# Potential impacts of black carbon on the marine microbial community

Andrea Malits, Raffaela Cattaneo, Eva Sintes, Josep M. Gasol, Gerhard J.

Herndl, Markus G Weinbauer

► To cite this version:

Andrea Malits, Raffaela Cattaneo, Eva Sintes, Josep M. Gasol, Gerhard J. Herndl, et al.. Potential impacts of black carbon on the marine microbial community. Aquatic Microbial Ecology, 2015, 75 (1), pp.27-42. 10.3354/ame01742 . hal-03502745

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# Potential impacts of black carbon on the marine microbial community

Andrea Malits<fnref n="1" />,<fnref n="2" />*, Raffaela Cattaneo<fnref n="1" />,<fnref n="2" />, Eva Sintes<fnref n="3" />,<fnref n="4" />, Josep M. Gasol<fnref n="5" />, Gerhard J. Herndl<fnref n="3" />,<fnref n="4" />, Markus G. Weinbauer<fnref n="1" />,<fnref n="2" />

**ABSTRACT:** Black carbon (BC) is the carbonaceous residue of the incomplete combustion of fossil fuels and biomass and encompasses a range of chemically heterogeneous substances from partly charred plant material to highly condensed soot aerosols. We addressed the potential role of BC aerosol deposition on marine microbial processes in the ocean by investigating the effects of BC reference material (and its exposure to simulated solar radiation) on viral and bacterial activity in batch cultures with aged seawater. Viruses and bacteria were rapidly adsorbed to BC. No difference between the effect of irradiated and non-irradiated BC on free viral parameters was observed. Bacterial leucine incorporation was higher in the BC treatments than in the BC-free controls. The stimulated bacterial production in the dark BC treatments might be caused by the reduction of viral infection due to adsorption of organic material or by direct use of BC material. Viral production was significantly lower in BC-amended treatments than in BC-free controls, and the estimated fraction of infected cells decreased with increasing BC concentration. Moreover, bacterial activity in the solar-radiation-exposed BC treatments was higher than in the dark BC treatments, indicating that radiation made BC more accessible to bacteria. Our data reveal that BC has the potential to stimulate bacterial activity in the water column, particularly after exposure to solar radiation. Rising BC levels in the atmosphere due to increasing anthropogenic emissions could have far-reaching effects, including potential stimulation of seawater heterotrophy and CO₂ production, through its effects on bacteria and viruses.

**KEY WORDS:** Black carbon aerosols · Light exposure · Viral lysis · Bacterial production

Resale or republication not permitted without written consent of the publisher

## INTRODUCTION

Black carbon (BC) refers to the carbonaceous products of incomplete combustion of vegetation and fossil fuels, and is defined as a continuum of compounds from partly charred plant material to highly condensed soot aerosols (Goldberg 1985, Schmidt &

Noack 2000, Mannino & Harvey 2004). These thermogenic compounds are chemically heterogeneous and are all characterized by high carbon content and aromatic structures, which are particularly resistant to biodegradation (Goldberg 1985, Schmidt & Noack 2000). BC particles constitute aggregates of small carbon spheres (Cattaneo et al. 2010), whose fractal

<footnote n="1">¹Sorbonne Universités, UPMC Univ Paris 06, UMR 7093, LOV, Observatoire océanographique, 06230 Villefranche/mer, France</footnote>

<footnote n="2">²CNRS, UMR 7093, LOV, Observatoire océanographique, 06230 Villefranche/mer, France</footnote>

<footnote n="3">³Department of Biological Oceanography, Royal Netherlands Institute for Sea Research (NIOZ), PO Box 59, 1790 AB Den Burg, The Netherlands</footnote>

<footnote n="4">⁴Department of Limnology and Oceanography, Faculty Center of Ecology, Althanstr. 14, 1090 Vienna, Austria</footnote>

<footnote n="5">⁵Institut de Ciències del Mar, CSIC, Departament de Biologia Marina i Oceanografia, Passeig Maritim de la Barceloneta, 37-49, 08003 Barcelona, Catalonia, Spain</footnote>
morphology, i.e. their high porosity, offers an impor-
tant surface area for the adsorption of organic matter
(Cornelissen et al. 2005, Koelmans et al. 2006). BC
particles occur in a wide size range, and small soot
particles (<1 µm) easily become airborne and can
remain in the atmosphere for weeks and spread over
remote areas including the open ocean (Masiello
2004, Hadley et al. 2007). During atmospheric trans-
port, BC aerosols efficiently absorb solar radiation
and are the second strongest contributor to current
global warming (Ramanathan & Carmichael 2008).

BC is introduced into the ocean by atmospheric
deposition of land-derived aerosols (Jurado et al.
2008), via river estuarine systems (Mitra et al. 2002,
Kim et al. 2004, Elmquist et al. 2008, Stubbins et al.
2010), and by marine diesel engine exhausts (Lack et
al. 2008). Dry and wet deposition of BC to the global
ocean is estimated to be 2 and 10 Tg C yr⁻¹, respec-
tively, with higher fluxes over the northern hemi-
sphere (Jurado et al. 2008). A portion of BC becomes
soluble over time and enters the dissolved organic
matter (DOM) pool (Kim et al. 2004). Dissolved BC
has been observed throughout different domains of
the ocean accounting for 1 to 9% of the DOM pool
with a strong coastal to open ocean concentration
gradient (Mannino & Harvey 2004, Dittmar & Koch
2006, Dittmar & Paeng 2009, Ziolkowski & Druffel
2010, Dittmar et al. 2012). In open ocean sediments,
BC is up to 14 000 yr older than the co-deposited bulk
organic carbon, suggesting that the small and light
BC particles are not used but are retained in the dis-
solved organic carbon (DOC) pool before being
deposited in marine sediments (Masiello & Druffel
1998). However, isotopic analysis revealed the petro-
genic origin of BC in marine sediments and sug-
gested a significant overestimation of combustion-
derived BC burial in sediments and underestimation
of degradation processes in the water column (Dick-
ens et al. 2004). Additionally, the low contribution of
BC to marine DOC in the open ocean, i.e. less than
3.5% (Dittmar & Paeng 2009, Ziolkowski & Druffel
2010), suggests that BC is more labile than formerly
believed. Recent studies on the photochemical (Stub-
bins et al. 2012) and microbial degradation of BC
(Zimmerman 2010) indeed challenge the previous
assumption on the refractory character of BC.

Despite the ubiquitous presence of BC in the
oceanic particulate organic matter (POM) (Flores-
Cervantes et al. 2009) and DOM pools, its effect on
marine systems and its role in the marine carbon
cycle remain largely enigmatic. It has been demon-
strated that BC reference material rapidly adsorbs
viruses and bacteria (Cattaneo et al. 2010) and stim-

ulates the aggregation of organic particles and bacte-
rial production (Mari et al. 2014). Also, there is evi-
dence that BC can have an influence on viral and
bacterial diversity (Cattaneo et al. 2010, Weinbauer
et al. 2012).

