Bacteria-sediment associations in an alpine, freshwater environment and their

effects on particle size, density and settling velocity

David C Barrett1 2 * and Kyle R. Hodder1

1
Prairie Environmental Process Laboratory, University of Regina, Regina, SK S4S

0A2, Canada, email: Kyle.Hodder@uregina.ca

2
Current address: Water and Climate Impacts Research Centre, University of

Victoria, Victoria, BC V8P 5C2, Canada, email: barrettd@uvic.ca

*Corresponding author

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doi: 10.1002/esp.4389
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Abstract

This study measures the presence of bacteria-sediment associations (BSA) in an

alpine, glacier-fed watershed in the Southern Coast Mountains of British Columbia,

Canada. The impact of BSA on the creation of flocculated particles and their settling

velocity are quantified using a laser transmissometer. Results from the study indicate

that BSA are present in the watershed and vary over both space and time. The

percentage of bacteria associated with sediment particles was found to range from

<1% to 40%. Major sources of planktonic bacteria such as agricultural land and

wastewater treatment outflow co-occur with large decreases in the BSA ratio.

Laboratory analysis demonstrates that an increase in the concentration of bacteria

was associated with a decrease in the volume concentration of small particles, and a

decrease in both estimated density and measured settling velocity for particles in

larger size classes; consistent with flocculated particles of increasing complexity

arising from combinations of primary particles and/or BSA. Paleoenvironmental

reconstructions using laminated lake sediments in alpine, glacier-fed systems benefit

from a fuller understanding of the geomorphologic processes by which they formed.

While bacteria are noted to enhance formation of flocculated particles in laboratory

systems, their impact upon geomorphic processes in natural systems have yet to be

fully explored.

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Introduction

Alpine, glacial meltwater at the source differs in temperature, if not nutrient

concentration and availability, from the increasingly distal and/or lowland sites into

which that meltwater flows. Glaciers support microbially-dominated food webs that

thrive in the presence of liquid water and sediment (Hodson et al., 2008) and

bacteria are widely reported from glacier-fed environments, including fjords

(Jankowska, 2005; Syvitski et al., 1989), subglacial meltwater (Sharp et al., 1999),

glacier-fed lakes (Mindl et al., 2007), supraglacial lakes (Liu et al., 2009), cryoconite

holes (Hodson et al., 2008), lake sediment cores (Wurzbacher et al., 2017) and

channel beds (Fegel et al., 2016; Battin et al., 2001). Alpine glaciers are sensitive to

hydroclimatic conditions (Hodder et al. 2007), and seasonal shifts in the availability

of liquid water and suspended sediment, especially during snow- and ice-melt and

the associated release of meltwater, are likely linked with corresponding shifts in

bacterial abundance. Indeed, glacier-derived rock-flour is known to enhance

bacterial growth in glacial and glacier-fed habitats (Mindl et al., 2007) especially at

those sites with greater temperature than at, or under, the glacier including proglacial

lakes, and meltwater at progressively more distal sites. The abundant sediment in

glacier-fed environments supports abundant microbial communities (Fegel et al.,

2016), consistent with the positive relationship reported between bacteria

populations and sediment concentration in subglacial meltwater (Skidmore et al.,

2000; Sharp et al., 1999).

Bacterial-transport dynamics are often framed in the vital water quality

perspective (Schindler, 2001; Tian et al., 2002; Wilkinson et al., 1995), omitting any

link between bacteria and geomorphic processes. One such process is the

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association of bacteria to sediment particles present in fluvial environments.

Although bacteria-sediment associations (BSA) are documented in urban

stormwater, agricultural runoff and lowland fluvial environments (Droppo et al., 1997;

Jamieson et al., 2005a; Schillinger and Gannon, 1985; Soupir and Mostaghimi,

2011), there remains a relative paucity of information on the interaction between

bacteria and suspended sediments in glacier-fed environments, as well as their

geomorphic significance. This situation persists despite the environmental ubiquity of

bacteria (Rothschild and Mancinelli, 2001) and the growing recognition of their

potential link with geomorphic processes over a variety of spatial and temporal

scales (Larsen et al., 2017; Reinhardt et al., 2010); the size of bacteria and their

perceived geomorphic power perhaps contribute to this situation (Viles, 2012; Viles

et al., 2008). Further, studies that have attempted to quantify the presence of BSA

have used a variety of methods (Jamieson et al., 2005a; Krometis et al., 2009;

Schillinger and Gannon, 1985; Soupir et al., 2010; Soupir and Mostaghimi, 2011),

which encumbers direct inter-study comparison. In many cases, researchers rely on

immediate transport of samples to a laboratory for analysis. Reliance upon

laboratory-proximal techniques may also contribute to the limited exploration of

bacteria on the formation of flocculated particles in remote, glacier-fed environments

at which standard laboratory facilities are not proximal.

