concentrations remained rather constant, although a slight decrease of all volatile fatty acids was observed, probably due to diffusional loss. Similar results were obtained with diluted standards in the same experimental set-up. The volatile fatty acid excretion by anoxic Anodonta cygnea is presented in Fig. 2. The total excretion by
I
A~AtC5
E
~'
Days
~k.~k.-..A-~k' ' ' - ' A ' A ~ A -
O~:~r---~
"°~
io
~'o
2'0
iC zo~
o
Days
Fig. 2. Volatile fatty acid excretion by anoxic Anodoma
cygnea at 16°C. Two experiments with 2 mussels each are indicated by panels [ and If; total wet wt was respectiveb 32.0 and 30.7 g (O: propionate, £x: acetate, O: butyrate, D: iso-butyrate, A: iso-valerate).
Anoxic mussels produce acetic and propionic acids The excretion rates of propionic acid over the first 4 days were for Mytilus edulis 3.3/~mol/g/day and for Anodonta cygnea 2.5 #tool/g/day. The propionic acid excretion rate for Anodonta, however, increased after about 6 days to 10 #tool/g/day. Similarly to Mytilus, Anodonta excretes butyric, iso-butyric and iso-valeric acid at a rate of about 100 times lower than propionic acid. Volatile fatty acids in mussel tissue Since volatile fatty acids are amphipatic and soluble in water, they will diffuse readily through all body compartments into the surrounding water. This does not imply that concentrations in water and tissues are equal, which holds for the free acid only. The total concentration difference will depend on the pH of separate compartments and can be derived from the well known buffer equation: pH=pK+log
~
.
(1)
When pHw and pHt are the pH of water and tissue respectively, we find the gradient from the pH difference: pHw - pHt = log
x HAw] ~- log ~-~ . (2)
Since water pH > 7.5 and p K -~ 4.8, the free acid in the water phase will be negligible. As for the tissue, pH may fall to lower levels (pH ~ 6.5), due to production of organic acids (de Zwaan and Wijsman, 1976), which will raise the free acid concentration and so accelerate diffusion. Since at low pH, the dissociated acid has a much lower concentration and the free acids tend to equilibrate between tissue and water compartment, the total tissue concentration may then fall even below the water concentration. Due to a constant production however, tissue concentration will remain at higher concentrations, mainly determined by total exchange area, diffusion and production rates. Thus it can be expected that the tissue volatile fatty acid concentrations will be of the same order of magnitude as the water concentration. Since in these experiments the biomass/water ratio was kept at 1/10 or lower, we may assume that the observed excretion rates are almost equal to the actual production rates of the animals.
301
Metabolic pathways In this study it is clearly demonstrated that acetic acid is excreted at almost the same rate as propionic acid, especially by anoxic Anodonta. Over the first 3 days no acetic acid was excreted by both species, which mdxcates that either pyruvate kinase was inhibited, or that pyruvate was converted to propionate via Krebs' cycle activity (de Zwaan, 1977). Since acetate most probably originates from pyruvate, we assume that regulation at the so-called "PEP-Branchpoint" changes after a few days, such that a significant carbon flux goes via the pyruvate kinase pathways. Propionic formation via the PEP-CK pathway is not in redox-balance; for each propionate molecule one extra electron-pair is needed• When the substrate for acetate production is assumed to be glycogen, then for each acetate molecule 2 electronpairs are formed. So propionate and acetate production would be in redox-balance at a ratio of 2: 1. This does not corroborate the observations (Figs 1, 2), so there may be an additional source for pyruvate other than glycogen, which would enable a higher production ratio• •
,,
,
REFERENCES G~ide G. and Wilps H. (1975) Glycogen degradation and end products of anaerobic metabolism in the freshwater bivalve Anadonta cygnea. J. comp. Physiol. 104, 79-85. Kluytmans J. H., Veenhof P. R. and Zwaan A. de (1975) Anaerobic production of volatile fatty acids in the sea mussel Mytilus edulis L. J. comp. Physiol. 104, 71-78. Kluytmans J. H. and Zandee D. I. (1983) Comparative study of the formation and excretion of anaerobic penetration products in bivalves and gastropods. Comp. Biochem. Physiol. 7511, 729-732. Schtttler U. (1978) Investigations of the anaerobic metabolism of the polychaete worm Nereis diversicolor M. J. Comp. Physiol. 125, 185-189. Seuss J., Hipp E. and Hoffmann K. H. (1983) Oxygen consumption, glycogen content and the accumulation of metabolites in Tubifex during aerobic-anaerobic shift and under progressing anoxia. Comp. Biochem. Physiol. 75A, 557-562. Surholt B. (1977) Production of volatile fatty acids in the anaerobic carbohydrate catabolism of Arenicola marina. Comp. Biochem. Physiol. 58B, 147-150. Zwaan A. de (1977) Anaerobic energy metabolism in bivalve molluscs. Oceanogr. Mar. Biol. A. Rev. 15, 103-187. Zwaan A. de and Wijsman T. C. (1976) Anaerobic metabolism in Bivalvia (Mollusca). Characteristics of anaerobic metabolism. Comp. Biochem. Physiol. 54B, 313-324.