Viruses are important mortality agents in the
ocean, structuring microbial communities and influ-
encing biogeochemical cycles (Fuhrman 1999, Suttle
2007) by converting cells into DOM and increasing
the recycling and retention of nutrients in the photic
zone (Wilhelm & Suttle 1999). Organic particles, in
turn, may provide shelter to bacteria from viral infec-
tion, although experimental results are controversial
(Suttle & Chen 1992, Noble & Fuhrman 1997, Rie-
mann & Grossart 2008, Sheik et al. 2014). As BC par-
ticles are a relevant type of organic particle, knowl-
edge on the potential impact of BC-rich aerosols on
production and viral-mediated loss of microorgan-
isms and the consequences for biogeochemical
cycles in the sea is needed.

To explore the potential mechanisms playing a role
in the response of prokaryotes and associated viruses
to BC, we set up 2 experiments in which we added
various concentrations of standard BC to a standard-
ized equilibrated community from aged seawater
from the North Sea. Exposure of BC to solar radia-
tion, a phenomenon that commonly occurs during
atmospheric transport, was simulated, and its effect
on BC bioavailability to bacteria was also investi-
gated.

Our experiments provide insight into the potential
effects of BC on the microbial community, such as: (1)
a stimulating effect of BC on bacterial heterotrophic
production, as BC is ultimately a carbon source, (2)
an increased bioavailability of BC due to previous
solar radiation mimicking atmospheric transport, (3)
attachment of bacteria and viruses, as BC are organic
particles with a high surface area, and (4) a deleteri-
ous effect on viral infectivity due to attachment and
loss of activity.

MATERIALS AND METHODS

Experimental set-up

Two experiments were performed in May 2005
with aged surface seawater from the open North Sea
collected in August 2004 from onboard the RV 'Pela-
gia' at the North Sea Oyster Grounds (54° 30' N, 04°
30' E) and kept in the dark at 20°C.

Aged seawater was chosen because it is not limited
in inorganic nutrients, but instead in organic carbon.
During aging, more degradable organic matter is eliminated which otherwise would obstruct the use of a more refractory carbon source such as BC. Also, other organic particles present in un-aged seawater might adsorb viruses and bacteria and bias the interaction between BC particles and viruses or bacteria. Additionally, aged seawater was used to ensure that the microbial community was in a steady state. Steady-state conditions were assessed prior to the experiments by measuring selected bacterial parameters for 3 consecutive days; these parameters were sampled every 13 ± 3 h and exhibited variations of ≤4% (data not shown). The rationale of the present study was to explore the potential mechanisms playing a role in the response of the microbial food web to BC rather than studying the effect of BC on any specific planktonic community. Therefore we used a standardized equilibrated community.

The first experiment was designed to assess the effect of a defined concentration of BC and consisted of duplicate incubations of seawater with and without receiving BC (experiment *BC-fix*). In the second experiment, a gradient of BC concentrations was tested in non-replicated incubations (experiment *BC-grad*). In both experiments, BC reference material (SRM 2975, Diesel Particulate Matter from the US Department of Commerce, National Institute of Standards and Technology [NIST], Gaithersburg, MD) was used. Soot carbon represents 95% of NIST diesel particulate matter (versus 5% organic carbon, Gustafsson et al. 1997), and the trace metal content of SRM 2975 is low (0.22 wt.%, Jensen 2006). The majority of BC reference material particles are within the size range of 1.5 to 4 µm equivalent spherical diameter (ESD). Details on the size distribution of SRM 2975 are given by Cattaneo et al. (2010).

Before being employed in the experiments, BC stock solutions (BC added to ultrapure [MilliQ, Millipore] water at concentrations of 20 mg l⁻¹ in experiment *BC-fix* and 100 mg l⁻¹ in experiment *BC-grad*) were either kept in the dark or exposed to artificial solar radiation in quartz tubes (2.8 cm inner diameter) for 15 h and 23 h, in *BC-fix* and *BC-grad*, respectively. Artificial solar radiation was supplied by 3 different types of light sources. Two HQI-T Powerstar (Osram) lamps provided photosynthetically active radiation (400–700 nm wavelength range), 2 TL 100W/10R fluorescent light tubes (Philips) were used to provide UV-A (320–400 nm), and 3 UVA-340 fluorescent light tubes (Q-Panel) supplied UV-A and UV-B (300–320 nm). The solar simulator was adjusted to 30–60% of the local maximum radiation intensity in late spring measured on a cloudless day (Pausz &

Herndl 2002). Thus, the dose rate received was similar to that expected in the top surface layer of the North Sea water column. For the dark control (unexposed) treatment, the BC stock was kept in the dark in glass tubes wrapped in aluminum foil in the same incubator. During the exposure to artificial solar radiation, BC stocks were kept in a flow-through water bath connected to a temperature control unit (LAUDA RCS/RC-6).

In experiment *BC-fix*, 10 ml of the light-exposed (BC-light treatment) and unexposed BC stock (BC-dark treatment) were added to 1 l of unfiltered aged seawater in duplicate acid-rinsed borosilicate bottles to obtain a final BC concentration of 200 µg l⁻¹. For experiment *BC-grad*, the BC stock solution was added to 1 l of aged seawater to obtain the following concentration gradient: 200, 500, 1000, and 2000 µg light-exposed BC l⁻¹ (BC-light treatments) and 500 and 2000 µg unexposed BC l⁻¹ (BC-dark treatments). The highest BC concentration measured to date in coastal seawater is 162 µg l⁻¹ (Mannino & Harvey 2004), i.e. similar to the concentrations added in experiment *BC-fix* (200 µg l⁻¹). Experiment *BC-grad* with up to 10-fold higher additions of BC than has been found in marine environments was designed to unveil the basic effects of BC on microbes, rather than simulating scenarios at ecologically relevant concentrations. Elevated concentrations of BC should also amplify the sorption dynamics and potentiate the interaction between particles and cells or viruses, respectively.

In both experiments, 10 ml of ultrapure water were added to the BC-free controls, and all bottles were incubated in the dark at 20°C for 4 d and sampled every 13 ± 6 h. We started sampling ($t_0$) approximately 10 min after adding the BC material. The incubation bottles were thoroughly mixed prior to sampling.

## Determination of DOC and inorganic nutrients

Samples for the measurement of dissolved inorganic nutrient concentrations ($NH_4^+$, $NO_3^-$, $NO_2^-$, $PO_4^{3-}$) were pre-filtered through 0.2 µm polycarbonate filters (GTTP, Millipore), and subsequently analyzed in a TRAACS 800 autoanalyzer system. $NH_4^+$ was detected with the indo-phenol blue method (pH 10.5) at 630 nm (Helder & De Vries 1979). $NO_2^-$, $NO_3^-$, and $PO_4^{3-}$ were determined following the Joint Global Ocean Fluxes Study recommendations (Gordon et al. 1993). $NO_2^-$ was detected after diazotation with sulfanilamide and N-(1-naphtyl)-ethylene
diammonium-dichloride as the reddish-purple dye complex at 540 nm. $NO_3^-$ was reduced in a copper cadmium coil to nitrite and then measured as nitrite. $PO_4^{3-}$ was determined via the molybdenum blue complex at 880 nm.

