A method for the experimental determination of light absorption by aquatic heterotrophic bacteria

Transcription

1 Journal of Plankton Research Vol.20 no.4 pp , 1998 A method for the experimental determination of light absorption by aquatic heterotrophic bacteria Giovanni M.Ferrari and Stelvio Tassan. Space Applications Institute, CEQ Joint Research Center, Ispra Site, Ispra (VA), Italy Abstract. A method has been set up for the experimental determination of the volume coefficient for light absorption in vivo by aquatic heterotrophic bacteria. The application described here is the absorption measurement of the bacterial fraction that passes through the commonly used GF/F filter and remains unaccounted for. The experimental samples were prepared by successive water nitrations through GF/F and 0.22 urn Millipore membranes. Light-transmission and light-reflection measurements of the filter-retained samples were performed using a dual-beam spectrophotometer equipped with an integrating sphere attachment. Sample absorption was derived from the data by a procedure that corrects for the contamination of the results due to the high degree of light scattering by the bacteria. The bacterial absorption was discriminated from fine detritus absorption by bleaching the bacterial respiratory pigments using a K^Og solution. The absorption amplification caused by multiple scattering in the filter was corrected for by an expression that was obtained experimentally. A test of the method, including error analysis, was performed on samples collected in both marine and inland waters. The relative contributions to light absorption by heterotrophic bacteria and various types of paniculate matter were also measured for a typical situation. Combining the measured volume absorption coefficients with backscattering coefficients computed by Mie theory yields a set of input data to multicomponent optical models that is needed to assess the contribution of these heterotrophic bacteria to the radiative transfer process. Introduction Accurate water optical models are an essential support to the interpretation of remote sensing data of sea colour (Prieur and Sathyendranath, 1981; Gordon and Morel, 1983). These models require as basic input data the absorption and backscattering coefficients of the optically relevant substances suspended and dissolved in the water body considered (Sathyendranath et al., 1989). The absorption coefficient of aquatic particulate matter is currently inferred from light-transmittance measurements on particle samples retained on GF/F glass fibre filters (Mitchell and Kiefer, 1988). The relative contribution of the absorption by particles that pass through the GF/F filter (0.7 um retention efficiency) may be important in oligotrophic water, where the concentration of heterotrophic bacteria relative to phytoplankton is higher (Stramski and Kiefer, 1991). In these situations, it would be an advantage to extend the water optical model so as to include specifically the contribution of the bacteria (consideration of picoplankton passing through the GF/F filter may also occasionally be required). A convenient filter to define the lowest diameter of the small particle fraction is the 0.22 urn Millipore cellulose membrane. Heterotrophic bacteria, with sizes peaking in the um range and numerical abundance in the range of 10 n cells m~ 3, may form a considerable part of the particulate organic carbon present in oligotrophic waters (Cho and Azam, 1990; Ulloa et al., 1992). The role of heterotrophic bacteria in radiative transfer in the ocean has received attention only in recent years. Most of the work has Oxford University Press 757

