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1 Environmental Microbiology

2 Grazing of particle-associated bacteria—an

3 elimination of the non-viable fraction

4 Q1 Maria-Judith Gonsalves ∗ , Sheryl Oliveira Fernandes, M. Lakshmi Priya,

5 P.A. LokaBharathi
6 Q2 Biological Oceanography Division, CSIR-National Institute of Oceanography, Dona Paula, Goa 403004, India
7

8 a r t i c l e i n f o a b s t r a c t
9

10 Article history: Quantification of bacteria being grazed by microzooplankton is gaining importance since
11 Received 5 February 2015 they serve as energy subsidies for higher trophic levels which consequently influence fish
12 Accepted 7 April 2016 production. Hence, grazing pressure on viable and non-viable fraction of free and particle-
13Q3 Available online xxx associated bacteria (PAB) in a tropical estuary controlled mainly by protist grazers was
Associate Editor: Welington Luiz de estimated using the seawater dilution technique. In vitro incubations over a period of 42 h
Araújo showed that at the end of 24 h, growth coefficient (k) of PAB was 9 times higher at 0.546
14 than that of free forms. Further, ‘k’ value of viable cells on particles was double that of free
15 Keywords: forms at 0.016 and 0.007, respectively. While bacteria associated with particles were grazed
16 Bacteria (coefficient of removal (g) = 0.564), the free forms were relatively less grazed indicating that
17 Ciliates PAB were exposed to grazers in these waters. Among the viable and non-viable forms, ‘g’
18 Grazing of non-viable fraction (PAB = 0.615, Free = 0.0086) was much greater than the viable fraction
19 Particle (PAB = 0.056, Free = 0.068). Thus, grazing on viable cells was relatively low in both the free and
20 Protists attached states. These observations suggest that non-viable forms of PAB were more prone
21 Viable to grazing and were weeded out leaving the viable cells to replenish the bacterial standing
stock. Particle colonization could thus be a temporary refuge for the “persistent variants”
where the viable fraction multiply and release their progeny.
© 2016 Published by Elsevier Editora Ltda. on behalf of Sociedade Brasileira de
Microbiologia. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).

gained importance. Predation by microzooplankton in aquatic 27

Introduction habitats is known to decrease bacterial abundance and stimu- 28

late mineralization of nutrients.3 The process could influence 29

22 Bacteria are capable of consuming more than 50% of pri- the morphological structure, taxonomic composition4 and 30
23 mary production in the form of dissolved organic matter.1,2 physiological status of bacterial communities.5 The graz- 31
24 Thus, they play an important role as energetic subsidies ers (microzooplankton) consist of holoplanktonic (protozoa, 32
25 for higher trophic levels. In recent years, studies focusing ciliates, flagellates, copepod nauplii, etc.) and meroplank- 33
26 on grazing of bacteria by bacteriophagous microfauna have tonic organisms (larval stages of benthic invertebrates:

∗
Corresponding author at: Aqua-geomicrobiology Laboratory, CSIR-National Institute of Oceanography, Dona Paula, Goa 403004, India.
E-mail: mjudith@nio.org (M. Gonsalves).
http://dx.doi.org/10.1016/j.bjm.2016.10.009
1517-8382/© 2016 Published by Elsevier Editora Ltda. on behalf of Sociedade Brasileira de Microbiologia. This is an open access article
under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: Gonsalves M, et al. Grazing of particle-associated bacteria—an elimination of the non-viable fraction. Braz J
Microbiol. (2016), http://dx.doi.org/10.1016/j.bjm.2016.10.009 BJM 172 1–6
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34 trochophores, veligers, etc.). Protozoans are known to play a up incubations with whole and diluted seawater to determine 87

35 major ecological role in the aquatic environment due to their bacterial abundance (total bacterial cells, PAB and free-living). 88

36 effectiveness in consuming a wide range of prey size classes A schematic representation of the experimental design is 89

37 and types.6 They are capable of consuming other protozoa,7 shown in Fig. S2. 90

38 phytoplankton8 and bacteria.9–11 Their grazing ability depends For preparing particle-free water (used for diluting whole 91

39 on the concentration of bacteria and digestion capacity of seawater), the filtration assembly was washed with 50% HCl 92

40 the grazer. Some ubiquitously distributed protists like ciliates and rinsed with Milli-Q water. Two litres of seawater was then 93

41 occasionally form a major component of the microzooplank- filtered through a 0.22 ␮m filter (pressure < 100 mm Hg). Thus, 94