The process by which ‘composite particles’ are formed has been variously

defined as flocculation (Droppo et al., 1997), coagulation (Lee et al., 2002) or

aggregation (Walling and Woodward, 2000) depending on the process inferred to be

responsible for genesis of the composite particle (Hodder and Gilbert, 2007). The

association of two or more primary particles is shared in common among these

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definitions, no matter which formative processes are involved (Hodder and Gilbert,

2007). Flocculated particles are present in a wide range of environments and many

studies have found bacteria to be present within flocs (Droppo, 2001; Droppo et al.,

1998; Eisma, 1986; Liss et al., 1996; Zabawa, 1978). Hodder and Gilbert (2007)

provided evidence of flocs in glacier-fed Lillooet Lake (the lake receiving meltwater in

this study), however, the presence of bacteria was not quantified. Flocculated

particles are also known to alter the hydrodynamic characteristics of suspended

sediment, most notably via effects on size and density in comparison with primary

particles (Bache and Gregory, 2010; Droppo et al., 1997; Gibbs, 1985; Hodder,

2009). Bacteria are a candidate process by which flocculated particles form, and

therefore a potential link in the process-network of alpine, glacier-fed systems

(Hodder et al., 2007). One key means by which to index the geomorphic effect of

bacteria is via the effect upon settling velocities of sediment in the system.

Bacteria often preferentially associate with sediment particles as sediment-

associated organisms are shielded from potential predators (Droppo et al., 2011).

The shielding that sediment-associated bacteria receive can slow decay rates, thus

allowing bacteria to persist longer in the environment (Russo et al. 2011). Flocs are

competent at transporting nutrients due to their increased surface area and assumed

presence of bacterial polymers, and thus provide an ideal microenvironment which

allow bacteria to persist (Wotton 2011).

Our goals in this study were to: (a) develop a method suitable for quantifying

BSA in a remote geomorphic setting; (b) document the presence of spatial and/or

temporal trends in BSA from glacier to distal runoff sites; and (c) examine the link

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between BSA, particle size and settling velocity of sediment as a direct link to

geomorphic processes in glacier-fed systems.

Study site

The focus of this study was the Lillooet River, from the headwater at Lillooet

Glacier, downstream to Lillooet Lake. Lillooet Glacier is located in the Coast

Mountain range of British Columbia and is the headwater for Lillooet River. The river

drains first into Silt Lake and eventually to Lillooet Lake, approximately 100 km

downstream (Figure 1). Aside from the channel of the Lillooet River itself, Silt Lake

and the floodplain of Lillooet River, there are no other lakes in the Lillooet River

valley upstream of Lillooet Lake (Hodder and Gilbert, 2007). Lillooet Glacier is the

largest of the valley glaciers in the region, terminating at an elevation of roughly 950

masl (Schiefer and Gilbert, 2008). Like most glaciers in the area, retreat of Lillooet

Glacier in recent years has been well documented (Schiefer et al., 2007; Schiefer

and Gilbert, 2007).

The glacier itself is inaccessible by road and as such, is considered to be free

of any significant level of direct human influence. Logging activities had progressed

to the vicinity of point F (Figure 1) at the time of sampling and are were also active in

the downstream vicinity as far as point M, near the town of Pemberton. There are

also agricultural areas in the Pemberton Meadows between Lillooet Lake and point I

(Figure 1; both livestock and crops). The confluence of the Green and Lillooet rivers

is found approximately 12 km upstream of Lillooet Lake, and the confluence with

Meager Creek is approximately 25 km downstream of Silt Lake. The majority of

sediment transported through the watershed has been assumed to be glacially-

derived (Desloges and Gilbert, 1994; Jordan and Slaymaker, 1991), but Friele et al.