2 G.M.FerTari and S.Tassan been carried out on high-concentration laboratory cultures, and has demonstrated the very large light scattering by these bacteria (Morel and Ahn, 1991; Ulloa et al., 1992). Although being lower than scattering, the light absorption of the heterotrophic bacteria can yield an appreciable contribution to the global absorption of oligotrophic waters in the blue region of the spectrum (Stramski and Kiefer, 1990). Cytochromes are the dominant light absorbers in heterotrophic bacteria (Stanier et al., 1988), particularly cytochrome c, which exhibits a typical 415 nm Soret absorption peak (Fruton and Simmonds, 1961). Its optical properties are influenced by the fact that the haematoporphyrin has two centres of asymmetry at the periphery of the cyclic structure (Jones and Poole, 1985). As demonstrated by Morel and Ahn (1991), bacteria conform well to Mie theory; thus, measured absorption coefficients for the bacterial fraction can be used in calculations of bacterial scattering, including backscattering. Hence, good measures of bacterial absorption provide key information for the improvement of models of radiative transfer in the water. This communication presents a new procedure for measurement of the volume absorption coefficient of the fraction of heterotrophic bacteria that passes through the GF/F filter. The procedure aims to offer a practical tool for routine experiments in support to remote sensing of water bodies. With some modification, and coupled to other experimental measurements (e.g. epifluorescence microscopy) and theoretical computations (e.g. by Mie theory), it can also provide information for basic studies of the optical properties of heterotrophic bacteria. The intrinsic difficulties that such a procedure must face are: (i) use of unenriched samples collected in situ, characterized by very low absorption values; (ii) measurement of absorption in the presence of a large scattering component; and (iii) the need to discriminate bacterial absorption from fine detritus and picoplankton absorption. Method Light absorption measurements of aquatic particles retained on filters are usually performed by dual-beam spectrophotometers, which yield the particle absorbance, A { [A f = log(l/7), where T is the transmittance of the sample (particles + filter) relative to that of a clean reference filter]. The filter-retained particle absorbance, A f, is converted, first to the corresponding particle suspension absorbance, A sus, by an empirical expression that corrects for the absorption amplification induced by multiple scattering in the filter (Cleveland and Weidemann, 1993), and then to the volume absorption coefficient, o(m" 1 ), by the equation: where X is the ratio of filtered volume to filter clearance area (m). The spectrophotometer used for this measurement was a Perkin-Elmer Lambda 758 (1)

3 Determination of light absorption 19 dual-beam unit, provided with a 60-mm-diameter, barium sulphate-coated integrating sphere attachment. The unit was operated in the wavelength range from 350 to 750 nm, with 1 nm spectral band width and 1 nm spectral increments. Water samples, collected in situ in the 2-5 m depth range, were pre-filtered by GF/F filters and then passed through a 0.22 um Millipore membrane (10.8 cm 2 clearance area), under 20 mm of Hg vacuum. Clogging of the glass fibre filter was prevented by subdividing the water volume into equal subvolumes, each passed through a separate GF/F filter. The particle-retaining membrane (sample) and a fresh Millipore membrane (reference) were cut into 10-mm-wide strips, wetted to saturation by the Millipore-filtered water, and supported by 1-mm-thick quartz backings. The particle absorbance determination was performed by the method described in Tassan and Ferrari (1995), referred to as the 'transmittance-reflectance' method in the following, which consists of the sequence of two measurements, carried out in the 'transmission mode' and in the 'reflection mode'. The method corrects for the effect of particle backscattering, and thus is suited to highly scattering samples such as the heterotrophic bacteria deposit on the Millipore membrane. For a complete description, the reader is referred to the original article. While laboratory cultures of heterotrophic bacteria do not include significant fractions of other matter, in situ collected samples of oligotrophic water, even if pre-filtered through the GF/F, usually contain matter other than bacteria, such as picoplankton and fine detritus (either inorganic and organic), the latter often being the dominant light absorber. This sets the requirement to discriminate bacterial absorption from the absorption of other matter deposited on the Millipore membrane. The standard procedure for pigment removal by methanol extraction (Kishino et al., 1985) is not effective with heterotrophic bacteria, since it removes <10% of the cytochrome absorption (Morel and Ann, 1991). In any case, this treatment cannot be applied to particles retained on the Millipore cellulose membrane, which is dissolved by methanol. The oxidizing agent NaCIO has proven to be very effective in bleaching pigments in phytoplankton cells (Tassan and Ferrari, 1995), and is applicable equally well to GF/F and Millipore filters (Ferrari and Tassan, 1996). It is not suitable for bleaching pigments in heterotrophic bacteria, however, because although it destroys the respiratory pigment absorption peaks, it also causes a yellowish deposit on the Millipore membrane, which gives a steep increase in absorption at wavelengths below 450 nm. It is very likely that the basic NaCIO solution oxidizes the Fe 2+ of cytochrome c to Fe 3+ with precipitation of Fe(OH) 3. This suggestion was supported by the observation that a Fe(OH) 3 deposit on a Millipore membrane displayed a very similar absorption spectrum. The reaction can be represented thus: 3NaClO + 2Fe {Cyt.} 2 " + 3H 2 O - 2Fe(OH) 3 + 3NaCl + 2{Cyt.) (2) where {Cyt.j indicates the part of the cytochrome that balances the positive iron charge, with the two electrons (= (Cyt.} 2 ~) or in the oxidized form (= {Cyt.}). 759