Samples for DOC measurements were filtered through Whatman GF/F filters using a glass filtration system. The Whatman GF/F filters and all of the glassware were combusted (450°C for 4 h) prior to the filtration. Eight ml of filtered samples were transferred to combusted glass ampoules, and 50 µl of 40% phosphoric acid were added to each sample. Afterwards, the ampoules were sealed and stored at 4°C in the dark and analyzed within 1 mo. DOC concentrations were determined in triplicate using a Shimadzu TOC-5000 analyzer (Benner & Strom 1993).

## Determination of total bacterial abundance, respiring bacteria, and ‘live’ versus ‘dead’ bacteria

Water samples for total bacterial abundance (1.8 ml) were fixed with paraformaldehyde (1%) and glutaraldehyde (0.05%), kept at room temperature for ca. 10 min, and then flash-frozen in liquid nitrogen. Within a few days, the samples were thawed, stained with SYBR Green I (Molecular Probes, Invitrogen) at 10× dilution for 10 min, amended with Fluoresbrite® Yellow Green (YG) beads of 0.98 µm diameter (Polysciences) as an internal standard, and analyzed in a Becton Dickinson FACSCalibur flow cytometer as described previously (Gasol & del Giorgio 2000).

Bacteria were detected in plots of 90° light scatter (SSC) and green DNA fluorescence (Fig. 1). Differences in the green fluorescence and SSC signature in the cytometric plot allowed us to separate different bacterial populations according to their nucleic acid content, i.e. low (LNA) and high nucleic acid (HNA) content, as previously described (Gasol et al. 1999).

In the samples with BC additions, we detected an additional population with a SYBR Green derived fluorescence comparable to HNA bacteria, yet with a much higher SSC (Fig. 1). The bacteria in that gate were operationally considered to be particle-associated bacteria (PAB) assuming that the increase in SSC without parallel increase in nucleic acid-derived fluorescence had to be due to the association of bacteria with particles.

The numbers of actively respiring bacteria were determined by the fluorogenic tetrazolium dye CTC (5-cyano-2,3-ditolyl tetrazolium chloride) labeling of

<figure><img src="image_3.png" /><figcaption>Fig. 1. Flow cytometric plot of side scatter (SSC) versus green florescence (FL1) of SYBR-Green-stained bacteria from experiment *BC-grad*. Examples are given of bacteria that had received black carbon (BC; lower panel) and bacteria from the BC-free control (upper panel). A secondary population of particle-associated bacteria (P+NA) with higher SSC and similar fluorescence to high nucleic acid (HNA) bacteria is evident in the lower panel. B: beads; LNA: low nucleic acid bacteria</figcaption></figure>

highly active cells (del Giorgio et al. 1997, Sherr et al. 1999, Sieracki et al. 1999). Five mM CTC of a daily prepared batch was added to water samples and incubated at 20°C for 3 h. After incubation, the samples were analyzed with a Becton Dickinson FAC-
SCalibur flow cytometer as described in detail elsewhere (Gasol & Arístegui 2007).

We also discriminated ‘live’ or membrane-intact from ‘dead’ or membrane-damaged bacteria using the nucleic-acid double staining protocol (Gregori et al. 2001) as explained in detail elsewhere (Falcioni et al. 2008). In brief, samples were dually stained with propidium iodine (10 µg ml⁻¹), a membrane-impermeable red dye, and SYBR Green I (Molecular Probes, Invitrogen), a membrane-permeable green dye, for 15 min, and then analyzed in a Becton Dickinson FACSCalibur flow cytometer at a low speed (ca. 15 µl min⁻¹). A dot plot of red (FL3 cytometric channel) versus green fluorescence (FL1 cytometric channel) allowed distinction of the ‘live’ cells from the ‘dead’ cells. ‘Live’ cells were considered those having more green than red staining, while ‘dead’ cells had more red than green.

## Bacterial heterotrophic production

Subsamples for the measurement of bulk leucine incorporation by bacteria were taken from the different treatments and treated using the method as outlined by Simon & Azam (1989). Two 5 ml samples and one 5 ml formaldehyde-killed blank per treatment were inoculated with ³H-leucine (20 nM final concentration, Amersham, specific activity 160 Ci mmol⁻¹) and incubated in the dark at 20°C for 2 h. Subsequently, the samples were fixed with formaldehyde (2% final concentration), filtered onto 0.2 µm Millipore GTTP polycarbonate filters, and rinsed 3 times with 10 ml of 5% ice-cold trichloroacetic acid. The filters were then transferred to scintillation vials and dried at room temperature. One ml of ethyl acetate and 8 ml of scintillation cocktail (Packard Gold Insta Gel) were added to each vial. The vials were counted in an LKB liquid scintillation counter after 18 h. The obtained disintegrations per minute (DPMs) were converted to leucine incorporation rates. Leucine incorporated into bacterial biomass was converted to bacterial carbon production using the empirical conversion factor 0.07 × 10¹⁸ cells mol⁻¹ Leu (Riemann et al. 1990) and assuming a C content of bacteria of 20 fg C cell⁻¹ (Lee & Fuhrman 1987). The application of this conversion factor resulted in similar bacterial heterotrophic production (BHP) estimates as with the theoretical factor of 1.55 kg C mol⁻¹ Leu that assumes no isotope dilution (Simon & Azam 1989). BHP divided by bacterial biomass provided estimates of the specific growth rate of bacteria (d⁻¹).

## Viral abundance

Viral abundance was enumerated by flow cytometry (FC), which currently cannot be combined with virus-aggregate disruption agents such as methanol, since it interferes with the staining dye (Weinbauer et al. 2009). Thus, bulk measurements represent likely free viral abundance (FVA). In specific samples, viruses attached to BC particles were enumerated by epifluorescence microscopy (see below).

For viral counts by FC, we followed the optimized protocol by Brussaard (2004). Subsamples (2 ml) were fixed with glutaraldehyde (0.5% final concentration), incubated at 4°C for 15 to 30 min, and subsequently frozen in liquid nitrogen and stored at -80°C. Upon thawing, viruses were stained with SYBR Green I (Molecular Probes, Invitrogen) at a final concentration of 0.5 × 10⁻⁴ of the commercial stock at 80°C for 10 min and quantified using a FACSCalibur (Becton and Dickinson) flow cytometer after dilution with TE buffer (10 mM Tris, 1 mM EDTA, pH 8).

## Viral production

Viral production (VP) was estimated for selected samples using the virus reduction technique (Weinbauer et al. 2010). The rationale behind the virus reduction approach is to reduce virus abundance and thereby essentially prevent new viral infection. Thus, the viruses produced originate from already infected cells. Bacteria from 200 ml of raw seawater were concentrated using a 0.2 µm pore size tangential flow system (VIVAFLOW 50). To obtain virus-free seawater, the 0.2 µm pore-size ultrafiltrate was passed through a 100 kDa cartridge (VIVAFLOW 50). The bacterial concentrates were brought up to the original volume with virus-free seawater and incubated in duplicate 50 ml Falcon tubes (BD Biosciences) at 20 ± 2°C for 24 h. At t₀ of the experiments, 2 additional tubes were amended with mitomycin C (Sigma) at a final concentration of 1 µg ml⁻¹ in order to induce the lytic cycle in prophages. Subsamples (2 ml) for viral abundance were taken every 3 to 4 h from each incubation, fixed with glutaraldehyde (0.5% final concentration), incubated at 4°C for 15 to 30 min, subsequently frozen in liquid nitrogen and stored at -80°C until counted by flow cytometry. Lytic VP was calculated as the increase in viral abundance over short time intervals (~4 h). An increase in viral abundance in the mitomycin C treatments represents lytic + lysogenic production (Paul & Weinbauer 2010, Weinbauer et al. 2010). VP was corrected for the changes in bacterial
abundance in the VP assays. Dividing the number of produced phages by an assumed burst size (BS) of 50 (Parada et al. 2006) yields the number of lysed cells and gives an estimate of the fraction of infected cells (FIC) when divided by the bacterial abundance at the start of the experiment (Weinbauer et al. 2002). Lysis rates were calculated by dividing VP by BS and were used to calculate virus-mediated mortality (VMM) as a percentage of bacterial standing stock (BSS) per day or as a fraction of BHP. Alternatively, VMM was related to FIC using the model by Binder (1999).