42 ton community12,13 and have a key position in plankton food to prepare ‘diluted’ samples for incubation, untreated ‘whole’ 95

43 webs as major consumers of pico- and nano-plankton.14,15 sample water was added to particle-free water in the ratio 1:4. 96

44 Availability and concentration of the prey size and type, The PAB were considered as those that would be retained 97

45 attached vs. unattached cells and viable vs. non-viable cells on a 3 ␮m pore-size filter (Fig. S2). Bacteria that passed through 98

46 are some of the factors affecting ciliate grazing rates. these filters were considered as free-living bacteria. An esti- 99

47 Among the planktonic bacterial community, particle- mate of the total and free-living bacterial population in 100

48 attached bacteria (PAB) have received more emphasis than the whole and diluted seawater was carried out. The abun- 101

49 free-living forms because of their relatively easier accessi- dance of PAB was derived by subtracting the free-living from 102

50 bility to filter feeders16,17 and their ability to improve the the total bacterial population. Controls were maintained 103

51 nutritional quality of the particles.18 The PAB can be directly for bottle effect and these were used to subtract from the 104

52 grazed by larger metazoans, bypassing consumption by proto- experimental values. Diluted and whole water samples were 105

53 zoan grazers and short-circuiting the microbial loop.16 Reports incubated for up to 42 h at 27 ± 2 о C under static conditions. 106

54 on pelagic bacterial community have demonstrated that it is Sub-samples were removed from the whole and diluted sea- 107

55 unwarranted to club particle-attached or unattached bacte- water bottles at 6 h intervals for the estimation of TC (total 108

56 ria as a single unit.19 To understand grazing preferences on bacterial cells), the total, viable and non-viable fraction of 109

57 the bacterial community in a tropical estuary dominated PAB and free-living forms. For the enumeration of total bac- 110

58 by protists, we conducted a seawater dilution experiment terial cells (TC), a 5 mL aliquot of water sample was fixed 111

59 to measure the coefficients of increase ‘k’ and grazing ‘g’.20 using 250 ␮L of buffered formalin (2% final concentration) as 112

60 The following queries were put forth: (1) do grazers exhibit described by Hobbie et al.28 For enumeration of viable cells 113

61 selective preference for free or particle-associated bacteria (VC), 5 mL of seawater samples were amended with 0.001% 114

62 (PAB) and (2) which physiological state (viable or non-viable) final concentration of yeast extract and 0.0016% final concen- 115

63 of free or particle-associated bacteria of the bacterioplank- tration of antibiotic cocktail solution containing piromedic, 116

64 ton will be eliminated by the grazers. Though previous work pipemedic and nalidixic acid which were in the ratio of 1:1:1.29 117

65 has highlighted the importance of PAB as food for graz- Samples were incubated statically in dark for 6 h. At the end 118

66 ers like microzooplankton16,17,21 for the first time we have of the incubation, the aliquots were fixed with 2% of buffered 119

67 demonstrated elevated grazing pressure on non-viable cells formalin. For TC and VC, 1 mL of sub-sample was filtered 120

68 of PAB and conservation of their viable forms for stock over a 0.2 ␮m black Isopore polycarbonate filter paper (Milli- 121

69 replenishment. pore Corp., MA, USA) and stained with acridine orange (final 122

concentration 0.01%, w/v). The samples were incubated for 123

2 min and then filtered. Bacterial cells retained on the filter 124
Materials and methods papers were counted using Nikon 50i epifluorescence micro- 125

scope equipped with a 100× oil immersion objective. Viable 126

70 Study area and sampling cells refer to those which enlarge using the direct viable count 127

(DVC) procedure.30 These enlarged cells comprise of a mix- 128

71 A Niskin sampler was used to collect water samples at 3 m ture of cells which have increased in size and actively dividing 129
72 depth in Dona Paula Bay (15◦ 27 N, 73◦ 48 E; Fig. S1) which is cells. Thus, in this study, only cells that enlarge or replicate are 130
73 located at the terminus of Zuari estuary in west coast of India. counted as viable while the non-viable forms are considered as 131
74 This semi-enclosed bay is undisturbed by seaport and riverine the ones which did not enlarge/replicate. Bacterial abundance 132
75 traffic. Details on the climatic conditions, annual variation in has been expressed as cells L−1 . 133
76 hydrographic parameters and biotic variables in the study area The specific rate of increase in bacterial cells ‘k’ and mor- 134
77 have been described elsewhere.22 The temperature, salinity tality/decrease ‘g’ of bacterioplankton were estimated. The 135
78 and pH of seawater during sampling were 24 ◦ C, 21 psu and constants k and g are coefficients of population increase and 136
79 6.3, respectively. grazing mortality, respectively.23 These constants also refer 137

to increase and removal not necessarily linked to growth 138

80 Grazing studies and death. Therefore, it would mean an increase in one 139

pool i.e., particle association by colonization, and decrease 140

81 The dilution technique23 is used for grazing studies in in another pool i.e., free-living forms. Both these coefficients 141