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(2005) also document significant sediment delivery from the Mt Meager volcanic

complex. The contribution of volcanic sediment is thought to be of greatest

significance immediately following major slides or mass wasting events, such as that

in 2010 (Guthrie et al., 2012). Regardless of the source, suspended sediment

concentrations in Lillooet River during nival and glacial melt periods are high (up to

1200 mg L-1; Gilbert 1975), and concentrations reaching up to 400 mg L-1 at Silt Lake

(Schiefer and Gilbert, 2008) and peaks of 800 mg L-1 at Lillooet Lake (Gilbert, 1975).

The Lillooet Valley is experiencing increasing anthropogenic modification due

to resource extraction, population growth and land-use change (Gilbert, 1975;

Schiefer and Gilbert, 2008). Logging in the valley is expected to enhance sediment

mobilization to the river (Jordan and Slaymaker, 1991). Fertile floodplain soils have

facilitated agriculture in the valley (Jordan and Slaymaker, 1991), and the population

of Pemberton tripled between 1996 and 2016 (Statistics Canada, 2012; 2017) and,

not coincidentally, a new wastewater treatment plant was installed in 2008 (Point O;

Figure 1).

Many glaciers and glacier-fed lakes in the Southern Coast mountains of

British Columbia have been extensively studied with a specific focus on

hydroclimatic reconstructions. The study site of the Lillooet Valley was chosen for

this study, in part, due to the number of prior investigations focusing upon

geomorphic processes at proglacial Silt Lake (Schiefer and Gilbert, 2008), within the

glacial valley (Reyes and Clague, 2004; Schiefer and Gilbert, 2007), in Lillooet Lake

(Desloges and Gilbert, 1994; Gilbert, 1975; Gilbert et al., 2006; Hodder and Gilbert,

2007; Menounos et al., 2005) and in the basin as a whole (Friele et al., 2005; Jordan

and Slaymaker, 1991). A notable omission from past studies undertaken in the river

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basin is the presence and geomorphic consequences of bacteria (Hodder et al.,

2007; Hodder 2009).

[Figure 1]

Figure 1 The Lillooet River valley in the Coast Mountains of British Columbia with sample locations along

Lillooet River labeled A through O. Inset: Sample locations between Lillooet Glacier and Silt Lake

labelled A through E. Water treatment facility located immediately upstream of sample location ‘O’.

Background data source: BC TRIM (2015).

Methods

Given the remote setting at the terminus of Lillooet Glacier, and sampling

sites throughout the Lillooet River valley, we combined fractional filtration and

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fluorescence microscopy such that sample collection and storage did not require

immediate laboratory support or analysis. We relied on established fractional

filtration methods at the field site (Auer and Niehaus, 1993; Krometis et al., 2009;

Soupir et al., 2008, 2010), combined with fluorescence microscopy to enumerate

bacteria back in the laboratory (Droppo et al., 2009; Franson et al., 1998). Records

of river discharge were obtained from the Water Survey of Canada monitoring station

on the Lillooet River near Pemberton (WSC station 08MG005).

Sample Collection

Water samples were obtained using both an ISCO 6712 automatic sampler at

location B, just downstream of the glacier snout (Figure 1) and by collecting grab

samples elsewhere. Although both methods might disrupt the largest flocs (Eisma,

1986; Hodder and Gilbert, 2007), smaller flocs are likely to remain undisturbed due

to their strong electrochemical and/or biologically cohesive bonds (Droppo et al.,

2009; Wotton, 2011). Grab samples were collected using 1L polycarbonate Nalgene

bottles and standard hand grab sample methods (CCME, 2011). All samples were

taken between 29 June and 6 July 2012 during glacier melt, and therefore between

the mean annual date of centre-of-volume (day 177 ±7.6) and peak (day 218 ±53.0)

in the flow of Lillooet River (Déry et al., 2009; using 1972-2006 flow data).

To investigate temporal trends in BSA, the automatic sampler collected 200

millilitre samples every 2 hours for a 48-hour period and then every 6 hours for an

additional 48 hours. Grab samples were retrieved at multiple locations along the

Lillooet River (Figure 1), including at the glacier and the first major sediment sink, Silt

Lake. Grab samples were also retrieved downstream of Silt Lake along the Lillooet

River at sites upstream of Lillooet Lake. These grab samples were taken to assess

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the spatial gradient in BSA, particularly as runoff from agriculture and/or wastewater

treatment join the river flow.

The automatic sampler was also employed to collect samples for suspended

sediment concentration (SSC) and particle-size distribution. Samples for SSC

analysis were collected every hour for a period of 40 hours at location B (Figure 1).