## BC particle size and attachment of viruses and bacteria

Associations between BC particles, bacteria, and viruses were investigated by epifluorescence and confocal laser scanning microscopy (CLSM), respectively, in experiment *BC-grad* following the protocol of Cattaneo et al. (2010). The size and number of colonized particles were high enough to allow enumeration only in the treatment with the addition of BC at the highest concentration (2000 µg l⁻¹). Slides were prepared using a slightly modified version of the Noble and Fuhrman procedure (Noble & Fuhrman 1998). Subsamples of 2 ml were fixed with glutaraldehyde (0.5% final concentration) at 4°C for 15 to 30 min, frozen in liquid nitrogen, and stored at -80°C until analysis. Thawed samples were filtered onto 0.02 µm pore size AlO₃ filters (25 mm diameter, Anodisc, Whatman) by low-pressure filtration and stained with SYBR Gold (Molecular Probes, Invitrogen) diluted 1000-fold in autoclaved and 0.2 µm filtered MilliQ water. The filters were transferred onto slides with a mounting solution (0.1% *p*-phenylendiamine; freshly made from a frozen 10% aqueous stock Sigma-Aldrich, P-1519) in 50% glycerol-50% phosphate-buffered saline (PBS, 0.05 M Na₂PO₄, 0.85% NaCl, pH 7.5) and Vectashield (1:6 v:v; Vector, Burlingame). The slides were stored at -20°C until examined under the CLSM.

Initially, abundances of free and BC-attached viruses and bacteria were assessed at 1200-fold magnification with an epifluorescence microscope (Axio-phot, Carl Zeiss). At least 25 microscopic fields were inspected. Colonized BC particles were then investigated with a CLSM (Leica SP2) equipped with an argon neon laser (excitation: 488 nm; emission spectrum: 530–550 nm). For each particle, stacks of images were acquired and 3-dimensional surfaces were measured for each particle as previously described (Cattaneo et al. 2010). The surface area of

particles was converted into ESD (i.e. the diameter of spheres with equivalent volume to nonspherical-shaped particles; Peduzzi & Weinbauer 1993). We quantified the abundance of attached viruses and bacteria, and manual counting was preferentially used over automated counting to ensure the required accuracy of the measurements (Luef et al. 2009, Cattaneo et al. 2010).

## Statistics

All statistical analyses were performed with JMP 7.0 (SAS). The Shapiro-Wilk *W*-test was used to check for normal distribution of data. Analysis of covariance (ANCOVA) and 1-way ANOVA for normal distributions and Kruskal-Wallis tests for non-normal distributions were used to evaluate the differences between treatments. Spearman rank correlation for nonparametric data was performed to determine the relationships between the various parameters measured.

# RESULTS

## Effects of BC on nutrients and DOC

The addition of BC resulted in differences in inorganic nutrients and DOC concentrations between treatments already at $t_0$: light-exposed BC additions produced a significant increase in DOC concentration in experiment *BC-fix* (ANCOVA, *p* < 0.05) compared to the BC-free control. Phosphate concentrations were higher in BC incubations than in the BC-free control, although the differences were only significant in experiment *BC-grad*. In both experiments, average ammonium concentrations were 5- to 6-fold higher with BC additions than in the controls (ANCOVA, *p* < 0.05, Table 1). In most cases, nutrient concentrations did not change significantly over the course of the experiments, but ammonium and nitrate concentrations in experiment *BC-fix* and nitrite concentration in experiment *BC-grad* increased (ANCOVA, Table 1).

## Total BA and the number of active bacteria

In both experiments, BA did not vary significantly with time in the BC-free control nor in the treatment with the addition of 200 µg BC l⁻¹ (ANCOVA, Table 2, Fig. 2). However, BA increased with time and BC con-
<table><caption>Table 1. Mean ± SD values of the nutrient and dissolved organic carbon (DOC) concentrations (µM) analyzed in each of the different treatments as well as results of analyses of covariance with time (*BC-fix*) and with time and black carbon (BC) concentration (*BC-grad*) as covariates. Values are averaged over the experimental time (n = 24). Significantly different values are highlighted in **bold**: **p < 0.001, *p < 0.05; ns: not significant. Treatments are as follows: BC-free: BC-free control; BC-dark: with 200 (*BC-fix*) or 500 and 2000 (*BC-grad*) µg BC l⁻¹ maintained in the dark; BC-light: with 200 (*BC-fix*) or 200, 500, 1000, and 2000 (*BC-grad*) µg BC l⁻¹ exposed to light</caption><thead><tr><th>Experiment</th><th>Treatment</th><th>PO<sub>4</sub><sup>3-</sup></th><th>NH<sub>4</sub><sup>+</sup></th><th>NO<sub>2</sub><sup>-</sup></th><th>NO<sub>3</sub><sup>-</sup></th><th>DOC</th></tr></thead><tbody><tr><td rowspan="3">BC-fix</td><td>BC-free</td><td>0.11 ± 0.02</td><td>0.14 ± 0.03<sup>**</sup></td><td>0.01 ± 0.005</td><td>0.08 ± 0.02</td><td>81.3 ± 3.4</td></tr><tr><td>BC-dark</td><td>0.16 ± 0.03</td><td><strong>0.87 ± 0.23</strong></td><td>0.02 ± 0.003</td><td>0.09 ± 0.03</td><td>80.9 ± 4.7</td></tr><tr><td>BC-light</td><td>0.15 ± 0.03</td><td><strong>0.93 ± 0.42</strong></td><td>0.02 ± 0.01</td><td>0.11 ± 0.04</td><td><strong>91.1 ± 13.6*</strong></td></tr><tr><td>Covariate</td><td>Time</td><td>ns</td><td>*</td><td>ns</td><td>*</td><td>ns</td></tr><tr><td rowspan="3">BC-grad</td><td>BC-free</td><td>0.10 ± 0.00<sup>*</sup></td><td>0.17 ± 0.10<sup>*</sup></td><td>0.02 ± 0.02</td><td>0.07 ± 0.02</td><td>80.3 ± 3.8</td></tr><tr><td>BC-dark</td><td><strong>0.14 ± 0.02</strong></td><td><strong>0.76 ± 0.50</strong></td><td>0.04 ± 0.01</td><td>0.12 ± 0.05</td><td>83.0 ± 6.1</td></tr><tr><td>BC-light</td><td><strong>0.16 ± 0.03</strong></td><td><strong>0.90 ± 0.59</strong></td><td>0.04 ± 0.01</td><td>0.10 ± 0.03</td><td>83.6 ± 9.0</td></tr><tr><td rowspan="2">Covariate</td><td>Time</td><td>ns</td><td>ns</td><td>*</td><td>ns</td><td>ns</td></tr><tr><td>BC concentration</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr></tbody></table>