4 CM-Ferrari and S.Tassan An alternative to the use of NaCIO is to use the oxidizing agent potassium persulphate (K^Os). This forms an acid solution and its action probably produces soluble Fe 3 * as ferric sulphate, thus: 3K 2 S2O 8 + 2Fe {Cyt.} 2 " -+ 3K 2 SO 4 + Fe2(SO 4 ) 3 + 2{Cyt.} (3) A 10% K 2 S2O 8 solution was therefore used to bleach the respiratory pigments, although the reaction rate was much slower than that of NaCIO (several hours versus a few minutes, depending on sample type). The absorption of the K 2 S2O 8 solution is negligible over the nm wavelength range. Similarly to NaCIO, K 2 S2Og was observed to have a negligible effect on the detritus absorption spectrum, over and beyond the standard time required for the complete bleaching of the pigments. This is probably due to the fact that the oxidation is much faster for pigments than for other organic matter present in the detritus. The discrimination between the absorbance caused by heterotrophic bacteria and that due to detritus was achieved by measuring the absorbance of the same filter-retained particle sample before and after addition of the K 2 S 2 O 8 solution. Subtraction gave the absorbance spectra due to the bacteria. In the following, this absorbance difference is either called 'bacterial', 'respiratory pigment' or 'cytochrome absorbance'. Even if these terms are not strictly interchangeable, the approximation is justified in practice, as is shown by the test presented here (Figure 2). The potassium persulphate is effective in discriminating cytochrome from detritus absorption, but not from absorption by picoplankton pigments, which are also bleached by this agent. The presence of picoplankton cells in the Millipore membrane is evident from the chlorophyll a absorption peak observed at -675 nm. Based on the magnitude of this peak, the chlorophyll absorption was reconstructed over the entire wavelength range using a mean normalized spectrum (Bricaud and Stramski, 1990), and the reconstructed spectrum was subtracted from the cytochrome 'apparent' absorption. This approximate correction is considered to be adequate in view of the small relative magnitude of the chlorophyll absorption term. The empirical expression needed to convert the measured absorbance of the filter-retained bacteria, A t, to the corresponding absorbance of the bacterial suspension, A sus, was determined by the procedure described in Tassan and Ferrari (1995). Briefly, the procedure consists of transmittance measurements of the suspension and transmittance-reflectance measurements of the filter-retained bacteria. Subtracting the results of measurements carried out before and after bleaching of the respiratory pigments yields the absorbance due to pigment absorption. The A sus, A f values obtained by this procedure are free from spurious absorption contributions due to light scattering by the bacteria. Results and discussion The determination of the (A^, A f ) relationship was performed using a concentrated suspension of particles with diameter in the um range, prepared 760