82 the present work. The technique has been widely used to may vary with time of day without affecting our comparisons 142

83 reduce the number of predators by creating a gradient.24 of increase in bacterial numbers in the dilution over a fixed 143

84 The method has also been used to estimate growth rates of period of incubation. Thus, the abundance and grazing mortal- 144

85 phytoplankton/bacterioplankton25,26 as well as grazing pres- ity of bacterioplankton was inferred from observed changes in 145

86 sure exerted by microzooplankton on these forms.26,27 We set population. 146

Please cite this article in press as: Gonsalves M, et al. Grazing of particle-associated bacteria—an elimination of the non-viable fraction. Braz J
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Whole seawater
16.0 A Table 1 – Coefficients of increase and removal of
Abundance (x 10 9 cells L-1)

14.0 bacterioplankton.
12.0 Bacterial Coefficient of Coefficient of k−g
10.0 fraction increase (k) removal (g)
8.0
PAB TC 0.546 0.564 −0.018
6.0
PAB VC 0.016 −0.056 0.072
4.0 PAB NVC 0.528 0.615 −0.087
2.0 Free TC 0.064 −0.077 0.141
0.0 Free VC 0.0066 −0.068 0.0746
0 6 12 18 24 30 36 42 Free NVC 0.074 0.0086 0.0654

Note: PAB, particle-associated bacteria; TC, total bacterial cells;

Diluted seawater
16.0 VC, viable bacterial cells; NVC, non-viable cells. Coefficient of
B increase includes actively replicating/enlarged cells and net col-
Abundance (x 10 9 cells L-1)

14.0
onization/exchange from the free to the particle-associated pool
12.0
and vice versa. The coefficient of increase of non-viable forms
10.0 was higher than that of viable ones as these include non-
8.0 replicating/non-enlarged cells.
6.0
4.0
Likewise, the peak in PAB abundance in whole and diluted 172
2.0
seawater was observed at the end of 36 and 24 h, respec- 173
0.0
tively. The maximum abundance of PAB in whole seawater 174
0 6 12 18 24 30 36 42
was 8.10 ± 0.73 × 109 cells L−1 (Fig. 1A). 175
Time (h)
The viable PAB abundance was not affected both in 176
TC PAB Free-living bacteria whole (R2 = 0.6937, p < 0.05, n = 5; Fig. 2B) and diluted samples 177

(R2 = 0.2204). In diluted seawater, PAB abundance was higher 178

Fig. 1 – Abundance of total, particle-associated and
by one order i.e., 1.20 ± 0.04 × 1010 cells L−1 (Fig. 1B). 179
free-living bacteria in whole (A) and diluted (B) seawater.
The non-viable fraction increased over time (R2 = 0.6845, 180

p < 0.05, n = 5; Fig. 2C) with a k value of 0.528 (Table 1) in com- 181

parison to the viable forms (k = 0.016). However, removal of 182

147 Microzooplankton density and diversity non-viable PAB by grazers was far greater (g = 0.615; Table 1) 183

than that of viable cells (Table 1). Free-living cells were far 184

148 Due to low density of plankton in the study area, 10 L of sea- lesser in abundance compared to PAB and were slow growers 185

149 water was obtained for the enumeration of microzooplankton as observed from the low k values (Table 1). Their numbers 186

150 density and diversity. The water sample was filtered through in whole seawater increased from 0.81 ± 0.07 × 109 cells L−1 187

151 a 200 ␮m mesh to exclude mesozooplankton. Further, this fil- at the beginning of the incubation period to a maximum 188

152 tered water was slowly passed through a net of 20 ␮m mesh of 6.25 ± 0.66 × 109 cells L−1 at the end of 30 h (Fig. 1A). The 189

153 size. The seawater was then concentrated to 1 L by siphoning increase in abundance of free-living non-viable cells was seen 190