<table><caption>Table 2. Mean ± SD values of organism abundance and activity analyzed in each of the different treatments as well as results of analyses of covariance with time (*BC-fix*) and time and black carbon (BC) concentration (*BC-grad*) as covariates. Values are averaged over experimental time. Significantly different values are highlighted in **bold**: **p < 0.0001, *p < 0.001, *p < 0.05. Treatments as in Table 1. BHP: bacterial heterotrophic production; BA: total bacterial abundance; PAB: particle-attached bacteria; CTC: 5-cyano-2,3-ditolyl tetrazolium chloride reducing cells; LIVE: membrane-intact cells using the nucleic acid double staining protocol (see Materials and Methods); FVA: free viral abundance</caption><thead><tr><th>Experiment</th><th>Treatment</th><th>BHP µg C l<sup>-1</sup> d<sup>-1</sup></th><th>BA × 10<sup>5</sup> ml<sup>-1</sup></th><th>PAB %</th><th>CTC %</th><th>LIVE %</th><th>FVA × 10<sup>6</sup> ml<sup>-1</sup></th></tr></thead><tbody><tr><td rowspan="3">BC-fix</td><td>BC-free</td><td>1.7 ± 0.2</td><td>2.0 ± 0.2</td><td>-</td><td>62 ± 11</td><td>83 ± 4<sup>***</sup></td><td>12.7 ± 0.5<sup>*</sup></td></tr><tr><td>BC-dark</td><td>1.9 ± 0.1</td><td>2.2 ± 0.2</td><td>-</td><td>64 ± 13</td><td><strong>78 ± 6</strong></td><td><strong>11.3 ± 0.8</strong></td></tr><tr><td>BC-light</td><td><strong>3.0 ± 0.8<sup>***</sup></strong></td><td>2.2 ± 0.2</td><td>-</td><td><strong>56 ± 13<sup>*</sup></strong></td><td><strong>75 ± 6</strong></td><td><strong>11.5 ± 0.7</strong></td></tr><tr><td>Covariate</td><td>Time</td><td>*</td><td>ns</td><td>-</td><td>*</td><td>ns</td><td>ns</td></tr><tr><td rowspan="3">BC-grad</td><td>BC-free</td><td>1.7 ± 0.4</td><td>1.5 ± 0.1</td><td>6 ± 1<sup>***</sup></td><td>49 ± 23</td><td>72 ± 4</td><td>12.7 ± 0.7<sup>**</sup></td></tr><tr><td>BC-dark</td><td>2.4 ± 1.2</td><td>1.8 ± 0.4</td><td><strong>49 ± 10</strong></td><td>54 ± 11</td><td>76 ± 6</td><td><strong>7.3 ± 1.7</strong></td></tr><tr><td>BC-light</td><td>2.6 ± 1.5</td><td>1.8 ± 0.3</td><td><strong>47 ± 10</strong></td><td>54 ± 16</td><td>75 ± 5</td><td><strong>8.2 ± 2.2</strong></td></tr><tr><td rowspan="2">Covariate</td><td>Time</td><td>*</td><td>**</td><td>*</td><td>***</td><td>***</td><td>ns</td></tr><tr><td>BC concentration</td><td>***</td><td>***</td><td>***</td><td>ns</td><td>*</td><td>***</td></tr></tbody></table>

concentration in the other treatments (ANCOVA, p < 0.0001, Table 2, Fig. 2B), reaching 78% higher BA in the light-exposed and 68% higher BA in the dark-exposed BC treatment at 2000 µg BC l⁻¹ with respect to initial values. The percentage of actively respiring cells that reduced 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was significantly lower in the BC-light treatment than in all other treatments in experiment *BC-fix* (ANCOVA, p < 0.05, Table 2). The percentage of membrane-intact cells (LIVE) was significantly lower in the BC treatments than in the control in *BC-fix*, but increased slightly with BC concentration and time in *BC-grad* (ANCOVA, p < 0.0001, Table 2).

## Abundance of free viruses

Initial free viral abundance (FVA) as measured by flow cytometry was 1.26 × 10⁷ and 1.16 × 10⁷

viruses ml⁻¹ in experiments *BC-fix* and *BC-grad*, respectively. In the BC-free controls, these values remained roughly stable from t₀ throughout the experiment *BC-fix* (Fig. 3A) while in *BC-grad* they increased by 8% until t₈h and then stayed approximately unchanged until the end (Fig. 3B). Upon BC amendment, FVA decreased from t₀ in both experiments and remained roughly constant after the first sampling 8 h later (Fig. 3). In *BC-fix*, this decrease amounted to 13% with respect to the BC-free controls. After 80 h, FVA increased in all treatments and finally leveled off at ca. 1.34 ± 0.09 (range) × 10⁷ viruses ml⁻¹ in the controls, and at 1.27 ± 0.03 × 10⁷ and 1.29 ± 0.03 × 10⁷ viruses ml⁻¹ in the light and dark BC treatments, respectively. Average FVA was significantly lower in the BC treatments than in the BC-free controls and was not influenced by BC light exposure (Table 1, Fig. 3A, ANCOVA, p < 0.05).
<figure><img src="image_4.png" /><figcaption>Fig. 2. Bacterial abundance in experiments (A) *BC-fix* and (B) *BC-grad* with addition of light-exposed (BCL) and non-exposed (BCD) black carbon (BC) and under control treatments (BCfree). Values are averages from duplicate treatments and error bars are the ranges in experiment *BC-fix* and in the BC controls of experiment *BC-grad*. BC concentrations are indicated (µg l⁻¹)</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>Fig. 3. As in Fig. 2, but for the abundance of free viruses in experiments (A) *BC-fix* and (B) *BC-grad*. Where not visible, error bars are contained within the symbol</figcaption></figure>

In experiment *BC-grad*, FVA averaged over the experimental time decreased with increasing BC concentrations (ANCOVA, *p* < 0.0001) but was not influenced by BC light exposure (Table 2, Fig. 4B).

The virus-to-bacteria ratio (VBR) ranged from 21 to 109 and averaged 56 ± 16 without differences between experiments. VBR was significantly lower with BC addition only in the experiment *BC-grad* (47 vs. 88, ANCOVA, *p* < 0.05,
<figure><img src="image_6.png" /><figcaption>Fig. 4. (A) Percentage of particle-attached bacteria (PAB) and (B) abundance of free viruses (FVA) in relation to black carbon (BC) concentration in experiment *BC-grad*. When interpolating all data, it is apparent that BC particles were becoming saturated at a BC concentration of 1000 µg BC l⁻¹, and at most about half of the bacteria were becoming attached. PAB increased with BC concentration, while FVA decreased with BC concentration: all PAB = 20% + 0.02 BC concentration (R² = 0.67); light BC treatment: PAB = 18% + 0.02 BC concentration (R² = 0.69); dark BC treatment: PAB = 13% + 0.02 BC concentration (R² = 0.77); Virus: all FVA = 1.17 × 10⁷ – 3344 BC concentration (R² = 0.87); light BC treatment: FVA = 1.20 × 10⁷ – 3721 BC concentration (R² = 0.88); dark BC treatment: FVA = 1.21 × 10⁷ – 3395 BC concentration (R² = 0.88)</figcaption></figure>

covariates: BC concentration: *p* < 0.0001, time: non-significant).