The automatic sampler intake was placed directly in a main branch of the Lillooet

River, with the intake suspended ~5 cm above the river bottom in a flow with an

estimated surface velocity of >2.2 ms-1 (‘float method’; Goudie 1990). The intake

sampler was positioned facing downstream, following Grangeon et al. (2012), in an

attempt to limit the capture of bedload. Additionally, the river depth at the location of

the auto-sampler was shallow (<1 m), though not measured due to safety concerns.

Quantifying BSA

Fractional filtration was used to separate free-phase (planktonic) bacteria from

those associated with sediment particles. Initial processing of all samples was

undertaken in the field with further analysis completed upon return to the laboratory.

Bacteria associated with suspended sediment was operationally defined as those

which would not pass through an 8-μm membrane filter, after triple rinsing of filtration

apparatus consistent with previous research (Krometis et al., 2009; Mahler et al.,

2000; Schillinger and Gannon, 1985), and that only planktonic bacteria pass through

the membrane. After completing fractional filtration, a mixture of glutaraldehyde

(0.5% w/v) and phosphate buffer (KH2PO4) was used to fix cells (Franson et al.,

1998) for transport to the laboratory. Bacteria-associated samples were transported

in vials including filtration membranes, fixative and buffer solution. Planktonic

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bacteria were obtained from sample filtrate and transported in vials composed of

filtrate, fixative and buffer solution.

Upon return to the laboratory, each sample was subsequently vacuum-filtered

through a 0.2 μm polycarbonate membrane to retain planktonic organisms; this pore

size at the lower end of the 0.1 to 0.4 μm range of pore sizes traditionally employed

for physical removal of microbial cells during treatment of drinking water and

wastewater (i.e. the ‘recognized standard sterilization filter’; Wang et al., 2007). To

ensure capture of all sediment-associated organisms, the 8 μm membrane was

rinsed with phosphate buffer. Additionally, the filtration apparatus was rinsed with

phosphate buffer before and after sample addition to maximize organism capture.

Enumeration of the bacteria was completed using fluorescence microscopy and

semi-automated cell counting. A solution of acridine orange fluorochrome was added

to the cells (Hobbie et al., 1977; Jones, 1974) at a final concentration of 0.05% (w/v).

An exposure period of two minutes was used to ensure adequate attachment to

bacterial nucleic acids (Hobbie et al., 1977; Jones, 1974; Kepner and Pratt, 1994).

Between samples, the filtration equipment was flame sterilized to prevent cross-

contamination.

Air-dried 0.2 μm membranes were mounted on microscope slides using

Cargille type-A immersion oil (refractive index = 1.694) and examined at 1000x

magnification. Fluorescence was induced using a fluorescent lamp and a 465-495

nm excitation filter, and digital images were captured using a microscope-mounted

Olympus DP70 camera. A visual comparison of at least 10 fields of view was

undertaken to establish that bacteria were equally distributed on the membrane, and

a minimum of three fields of view was subsequently captured for each slide.

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 The digital images were then processed using ImageJ

(http://rsbweb.nih.gov/ij/) to obtain total bacteria counts. Each individual image was

manually verified visually to ensure all visible organisms were counted. Once total

bacterial counts were measured, count of organisms per unit volume of sample were

calculated. No attempt was made to identify the species of bacteria present in any

sample. The BSA ratio was calculated using the concentration of planktonic

organisms and sediment-associated organisms using Equation 1.

Equation 1

(1)

Measurement of particle size and SSC

Each of the 40 SSC samples was processed using the LISST device, after

ensuring an optical transmission of >30% (Sequoia Scientific, 2014). All grab

samples obtained were also processed using the LISST-C device to obtain particle-

size distributions (data not shown). Samples were vacuum filtered through pre-

weighed 0.4 μm polycarbonate membranes, air-dried for 24 hours and subsequently

weighed using a precision balance (±0.2 mg) to determine SSC.