154 out excess water with the help of a Tygon tubing (Saint-Gobain to be strongly dependent on the incubation time (R2 = 0.7725, 191

155 Performance Plastics Corporation, USA). The dipped end of the p < 0.05, n = 5; Fig. 2F). In diluted seawater, a steady increase in 192

156 tubing was covered with 20 ␮m mesh. Thereafter, the sample free-living bacterial cells was observed up to 36 h where they 193

157 was preserved in 1% acid Lugol’s solution and left to set- numbered about 3.34 ± 0.22 × 109 cells L−1 (Fig. 1B). Increase 194

158 tle for 24 h and further concentrated to 10 mL by siphoning in the abundance of viable free-living cells over time was 195

159 out the excess water. One millilitre of concentrated sample marginally higher (R2 = 0.3756, n = 5; Fig. 2E) than the non- 196

160 was transferred into a Sedgwick-Rafter chamber to enumerate viable fraction (R2 = 0.3653, n = 5; Fig. 2F). Removal of free-living 197

161 microzooplankton abundance. Counting was done using an forms by grazers was found to be negligible (Table 1). 198

162 inverted phase microscope fitted with a Whipple grid. About

163 30–40 fields representing 150–200 individuals were counted. Microzooplankton density and diversity 199

164 The microzooplankton were identified to genus and/or species

165 level. Their density has been expressed as individuals L−1 . Leprotintinnus nordquisti and Stenosemella sp. dominated the 200

microzooplankton community in the Dona Paula bay at an 201

abundance of 25 and 24% of the total, respectively (Fig. S3). 202

Results
Other important microzooplankton included Favella sp. (12%), 203

Dictyocysta sp. (8%), Codonellopsis sp. (8%) and Tintinnopsis 204

166 Grazing of particle-associated and free-living bacteria
uryguayensis (5%). 205

167 The abundance of bacterial cells in whole seawater increased

168 steadily over time and peaked at the end of 36 h to reach Discussion
169 1.16 ± 0.06 × 1010 cells L−1 (Fig. 1A). In the diluted sample, peak
170 in cell abundance was observed at 24 h of incubation and was Bacteria are relevant members of the planktonic food web, 206

171 marginally higher at 1.51 ± 0.09 × 1010 cells L−1 (Fig. 1B). both in terms of biomass and production share. However, 207

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12.0 6.0
A D

Free-living_TC (x 109 cells L-1)

PAB_TC (x 10 9 cells L-1)

10.0 5.0

8.0 4.0
y=0.426x – 1.46
2
6.0 R =0.7307 3.0
y=–0.0087x + 2866 y=0.1323x+0.986
2 2
4.0 R =0.017 R =0.622
2.0 y=0.0847x+0.468
2
2.0 R =0.6238
1.0

0.0
0.0
0 6 12 18 24
0 6 12 18 24
2.0 3.0
B E

Free-living_VC (x 10 9 cells L-1)

PAB_VC (x 10 9 cells L-1)

1.6 2.5

2.0
1.2 y=0.0255x + 0.258
y=0.0602x + 0.116 2
2
R =0.6937 R =0.2204
1.5
0.8
y=0.0737x + 0.128
1.0 2
0.4 R =0.4477
0.5
y=0.0183x + 0.196
0.0 2
R =0.3756
0 6 12 18 24 0.0
Time (h) 0 6 12 18 24
12.0 3.0
C Free-living_NVC (x 10 9 cells L-1) F
PAB_NVC (x 10 9 cells L-1)

10.0 y=0.4242x – 1688 2.5

2
R =0.6845
8.0 2.0

6.0 y=0.0693x + 0.572

1.5 2
y=–0.0777x + 3.16 R =0.7725
y=0.0598x + 0.208
4.0 2
R =0.3449 1.0 2
R =0.3653
2.0
0.5
0.0
0.0
0 6 12 18 24
0 6 12 18 24
Time (h) Time (h)

Whole seawater Diluted seawater

Fig. 2 – Regression analysis of total (A), viable (B) and non-viable (C) particle-associated bacteria (PAB) in whole () and
diluted () seawater. Regression plots for total, viable and non-viable free-living bacteria are shown in (D), (E) and (F),
respectively. Regression line for whole seawater samples has been depicted using a solid line whereas the dashed lined is
for diluted seawater samples. The R2 values denoted in the figures have been converted to the correlation coefficient (r).
Subsequently, the level of significance (p) for a two-tailed test was inferred from a standard table (Wheater and Cook,
2000)43 and was accepted at p < 0.05.