## BC-attached viruses and bacteria and size of the BC particles

In *BC-grad*, the size of BC particles with attached bacteria and viruses as analyzed by CLSM ranged from 4.6 to 22.4 µm ESD (average: 9.5 ± 5.4 µm), but 58% of the particles were smaller than 10 µm ESD throughout the experiment except $t_0$ (details not shown). Fifty-six percent of the particles had fewer than 10 attached bacteria. The abundance of attached bacteria was positively related to BC particle size (BA = -0.03 + 0.94 ESD, n = 16, R² = 0.38, Table 3), and the average number of bacteria per ESD was 0.9 ± 0.7 µm⁻¹. The number of BC-attached viruses ranged between 4 and 64 viruses per particle. A positive correlation was observed between the abundance of BC-attached viruses and BC particle size, while the number of BC-attached viruses per µm² was negatively correlated with BC particle size (Table 3).

At the end of the incubation, in the treatment with the addition of BC at the highest concentration

<table><caption>Table 3. Spearman rank correlation analysis of microbial abundances with black carbon (BC) particle size from the treatments with the addition of 2000 µg BC l⁻¹ kept in the dark in experiment *BC-grad*. VBR: virus-to-bacteria ratio; ns: not significant</caption><thead><tr><th></th><th>p</th><th>n</th><th>p</th></tr></thead><tbody><tr><td>Abundance of attached:</td><td></td><td></td><td></td></tr><tr><td>viruses particle<sup>-1</sup></td><td>0.640</td><td>18</td><td>&lt;0.05</td></tr><tr><td>viruses µm<sup>-2</sup></td><td>-0.612</td><td>18</td><td>&lt;0.05</td></tr><tr><td>bacteria particle<sup>-1</sup></td><td>0.715</td><td>16</td><td>&lt;0.05</td></tr><tr><td>bacteria µm<sup>-2</sup></td><td>-0.441</td><td>16</td><td>ns</td></tr><tr><td>VBR µm<sup>-2</sup></td><td>-0.738</td><td>16</td><td>&lt;0.05</td></tr></tbody></table>

(2000 µg l⁻¹), BC-attached viruses assessed by epifluorescence microscopy accounted for 40% of total viral abundance, and attached bacteria accounted for 50% of the total bacterial abundance. The percentage of particle-attached bacteria measured using the FC approach was only recorded in *BC-grad* and ranged from 4–8% in the BC-free controls (average 6 ± 1%, considered to be the background counts of the technique), to 44–63% (average 57 ± 6%) at 2000 µg BC l⁻¹. The percentage of particle-attached bacteria increased significantly with increasing BC concentration (Fig. 4A, Table 2). Furthermore, per-
centage of particle-attached bacteria correlated positively and significantly with bacterial production (ρ = 0.335, n = 55, p < 0.05) and with the % HNA (ρ = 0.310, n = 55, p < 0.05). No effect of exposure to artificial solar radiation of BC on the percentage of particle-attached bacteria was detected.

## Bacterial heterotrophic production

In *BC-fix*, the addition of irradiated BC led to a significant increase in BHP with respect to the other treatments after 24 h (ANCOVA, p < 0.0001), and this level of activity was maintained throughout the experiment (Fig. 5A). In contrast, BHP in the BC-dark treatment was not significantly different from the BC-free controls and increased by only 32% (vs. 148% in treatment BC-light, Fig. 5A).

In *BC-grad*, BHP increased significantly with increasing BC concentrations in all BC treatments (Table 2, Fig. 5B). BHP amended with light-exposed BC was up to 12 times higher than the initial BHP of 0.7 µg C l⁻¹ d⁻¹, whereas BHP in the dark BC treatment was only higher than the BC-free control at the highest BC concentration (2000 µg BC l⁻¹), reaching 5 µg C l⁻¹ d⁻¹. Time-averaged BHP for the time interval of 2 to 3 d was significantly higher with the addition of light-exposed BC than in the other treatments

(ANCOVA, p < 0.05). The specific growth rate of bacteria showed the same trend as BHP in both experiments (details not shown).

## Viral production and infection

VP was measured at t₀ and after 2 d in all treatments of experiment *BC-fix*. In *BC-grad*, VP was measured only in some of the treatments (Table 4). Tangential flow filtration through a 100 kDa cartridge removed viruses efficiently, resulting in <1% of the original viral abundance. At t₀, viral abundance in the VP incubations ranged between 9 and 40% of the original viral abundance. These viruses were introduced along with the bacterial concentrate into the incubations for estimating VP.

In *BC-fix*, lytic VP increased from 1.3 ± 0.3 × 10⁶ viruses ml⁻¹ d⁻¹ at t₀ to 2.7–24.3 × 10⁶ viruses ml⁻¹ d⁻¹ after 2 d of incubation (Table 4). VP was significantly lower in the dark BC treatments compared to the BC control and the light-exposed BC treatments (Table 4, Kruskal-Wallis test, p < 0.05). At t₀, 12 ± 2% bacteria were infected, corresponding to an estimated viral lysis rate of 2.5 ± 0.6 × 10⁴ bacteria ml⁻¹ d⁻¹. At t₂d, significantly fewer bacteria were infected in the dark BC treatment than in the BC control and in the light BC treatment, and estimated viral lysis rates were signifi-