Measuring settling velocities

Settling velocity experiments were undertaken in a walk-in 4°C cold room to

approximate thermal conditions in glacier-fed runoff using a Sequoia Scientific

LISST-STX fitted with a 50% optical path reduction module. The LISST device, and

the settling column, were packed in layers of 38 mm foam insulation board (R value

= 5.63) to insulate the equipment from drafts, and all supplies and samples were

stored in the same cold room. Ambient temperature was recorded in the room

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(HOBO U22-001, ±0.21°C), and by the LISST-STX (±0.01°C), during the

experiments. Varying concentrations of bacterial solution inoculum were added to a

constant concentration of suspended sediment. A mechanically homogenized bottom

sediment sample from Lillooet Lake was sub-sampled, suspended in reverse

osmosis (RO) water and immersed in an ultrasonic bath for 1 minute (Sperazza et

al., 2004). The prepared sediment was then added to the settling chamber for a final

concentration of 120 mg L-1; this concentration selected as representative of values

measured in Silt Lake (Schiefer and Gilbert, 2008), as well as other glacier-fed lakes

in the region (Gilbert and Desloges, 1987), and near the maximum concentration that

allowed for adequate optical transmission in the LISST-STX device.

Settling velocity estimates provided by the LISST-ST unit rely, in part, on the

evolution in a well-mixed distribution of particles over time such that the

concentration for any particle-size class shifts from an initial concentration to zero as

particles reach, and pass through, the laser beam. Assuming particles of a given size

possess equal mass density, each will reach the base of the chamber at a unique

settling velocity. The concentration history in each size class will show a steady

concentration until all particles have reached the laser beam, followed by a slope to

zero as particles settle through the beam (creation of a ‘knee’ in concentration

history; Sequoia Scientific). An increase in the concentration of any size class over

the observation period, such as via the creation of BSA during an experiment,

violates this approach. Therefore, we deliberately excluded any settling velocity

estimates for size classes in which the concentration history increased over the

observation period; in general, we found smaller size classes consistently revealed a

‘knee’ in the concentration history.

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 Fixed quantities of bacteria inoculants were created using Polyseed®

bacterial capsules (http://polyseed.com) and a nutrient water solution created

following manufacturer protocols. Polyseed contains a range of naturally occurring

microorganisms in granular form, with a minimum viable count of 4.00 * 10 9 cfu g-1.

Containers and equipment exposed to bacteria inoculant were triple rinsed with RO

water before being sterilised with triclosan (5-Chloro-2-2,4-dichlorophenoxy-phenol)

soap and 10% HCl solution. Fixed volumes of inoculant were added to the settling

chamber along with the sediment slurry. Settling experiments were initiated

immediately following the addition of bacteria and sediment slurry. Each settling

experiment run consisted of 83 size-distribution profiles collected at logarithmically-

spaced intervals over a 22-hour period (Sequoia Scientific, 2014), and each settling

experiment was repeated three times.

Results

Lillooet River

The majority of in situ samples examined were collected from the main stem

of the Lillooet River approximately 2 km downstream of the Lillooet Glacier snout

(Location B; Figure 1). The initial sampling interval of 2 hours revealed changes in

the BSA ratio over 48-hours, as did samples taken every 4 hours thereafter (Figure

2). Ratios of sediment-associated to planktonic ranged from a minimum of 0.04 to a

maximum of 0.40 during this 72-hour sampling period. Total microorganism counts

also varied during this time period (Figure 2). Peaks in the sediment-association ratio

occurred at or around 0600 hours each day. It is noted that additional peaks,

including a diurnal trend with peaks overnight, are visible. Further study of the

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hydrologic conditions and sediment fluxes in the region over a longer sampling

period, would be required to determine if these cyclical trends continue.

In the Lillooet River transect, the ratio of sediment-associated to planktonic

bacteria ranged from a minimum of 0.063 to a maximum of 0.38 (Figure 3). Samples

that were taken at locations immediately downstream of the wastewater treatment

plant, a presumed bacteria source (Bernhard et al., 2003), were associated with a

lower BSA and higher total-bacteria counts than at other locations (Figure 3). The

BSA ratio increased from Lillooet Glacier to Silt Lake (sites A through E; Figure 3),

but no discernible trend was evident in the BSA ratio over the entire transect from

glacier to Lillooet Lake (sites A through O; Figure 3). However, the total organism

count measured in samples increased in the downstream direction; linearly

correlated to distance from Lillooet Glacier (R2=0.51). All fifteen of the grab samples

taken along the Lillooet River were processed through the LISST device to obtain

particle-size distributions of the suspended sediment in the river. The concentration

of the very finest measurable particles (2.73 μm) was inversely related to BSA ratio,

whereas the concentrations of 88.2 μm particles was directly related to BSA ratio.