208 bacterial mortality/loss occurs largely due to protist grazing31 At the time of sampling in the Dona Paula bay, tintinnid cil- 227

209 whereas, viral lysis accounts for ∼10–20% of the loss.32 In iates viz., L. nordquisti and Stenosemella sp. were dominant 228

210 aquatic systems, PAB are known to constitute about 60% of the (Fig. S3). There was a considerable bacterial load in the study 229

211 total bacterial counts. In the present study, microscopic exam- area which implies that the bacterivorous ciliates did not face 230

212 ination revealed that PAB formed up to 76% of the TC (Fig. 1B) in any limitation for food. It may be noted that the experimental 231

213 whole seawater whereas, the free-living bacterial cells varied approach used in the present study did not directly measure 232

214 between 26 and 82% (Fig. 1C). Our attempt to demonstrate the grazing. Therefore, all references to grazing are inferred from 233

215 effects of grazing on particle-attached and free bacterial pop- indirect observations. We intend to study the impacts of graz- 234

216 ulation in natural samples using dilution method, estimated ing on bacteria by ciliates in our future research. 235

217 an increase and removal coefficients of PAB and free-living An increase of viable cells in both diluted and whole 236

218 bacterial cells. This approach minimized the grazing pressure water samples (Fig. 2B and E) was observed. The increase 237

219 on bacterial cells as evident from a peak in their abundance was more prominent in the latter with a negative coefficient 238

220 within 24 h of sample incubation as compared to the undi- of removal suggesting avoidance/escape. More viable cells 239

221 luted sample where the peak was at 36 h (Fig. 1B). Based on perhaps get added from the free pool besides the increase 240

222 the g values (Table 1), the grazing pressure on PAB was far by multiplication on particles. In contrast, the coefficient of 241

223 greater than on free-living cells. In aquatic systems, bacte- increase for PAB NVC was marginally lesser than their rate 242

224 ria attached to detrital particles serve as an important food of removal (Table 1) indicating their elimination as soon as 243

225 source for zooplankton33 e.g., ciliates which transfer signifi- they accumulate. This in turn leaves the viable cells relatively 244

226 cant amount of bacterial production to higher trophic levels.34 intact. 245

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246 Not only do the free viable cells but also the particle-
247 associated viable ones escape grazing. We observed negative
Acknowledgements
248 grazing coefficients for viable cells of PAB and free-living
249 bacteria. Attached bacterial communities are known to release This paper was supported by grants from the Office of Naval 299

250 progeny into the surrounding water.35 Consequently, a general Research, Washington, D.C., USA. The authors are thankful 300

251 exchange of attached and free bacteria36 in the viable state to the Director, NIO for the facilities. The authors thank the 301

252 could be expected. As the coefficient of increase of PAB was two anonymous reviewers for their valuable comments. This 302

253 more than 2× higher than that of free-living forms (Table 1), is NIO contribution no. 5885. 303

254 it is apparent that free cells attach and multiply on particles.

255 The adherence of bacteria to detrital particles and ability to Appendix A. Supplementary data
256 rapidly reproduce is an important response for survival in
257 the pelagic marine environment.37 It is possible that bacte-
Supplementary data associated with this article can be found, 304
258 rial cells first lose their viability and when the accumulation
in the online version, at doi:10.1016/j.bjm.2016.10.009. 305
259 reaches a threshold, grazing eliminates them. From our exper-
260 iments, it can be noted that the percentage of non-viable PAB 306

261 and free-living cells is relatively more than the VC (Fig. 2). For references
262 example, at the end of 6 h of incubation (Fig. 2B), the viabil- 307

263 ity of PAB in whole and diluted seawater is <21% of the TC;
264 whereas, the NVC can constitute almost 90–99%. When the 1. López-Urrutia A, Morán XAG. Resource limitation of bacterial 308

265 NVC reaches such a threshold, the low numbers of remaining production distorts the temperature dependence of oceanic 309

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Please cite this article in press as: Gonsalves M, et al. Grazing of particle-associated bacteria—an elimination of the non-viable fraction. Braz J
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Please cite this article in press as: Gonsalves M, et al. Grazing of particle-associated bacteria—an elimination of the non-viable fraction. Braz J
Microbiol. (2016), http://dx.doi.org/10.1016/j.bjm.2016.10.009 BJM 172 1–6