<figure><img src="image_7.png" /><figcaption>Fig. 5. Bacterial heterotrophic production in experiments (A) *BC-fix* and (B) *BC-grad* with addition of light-exposed (BCL) and non-exposed (BCD) black carbon (BC) and under control treatments (BCfree). Values are averages from duplicate treatments and error bars are the ranges in experiment *BC-fix* and in the BC controls of experiment *BC-grad*. BC concentrations are indicated (µg l⁻¹)</figcaption></figure>
<table><caption>Table 4. Viral production (VP, viruses ml⁻¹ d⁻¹), frequency of infected cells (FIC, %), bacterial lysis rates and virally mediated loss of bacterial standing stock (VMM<sub>SS</sub>, % d⁻¹) and viral mediated mortality related to FIC (VMM, %) by a model (Binder 1999). Data are given as means ± range of duplicate measurements. Significantly different values for treatments are highlighted in **bold** (p < 0.05, Kruskal-Wallis test). Treatments as in Table 1</caption><thead><tr><th>Experiment</th><th>Time (d)</th><th>Treatment</th><th>VP × 10<sup>6</sup> ml<sup>-1</sup> d<sup>-1</sup></th><th>FIC %</th><th>Lysis rate × 10<sup>4</sup> ml<sup>-1</sup> d<sup>-1</sup></th><th>VMM<sub>SS</sub> % d<sup>-1</sup></th><th>VMM %</th></tr></thead><tbody><tr><td rowspan="4">BC-fix</td><td>0</td><td></td><td>1.3 ± 0.2</td><td>12 ± 2</td><td>2.5 ± 0.4</td><td>12 ± 2</td><td>25 ± 4</td></tr><tr><td rowspan="3">2</td><td>BC-free</td><td>9.9 ± 2.9</td><td>20 ± 5</td><td>19.7 ± 5.8</td><td>101 ± 26</td><td>46 ± 17</td></tr><tr><td>BC-dark</td><td><strong>3.6 ± 0.9</strong></td><td><strong>10 ± 3</strong></td><td><strong>7.2 ± 1.8</strong></td><td><strong>34 ± 9</strong></td><td>21 ± 8</td></tr><tr><td>BC-light</td><td>15.4 ± 9.5</td><td>27 ± 16</td><td>30.8 ± 19.1</td><td>140 ± 85</td><td>88 ± 68</td></tr><tr><td rowspan="4">BC-grad</td><td>0</td><td></td><td>2.0 ± 0.9</td><td>8 ± 3</td><td>4.0 ± 1.8</td><td>28 ± 12</td><td>16 ± 7</td></tr><tr><td rowspan="3">2</td><td>BC-free</td><td>15.2 ± 1.8</td><td>25 ± 2</td><td>30.5 ± 3.5</td><td>183 ± 21</td><td>63 ± 8</td></tr><tr><td>BC-dark 500</td><td><strong>2.2 ± 2.2</strong></td><td><strong>3 ± 3</strong></td><td><strong>4.4 ± 4.4</strong></td><td><strong>24 ± 24</strong></td><td><strong>6 ± 6</strong></td></tr><tr><td>BC-light 500</td><td><strong>6.2 ± 0.6</strong></td><td><strong>9 ± 1</strong></td><td><strong>12.3 ± 1.2</strong></td><td><strong>71 ± 7</strong></td><td><strong>19 ± 2</strong></td></tr><tr><td></td><td></td><td>BC-light 2000</td><td><strong>1.3 ± 1.3</strong></td><td><strong>2 ± 2</strong></td><td><strong>2.7 ± 2.7</strong></td><td><strong>14 ± 14</strong></td><td><strong>3 ± 3</strong></td></tr></tbody></table>

cantly lower in BC-dark (7.2 ± 1.8 × 10⁴ bacteria ml⁻¹ d⁻¹) than in the other treatments (26.0 ± 15.1 × 10⁴ bacteria ml⁻¹ d⁻¹, Table 4, Kruskal-Wallis test, p < 0.05). Virus-mediated mortality of bacterial standing stock per day (VMM<sub>SS</sub>) varied between 12% d⁻¹ at t₀ and >100% d⁻¹ in the light-exposed BC treatment at t₂d and was significantly lower in the dark BC treatment than in the light-exposed BC treatment and the BC control (Table 4, Kruskal-Wallis test, p < 0.05). The estimated virally mediated loss of BHP ranged from 33% at t₀ to >100% in the light-exposed BC treatment and BC-free control without significant differences between treatments. Following the model by Binder (1999), VMM ranged from 7% in the dark-exposed BC treatment to >100% in the light-exposed BC treatment without differences between treatments.

In BC-grad, lytic VP increased from 2.0 ± 0.9 × 10⁶ at t₀ to 15.2 ± 1.8 × 10⁶ viruses ml⁻¹ d⁻¹ at t₂d in the BC-free control and was significantly higher than in the BC-amended treatments (3.2 ± 2.8 × 10⁶ viruses ml⁻¹ d⁻¹, Table 4, Fig. 6, Kruskal-Wallis test, p < 0.05). At t₀, 8 ± 3% of bacteria were infected. At t₂d, FIC was significantly higher in the BC-free control than in the BC-amended treatments (Table 4, Kruskal-Wallis test, p < 0.05). Consequently, estimated viral lysis rates were, on average, lower with BC addition (6.5 ± 5.7 × 10⁷ bacteria ml⁻¹ d⁻¹) than in the BC-free control (Table 4, Kruskal-Wallis test, p < 0.05). The resulting VMM<sub>SS</sub> values were significantly lower in the presence of BC (on average 36 ± 33% d⁻¹) compared to the control (>100% d⁻¹, Table 4, Kruskal-Wallis test, p < 0.05). According to the model by Binder (1999) VMM was significantly lower with BC addition (9 ± 9%) than in the BC-free control (63 ± 8% Table 4, Kruskal-Wallis test, p < 0.05). No lysogens could be induced by mitomycin C in any of the experiments.

<figure><img src="image_8.png" /><figcaption>Fig. 6. Viral production (VP, viruses ml⁻¹ d⁻¹) and the fraction of infected cells (FIC, %) in experiment BC-grad from Day 2 as a function of black carbon (BC) concentration. Values are averages of duplicates from the virus reduction approach to measure VP and FIC, and error bars are the ranges. BCL: light-exposed BC; BCD: non-exposed BC</figcaption></figure>

DISCUSSION

The effects of BC on bacterial abundance, production, and viral-mediated loss of bacterial production were assessed in this study using BC reference material and aged marine microbial communities. The main findings were that (1) BC addition increased bacterial biomass accumulation and reduced VP and infection and (2) exposure of BC to UV light increased bacterial production. These results suggest a stimulating effect of BC on aggregation processes and bacterial production and a suppressing effect on the viral shunt and associated nutrient regeneration with the potential to increase system heterotrophy.
## Interaction between microorganisms and BC particles

With the help of flow cytometry, we detected an additional bacterial population characterized by high SSC (Fig. 1). Since this population with high SSC but comparable SYBR green derived fluorescence to HNA bacteria was not observed in the BC-free controls, it is likely that it consisted of bacteria associated with particles. The strong correlation between this bacterial FC population and BC concentration (Fig. 4A) supports the notion that these were BC-associated bacteria. Also, the similarity of the data on the fraction of particle-associated bacteria data obtained by FC and by epifluorescence microscopy supports this idea. The values of the side scatter in the FC cytograms for this type of particle increased during the course of the experiment (data not shown). Assuming that light scatter is proportional to the size of the particles (Koch et al. 1996), the data indicate that particles were increasing in size. Hence, a progressive aggregation of BC including colloids and bacterial cells into larger particles might have taken place. Different stages of association between bacteria and BC material, which range from small BC colloids attached to bacteria to bacteria completely entrapped in BC have been shown previously (Cattaneo et al. 2010), suggesting BC-driven aggregation and particle formation. This is supported by findings that BC reference material stimulated the formation of a specific class of organic aggregates, the transparent exopolymeric particles (Weinbauer et al. 2012, Mari et al. 2014). Also, *in situ* evidence suggests that soot deposition can increase coagulation and aggregation of organic matter (Mari et al. 2014).

Viral abundance per unit surface of BC decreased with BC particle size similarly to patterns previously observed in BC reference material (Cattaneo et al. 2010) and in other suspended material (Simon et al. 2002, Luef et al. 2007, Mari et al. 2007). The decreasing concentration of attached viruses with increasing particle size can be assigned to the fractal structure of BC particles (Slowik et al. 2007) which translates into increasing porosity with increasing particle size. Increasing porosity implies a higher content of pore water with potentially lower microbial and viral densities.