At sample location B (Figure 1), the recorded SSC values ranged between

160 – 675 mg L-1 with a mean value of 281 mg L-1 (Figure 4); magnitudes consistent

with those measured at Silt Lake by (Schiefer and Gilbert, 2008). No discernible

temporal trend in SSC values was evident in the measurements.

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[Figure 2]

Figure 2 Temporal changes in bacteria-sediment associations (BSA; solid line) and total organism count

(dashed line) at the Lillooet River location (Point B; Figure 1) over a 72-hour sampling period.

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[Figure 3]

Figure 3 Lillooet River transect from glacier snout to downstream of Pemberton, showing BSA ratio

(grey) and total organisms per litre (white). Bottom plot illustrates sample locations relative to distance

downstream of Lillooet Glacier and elevation above sea level.

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[Figure 4]

Figure 4 Suspended sediment concentrations of the Lillooet River, proximal to Lillooet Glacier (Point B,

Figure 1), measured over a 40-hour period. Outliers resulted when the sampler intake was inadvertently

moved during sampling.

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Settling velocity and particle-size distribution

Standardized departures were calculated relative to the mean concentration

for each size-class across all experiments as the measured concentration of

particles varied over six-orders of magnitude. Standardized departures facilitate side-

by-side comparison of relative change in volume concentration between classes

using a common legend, and are shown according to sample number (i.e. time) and

Polyseed dilution (Figure 5). Negative (positive) standardized departures indicate a

lower (higher) volume concentration relative to the mean concentration in that size

class across all experiments. Volume concentration in the 3.48, 6.76, and 13.12 m

size classes fell during each run (Figure 5), ideal for estimation of settling velocity.

The switch from positive to negative standardized departures in the 3.48, 6.76, and

13.12 m size classes generally occurred earlier during each run with greater

bacteria concentration. Volume concentration in the other size classes exhibits far

less consistency between runs. However, and notwithstanding this inconsistency, the

360.34 m size class exhibits positive departures as bacteria concentration

increased, and during each run. In other words, the largest size class exhibits an

opposite, if more variable, trend from that exhibited in the smallest size classes.

Finally, volume concentration in the 25.48, 49.4, 95.82 and 185.82 m size classes

shows a U-shaped (i.e. positive-negative-positive departures) trend in the absence of

bacteria.

Both (a) settling and (b) flocculation of sediments in the 3.48, 6.76, and 13.12

m size classes are consistent with the observed shift in volume concentration over

the course of each experimental run (Figure 5). Both settling and flocculation can

result in a loss of volume concentration in any single size class over time, and we

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infer both processes occurred simultaneously. The trends in volume concentration in

the 25.48, 49.4, 95.82 and 185.82 m size classes are consistent with: (a) the initial

loss of primary (i.e. unflocculated) particles present in the solution (i.e. the initial

positive-to-negative trend), followed by (b) formation of flocculated particles in these

size classes (i.e. the subsequent negative-to-positive trend) concurrently with the

loss of volume concentration in the 3.48, 6.76, and 13.12 m size classes (i.e. the

positive-to-negative trend). In the presence of bacteria, the creation of BSA from

smaller particles is consistent with: (a) the ever-earlier switch from positive-to-

negative departures in the 3.48, 6.76, and 13.12 m size classes during each run

with greater bacteria concentration (i.e. small particles disappear earlier at greater

bacteria concentration); and (b) the negative relationship between bacteria

concentration and volume concentration in the 3.48 m size class (i.e. the

concentration of smallest particles is inversely related to bacteria concentration); and

(c) the positive departures in the 360.34 m size class with increased bacteria

concentration (i.e. concentration of the largest particles is directly related to bacteria

concentration, and these particles form during the course of each experimental run).

Although volume concentration departures among the 25.48, 49.4, 95.82 and

185.82 m size classes show far less consistency between runs than is exhibited

between other size classes, they do consistently reveal: (a) a U-shaped trend in

departures in the absence of bacteria, and (b) negative departures in runs of ≥20 ml

of Polyseed. The latter aspect, taken together with (a) the simultaneous negative

departures in the 3.48, 6.76, and 13.12 m size classes, and (b) the positive

departures in the 360.34 m size class, strongly suggest creation of the largest BSA

is linked with bacteria concentration under these conditions. Particles in the largest
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size class may be forming from combinations of particles in any of the smaller size

classes along with bacteria. In other words, particles of increasing complexity arise

from combinations of primary particles and/or BSA with void spaces occupied by

water with the corollary that floc density must be generally lower than that of the

primary particles. The far greater variability in volume concentration exhibited

between runs among the 25.48, 49.4, 95.82 and 185.82 m size classes departures

may also relate to the increasingly complex combinations of primary particles and/or

BSA with void spaces – both of which are associated with larger particles.