5 Determination of light absorption by resuspending particles deposited on Millipore membranes (water collected in Lake Varese, Italy). The suspension sample was contained inside a quartz cuvette with 1 cm optical path length. Bleaching of the respiratory pigments was achieved by addition of a 10% solution of potassium persulphate (0.5 cm 3,10 h treatment time). The pigment absorbance spectrum obtained subtracting measurements carried out before and after the K 2 S2Og treatment showed typical cytochrome peaks at -410,500 and 550 nm. A least squares fit to the data yielded the quadratic expression: A sus (\) = (0.405 ± 0.018) Af{\.) + (0.705 ± 0.36) Af(\) 2 (4) where the errors of the numerical constants are standard deviation estimates. Equation (4) is valid in the range 0 < Af(\) < 0.1, which is adequate to cover most practical situations. Although this relationship is likely to apply to a variety of bacteria with a diameter in the um range, we suggest repeating the determination of the empirical relationship (/t sus, A f ) for each experimental campaign of in situ measurements of bacterial absorption. Figure 1A displays typical Af(\) spectra yielded by the 'transmittancereflectance' method and by the standard 'transmittance' method (thick and thin solid line, respectively; 5 1 sample from the Adriatic Sea). The bacterial identification is provided by the characteristic Soret peak of cytochrome c; there is no evidence of picoplankton presence (no discernible 675 nm peak of chlorophyll a). The difference between the spectra yielded by the two methods is due to the spurious absorbance resulting from light scattering by the particles, which is not corrected for by the transmittance method. The magnitude of this term is consistent with the high observed scattering of heterotrophic bacteria. If the usual 'a posteriori' correction to the transmittance measurement, namely the subtraction of the 750 nm absorbance bias from the measured spectrum (Mitchell and Kiefer, 1988), is applied to the data, one obtains the curve marked by the dashed line. The considerable disagreement with the absorbance spectrum yielded by the 'transmittance-reflectance' measurement, which is larger than a factor of two at the wavelength of the Soret peak, shows how the standard transmittance measurement does not yield sufficiently accurate estimates of heterotropic bacterial absorption. Figure IB displays /1 SUS (X) derived by equation (4) from Af(k) measured by the 'transmittance-reflectance' method, before and after addition of the K2S2O8 solution (thin solid line and dashed line), as well as the corresponding cytochrome suspension absorbance (thick solid line). In this case, omitting bleaching of the pigments would have resulted in an overestimate of the Soret peak of cytochrome c by about a factor of two. The volume absorption coefficient of the cytochrome, at the 415 nm wavelength of the Soret peak, is [equation (1)]: a(415) = tor 1. The procedure for discrimination of the light absorption by heterotrophic bacteria through bleaching of the respiratory pigments is based on the assumption that absorption other than by pigments is negligible. In order to test this assumption, the bacterial population of a Millipore-retained particle sample was grown in a laboratory culture, so as to minimize the relative contribution of the 761

6 GJVLFerrari and S.Tassan FIG.1A ABSORBANCE (dimensionless) WAVELENGTH (nm) FIG.1B ABSORBANCE (dimensionless) WAVELENGTH (nm) Fig. 1. Result of measurements carried out on samples collected in the Adriatic Sea. (A) Filterretained particle absorbance spectra, as measured by the 'transmittance-rerflectance' method (thick solid line) and by the standard 'transmittance' method without and with subtraction of the -4K750) bias (thin solid line and dashed line). (B) Particle suspension absorbance spectra given by equation (4) from filter-retained particle absorbances measured by the 'transmittance-resectance' method for the original sample (thin solid line) and the K^Og bleached sample (dashed line). Respiratory pigment absorbance in the water suspension (thick solid line). absorption by the detritus contained in the water collected in situ, and then measured by the 'transmittance-reflectance' method before and after ^SjOg addition. The result of the test is presented in Figure 2. The very small difference between total and pigment absorbance spectra is proof that the pigment absorption is a sufficiently accurate estimate of the total bacterial absorption. The low absorbance values of Figure 1, which are typical of this type of measurement on in situ collected samples, induce a particular concern on the impact of the experimental error on the results obtained. A comprehensive error 762