### Effect of BC on viral production and infection

Free viral abundance was significantly lower in the BC treatments than in the controls (Table 2) and de-

creased with BC concentrations (Fig. 4B). Viruses were found attached to BC particles as in experiments performed with water from the Mediterranean Sea (Cattaneo et al. 2010). VP and the fraction of infected cells were also significantly lower in the BC-amended treatments (Table 4, Fig. 6) except for the BC-light treatment in *BC-fix*. In this treatment, the negative effects of BC on VP as experienced in the dark BC treatment (see discussion below) could have been compensated by an increased viral infection due to the stimulated bacterial production in BC-light (see below) with the net outcome of no detectable effect of BC at a concentration of 200 µg l⁻¹.

Our data suggest that adsorption onto BC particles inactivated viruses and prevented infection of attached cells, or reduced infection by reducing the abundance of free viruses and thus encounter rates. Similarly, in a study with water from the Danube River, free viruses were scavenged on particles (Kernegger et al. 2009). Such a mechanism could have resulted in the increased BHP in the BC treatments as also observed for suspended material in the Danube River. Interestingly, particle quality was a determining factor for microbial attachment in these freshwater experiments, and significantly fewer viruses attached to mineral than to organic particles (Kernegger et al. 2009). In accordance, the addition of mineral particles, such as Saharan dust (Weinbauer et al. 2009, Pulido-Villena et al. 2014) or clay particles (Salter et al. 2011) to marine coastal waters led to a negligible sorption of viruses to particles and increased VP rates in contrast to the response of the microbial community to BC addition in the present study.

VP in solar radiation-exposed BC treatments was significantly stimulated with respect to the non-exposed BC (Table 4). This could be traced to the stimulation of bacterial production in BC-light treatments, since viral infection and production are related to bacterial activity and production (Womack & Colwell 2000). Also, viral adsorption or inactivation by BC could have been reduced due to UV-exposure, thus allowing for higher infection rates.

### Effect of BC on bacterial production

The addition of BC stimulated bacterial production in both experiments (Table 2). The positive correlation found between particle-associated bacteria and heterotrophic bacterial production as well as HNA bacteria, which are often the more active bacterial fraction (e.g. Gasol et al. 1999), suggests that
attached bacteria were particularly active and
contributed significantly to the measured bacterial
production. Such an increased metabolic activity of
attached bacteria has been found following the up-
regulation of enzyme activity upon colonization of
organic aggregates (Riemann et al. 2000, Simon et al.
2002, Grossart et al. 2007). Historically, BC was as-
sumed to be highly refractory (Schmidt & Noack
2000), but an increasing body of literature shows abi-
otic (Decesari et al. 2002, Lehmann et al. 2005, Cheng
et al. 2006, Stubbins et al. 2010, 2012) and biotic oxi-
dation of BC in soils (Potter 1908, Shneour 1966,
Cheng et al. 2006, 2008, Zimmerman 2010) and sedi-
ments (Middelburg et al. 1999). For example, oxida-
tion of turbidite in the Madeira Abyssal Plain
removed about 77% and 64% of organic and soot
carbon, respectively, thus challenging the assumption
that BC is biologically and chemically recalcitrant
(Middelburg et al. 1999). However, since the quantifi-
cation of BC was beyond the scope of this study, there
is no direct evidence of BC decomposition or its use as
a carbon source during our experiments. The addition
of ammonium along with the BC material (Table 1)
could have stimulated bacterial production, since the
simultaneous addition of ammonium and organic car-
bon can stimulate bacterial production (Kirchman &
Rich 1997). However, the use of aged seawater and
the finding that ammonium concentrations did not
decrease with incubation time argues against such
a mechanism. Another mechanism explaining in-
creased bacterial activity could be the adsorption of
DOC to BC particles, which could create hot spots of
microbial activity similar to those observed in marine
snow particles (Azam & Malfatti 2007). It is well
known that soot particles can interact with organic
compounds (Rockne et al. 2000, Slowik et al. 2007)
and redistribute them in the environment (Ahrens &
Morrisey 2005). There is additional evidence (Catta-
neo et al. 2010, this study) that viruses, which are by
definition part of the DOC pool, are rapidly adsorbed
to BC. Overall, it is well known that organic com-
pounds adsorb very efficiently to materials such as
BC, generally exceeding adsorption for typical amor-
phous organic matter by a factor of 10 to 100 (Cor-
nelissen & Gustafsson 2005, Cornelissen et al. 2005).

In both experiments, heterotrophic bacterial pro-
duction was significantly higher after exposing BC
to solar radiation. Photo-oxidation can induce chemi-
cal changes in the highly aromatic structure of BC-
like molecules (Stubbins et al. 2010), thus offering
sites for degradation and increasing BC bioavailabil-
ity. Stubbins et al. (2012) reported a 20-fold decrease
in marine dissolved BC concentration after exposure

of North Atlantic Deep Water to simulated solar radi-
ation confirming the high photo-lability of dissolved
BC. Maki et al. (2001) found that the exposure of
biodegraded crude oil to sunlight significantly
decreased the aromatic fraction and that this material
favored the growth of seawater microbes as com-
pared to non-irradiated material (Maki et al. 2001).
Our data suggest that light exposure of BC in surface
waters can alter BHP. As aerosols are normally
exposed to solar radiation during atmospheric trans-
port with radiation levels potentially exceeding those
observed in surface waters, our results also suggest
that the distance from the BC source and its resi-
dence time in the atmosphere prior to the deposition
on the ocean surface could be an important para-
meter modulating the effect of BC on the marine
ecosystem.

CONCLUSIONS

Our study indicates that BC deposition in the ocean
can stimulate heterotrophic bacterial production
either by reducing the impact of viral lysis, by serv-
ing as a carbon source to bacteria, and/or by serving
as hotspots due to organic matter adsorption. Our
experiments also indicate that atmospheric transport
of BC, and the residence time in surface waters, will
likely influence the effect on the microbial commu-
nity, since exposure to solar radiation increased BC
availability. Fossil-fuel and biomass burning will
probably further increase in the future (Novakov et
al. 2003), and the impact of BC on the functioning of
the marine microbial food web is likely to become
more important, particularly in coastal oceans. One
of the potential consequences could be a shift in
the metabolic balance of the planktonic ecosystem
towards a more heterotrophic ocean.

*Acknowledgements.* We thank I. Lekunberri for help during the experiments. Previous reviewers contributed to improv- ing the original manuscript with detailed comments and cor- rections. Financial support was provided by the European Union in the framework of the BASICS project (EVK3-CT- 2002-00078), by the Eur-Oceans Network of Excellence (Project Number WP4-SYSMS-1021), and a Spanish grant from the ministry of education (SB2010-0079) to A.M. J.M.G. is supported by grant STORM from the Spanish MICINN.

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Editorial responsibility: Hans-Georg Hoppe,
Kiel, Germany

Submitted: June 12, 2014; Accepted: January 29, 2015

Proofs received from author(s):