Settling velocities were tabulated according to size class and Polyseed

dilution, and expressed relative to the Stokes’ velocity for spheres with a density of

2.65 g cm-3 (e.g. quartz) in 4°C water (Table 1) excluding settling velocity estimates

for any size class or run in which the concentration history increased during the

observation period. We use Stokes’ velocity as a convenient benchmark, and we

note that many of the assumptions upon which it is based are not met for flocculated

particles in finite, and turbulent, fluid volumes. Generally, settling velocity estimates

showed greater departure from Stokes’ velocity: (a) at greater concentrations of

bacteria within any given size class, and (b) at successively larger size classes. In

other words, larger size classes exhibited greater departure from Stokes’ velocity

with bacteria concentration; consistent with particles of increasing complexity arising

from combinations of primary particles and/or BSA. The 40-ml Polyseed run was an

exception to this trend, which exhibited the greatest departure from Stokes’ velocity

in the 6.76 and >49.4 m size classes. The larger the size class, the less likely

settling velocity could be obtained; this was undoubtedly the result of the combined

effects of an increase in volume concentration during the observation period

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preventing use of the settling technique (i.e. flocculation) and that the settling velocity

of these particles was below the minimum detection threshold of the device (i.e. very

low effective density).

The density of a median-sized particle in each size class was estimated using

measured settling velocity, assuming spherical particles in 4°C water for each

Polyseed dilution according to the Stokes’ equation (Table 2). This approach

provides an estimate of particle density, and so we interpret only the trend in density

values and not absolute magnitude. We use the trend in estimated densities to

provide proxy evidence for a change in particle complexity via flocculation assuming

settling velocity and (median) diameter are known. Density values are, in general,

greatest in the 3.48 m size class and lowest in the 185.82 m size class –

consistent with particles of increasing complexity arising from combinations of

primary particles and/or BSA which incorporate a greater volume, and greater

proportion, of void space within larger particles. The estimated density of the largest

particles approaches that of water. The estimated density of particles in each size

class also generally falls from one run to the next.

To illustrate the sensitivity of the density calculation to diameter, and why we

interpret only the trend in density values, and not absolute magnitude: The single

greatest density estimate (~4 g cm-3) was obtained for particles in the 3.48 m size

class at 40 ml Polyseed, which exceeds the density of primary particles we

anticipated in this environment. The size class with a median diameter of 3.48 m

ranges between 2.5 m and 4.85 m, and corresponding estimated densities for the

upper and lower end of diameters in this size class are 7.32 and 2.68 g cm-3 at a

temperature of 4°C. We have not measured the mineral composition of the

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suspended sediment samples in our study to permit an interpretation of the absolute

magnitude for estimated densities. However, the lower end of this estimated density

aligns with the phyllosilicates (illites ~2.7 g/cm3; chlorites ~3.0 g/cm3; kaolinites ~2.2

to 2.7 g/cm3) and other tectosilicates (e.g. plagioclase 2.6 to 2.8 g/cm3) that are

known to dominate the clay-size fraction of debris-flow deposits in the study basin

(Friele and Clague 2004). The sands of the lower Lillooet River are also known to

contain a large fraction of magnetically susceptible minerals, including hematite (~5.3

g/cm3) in sufficient quantity to warrant consideration of placer mining (Gilbert et al

2006), but we have no information about the presence of such minerals in the finer

sediments of the river. Nevertheless, the observed trend in estimated densities is

consistent with the flocculation process – even if the estimated density for the very

smallest particles exceeds our expectations.

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[Figure 5]

Figure 5 Standardized departures in volume concentration (colour; centred on zero) for each particle-size class (3.48,

6.76, 13.12, 25.48, 49.4, 95.82, 185.82 and 360.34 microns), each Polyseed dilution (0, 10, 15, 20, 30 and 40 ml) and

time step (0 to 22 hours) and averaged across all three runs; labelled time steps are the average volume concentration

up to, and including, the labelled step. Standardized departures in volume concentration were calculated separately

for each size class and relative to the mean volume concentration in that size class across all runs.