7 Determination of light absorption ABSORBANCE (dimensionless) WAVELENGTH (nm) 650 Fig. 2. Absorbance spectra of marine heterotrophic bacteria grown in a laboratory culture, as measured by the 'transmittance-reflectance' method: total absorbance (thin solid line) and respiratory pigment absorbance (thick solid line). analysis of the 'transmittance-reflectance' method is presented in Tassan and Ferrari (1995). In summary, the error in Af(\) is around ±0.005 (SD estimate). Differentiating equation (4) and combining the errors quadratically, also taking into account the computed correlation coefficient of the constants, yields J4 SUS (X) errors in the range from ±0.002 to ±0.004, as Af(k) increases from 0.02 to 0.1. Note that the same relative error affects A SUS (X) and the corresponding volume absorption coefficient, a(\), obtained by equation (1), since the uncertainty in theat term is negligible. The bacterial absorption error is a quadratic combination of the errors of the measurements carried out on the original and bleached samples. In the specific case (Figure IB), the error in the volume absorption coefficient of the cytochrome at 415 nm is estimated at around ±15%. The above analysis is not meant to be a complete statistical treatment, which would have not been coherent with the limited set of available data. However, the reliability of the a priori estimate of the error was supported by the dispersion observed a posteriori among the results of repeated measurements. Obviously, the error is reduced if the filtered volume is increased. Figure 3 shows absorbance spectra of the suspension of particles with a diameter in the range um, measured by the 'transmittance-reflectance' method (5 1 sample from Lake Varese). The spectrum of the total absorbance (thin solid line) exhibited the typical 675 nm peak of chlorophyll a, indicating the presence of picoplankton on the Millipore membrane. Thus, the cytochrome absorbance (dotted line) obtained as the difference in the total and bleached sample (dashed line) absorbance was corrected for the chlorophyll contribution, reconstructing the chlorophyll absorbance with the aid of the normalized absorption spectrum measured for the picoplankton Prochlorococcus (Morel et al., 1993). The corrected cytochrome absorbance (thick solid line) is only slightly different from the uncorrected spectrum (-12% at the 415 nm wavelength), confirming the validity of the simplified correction scheme adopted. The 763

8 CMFerrari and S.Tassan ABSORBANCE (dimensionless) WAVELENGTH (nm) Fig. 3. Particle suspension absorbance spectra obtained from 'transmittance-reflectance' measurements carried out on samples collected in Lake Varese (Italy): total absorbance (thin solid line), bleached sample absorbance (dashed line) and respiratory pigment absorbance before and after correction for chlorophyll absorption (dotted and thick solid lines). absorption spectrum is similar in shape to that of the bacteria in the Adriatic Sea water (Figure IB), but higher in magnitude [fl(415) = ± 12% nr 1 ]. The importance of absorption by heterotrophic bacteria and fine detritus in oligotrophic water (Gulf of Naples) is highlighted in Figure 4, discriminating the contribution by particles with a diameter in the um range and above 0.7 um. A 5 1 volume of water was filtered first through a series of GF/F filters and then through a 0.22 um Millipore membrane. The absorbance of the GF/Fretained and of the Millipore-retained particles was measured by the 'transmittance-reflectance' method before and after NaCIO and K^Og addition, respectively, converted to suspension absorbance by equation (4) (Millipore data) and equation (18) of Tassan and Ferrari (1995) (GF/F data), and then to volume absorption coefficient by equation (1). The displayed spectra are: GF/F pigment (thick solid line), GF/F detritus (dashed line), Millipore cytochrome (solid line), Millipore detritus (dotted line). The Millipore spectra are amplified by the right scale. At the 440 nm wavelength of the chlorophyll absorption maximum, a(pigment GF/F) = nr 1, a(detritus GF/F) = nr 1, ^(detritus Millipore) = m -1 and a(bacteria Millipore) = nr 1. In this case, neglecting the um particle fraction would underestimate the particle total absorption at 440 nm by -20%. The bacteria alone contribute around 8% of the total absorption at 440 nm. In conclusion, the proposed experimental method was tested to yield a satisfactory determination of the volume coefficient for light absorption in vivo by the fraction of heterotrophic bacteria with a diameter in the um nominal range, which are not retained by the GF/F filter routinely used for measuring the absorption of aquatic particulates, and thus are not explicitly accounted for. This information is of practical usefulness in the case of oligotrophic water, where these bacteria often contribute a sizeable fraction of the overall particle 764

9 Determination of light absorption VOLUME ABS.COEFF. (1/m) WAVELENGTH (nm) Fig. 4. Volume absorption coefficient spectra obtained from 'transmittance-reflectance' measurements carried out on samples collected in the Gulf of Naples, and retained on GF/F filters or on 0.22 urn Millipore membranes (left and right scales, respectively). The curves displayed are: GF/F pigment (thick solid), GF/F detritus (dashed), Millipore cytochrome (solid) and Millipore detritus (dotted). absorption. Combining the measured absorption coefficients with backscattering coefficients computed by Mie theory provides the set of optical parameters needed to include the contribution of this fraction of heterotrophic bacteria in multicomponent models describing the radiation transfer in water. The proposed experimental procedure can also be used to yield information of a more basic nature on the light absorption by heterotrophic bacteria. For this application, it is preferable to pre-filter the water sample through pierced membranes with retention efficiency close to the nominal pore size, so as to obtain a better defined and reproducible bacterial population. References Bricaud.A. and Stramski.D. (1990) Spectral absorption coefficients of living phytoplankton and nonalgal biogenous matter: a comparison between the Peru upwelling area and the Sargasso Sea. Limnol. Oceanogr., 35, Cho,B.C. and Azam,F. (1990) Biogeochemical significance of bacterial biomass in the oceans' euphotic zone. Mar. EcoL Prog. Ser, 63, ClevelandJ.S. and Weidemann,A.D. (1993) Quantifying absorption by aquatic particles: a multiple scattering correction for glass-fiber filters. Limnol. Oceanogr, 38, Ferrari.G.M. and Tassan,S. (1996) Use of the 0.22 \im Millipore membrane for light-transmission measurements of aquatic particles. /. Plankton Res., 18, Fruton J.S. and Simmonds.S. (1961) General Biochemistry, 2nd edn. Wiley, New York. Gordon,H.R. and Morel,A. (1983) Remote assessment of ocean color for interpretation of satellite visible imagery: a review. In Bowman,M. (ed.). Lecture Notes on Coastal and Estuarine Studies. Springer, Berlin, pp Jones.C.W. and Poole,R.K. (1985) The analysis of cytochromes. Methods MicrobioL, 18, KishinoJVI., Takahashi,M., Okami,N. and Ichimura.S. (1985) Estimation of the spectral absorption coefficients of phytoplankton in the sea. Bull. Mar. Sci, 37, Mitchell3G. and KieferJD.A. (1988) Chlorophyll a specific absorption and fluorescence excitation spectra for light-limited phytoplankton. Deep-Sea Res., 35,

10 G.M.Ferrari and S.Tassan Morel,A. and Ahn.Y. (1991) Optical efficiency factors of free-living marine bacteria: Influence of bactenoplankton upon the optical properties and particulate organic carbon in oceanic waters. /. Mar. Res., 48, Morel,A., Ahn.Y., Partenski,F., VaulotJ). and Claustre,H. (1993) Prochlorococcus and Synechococcus: A comparative study of their optical properties in relation to their size and pigmentation. / Mar. Res., 51, Prieur.L. and Sathyendranath.S. (1981) An optical classification of coastal and oceanic waters based on the specific spectral absorption of phytoplankton pigments, dissolved organic matter and other particulate materials. Limnol. Oceanogr, 26, Sathyendranath.S., Prieur,L. and Morel,A. (1989) A three-component model of ocean colour and its application to remote sensing of phytoplankton pigments in coastal waters. Int. J. Remote Sensing, 10, Stanier,R.Y., IngrahamJ.L., Wheelis,M.L. and Painter,P.R. (1988) The Microbial World, 5th edn. Prentice-Hall, London. Stramski,D. and Kiefer,A.D. (1990) Optical properties of marine bacteria. In Spinrad,R.W. (ed.). Ocean Optics 10, Proc SPIE1302, The International Society for Optical Engineering, pp StramskiJ). and Kiefer,D.A. (1991) Light scattering by microorganisms in the open ocean. Prog. Oceanogr., 28, Tassan.S. and Ferrari.G.M. (1995) An alternative approach to absorption measurements of aquatic particles retained on filters. Limnol. Oceanogr., 40, Ulloa.O., Sathyendranath,S., Platt.T. and Quinones.R. (1992) Light scattering by marine heterotrophic bacteria. /. Geophys. Res., 97, Received on June 2, 1997; accepted on December 5,