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Discussion

The results of this study provide evidence of previously undocumented

bacteria-sediment associations within an alpine watershed through the application of

a unique detection method suitable for remote environments. Samples that were

taken immediately downstream of areas with agricultural activity showed a decrease

in BSA ratio and an increase in total organism concentration (Figure 3), consistent

with organisms being supplied from these sites predominantly in planktonic, as

opposed to sediment-associated, form. The increase in planktonic organisms results

in a lower BSA ratio by elevating total organism count. We infer that greater BSA

ratios that appear at adjacent downstream sample sites are likely the result of an

increase in sediment-associated bacteria and not the result of changes in suspended

sediment concentration (Figure 3). Creation of BSA downstream of the input of

planktonic bacteria is consistent with in-stream flocculation processes.

Total organism concentration generally increased downstream (Figure 3);

likely associated with habitat gradients (temperature, nutrient availability; Battin et

al., 2001). The change in total organism concentration in the downstream direction is

not monotonic, but rather exhibits two clear peaks: at the wastewater treatment

outfall (site O), and in the middle of Pemberton Meadows (site L). In other words,

peaks in organism counts increase significantly near sources of planktonic bacteria

(Biswas and Pandy, 2016; Santhi et al., 2001). Local peaks in BSA ratio appear

adjacent to local, but upstream, peaks in total organism count; examples include

sites A and B, sites C and D/E, sites G and H and sites L and M. In other words, a

local peak in BSA ratio is preceded by a local, and upstream, peak in total organism

count. The single greatest BSA ratio across the river transect occurred at Silt Lake

(Site D/E; 0.38). This peak in BSA ratio could be related to a decrease in turbulence

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when compared to the riverine environment, thus potentially facilitating formation of

larger flocs. Further sampling is required to test this possibility.

BSA ranged from <0.05 to 0.4 over the duration of this study and changed

markedly over periods as brief as 4-hours (Figure 2) and distances < 1 km (Figure

3). Broadly, the greater the total organism count in the temporal plot, the lower the

lower the corresponding BSA ratio (Figure 2); consistent with the observation that a

local peak in BSA ratio is preceded by a local, and upstream, peak in total organism

count (Figure 3). The diurnal trend of peaks in BSA at around 06:00 may be related

to diurnal temperature/melt fluctuations (Hock, 2003; Röthlisberger and Lang, 1987)

and the release of organisms from subglacial (Sharp et al., 1999) and supraglacial

storage sites (Anesio et al., 2009); a longer observation period is needed to follow-up

on this possibility.

To our knowledge, this is the first study to measure a spatial gradient in BSA

for suspended particulates. In order to estimate a timescale for the creation of these

composite particles using the change in BSA and/or shifts in particle size across

space (for example between L and M; Figure 1), we would need to have sampled the

same stations repeatedly. However, the rate at which BSA are formed and therefore

the corresponding rate of bacteria-related flocculation (and, indeed, deflocculation)

might be variable and dependant on local conditions. For example, the decrease in

total organism concentration (Figure 3) between sites L and M may be concomitant

with flocs settling to the riverbed and becoming part of the bed sediments (temporary

or otherwise), removing the bacteria associated with them from suspension.

Resuspension of these bed sources relies on periods of high flow (Jamieson et al.,

2003, 2005b; Nagels et al., 2002); when combined with the decreased die-off rate of

bacteria associated with sediment (Davies et al., 1995; Pachepsky and Shelton,

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2011), periods of high flow can pose a significant health concern (Characklis et al.,

2005; Rehmann and Soupir, 2009). Furthermore, the marked shifts in BSA at the

same site over periods as short as approximately 4 hours further hint that the rate at

which BSA are formed (and, potentially, destroyed) might be more variable than we

first expected. Shifts in the concentration of suspended sediment over time (e.g.

Figure 4) may contribute to the shifts in BSA. However, we infer that changes in the

BSA ratio shown in Figure 2 over multi-hour periods are not exclusively the result of

changes in suspended sediment concentration given the multi-hour trends in

suspended sediment concentration observed at the same site (Figure 4).

Fecal coliform bacteria have a long axis of ≥ 2 μm (Qualls et al., 1983), and

thus, the negative relationship between particle concentration in the 2.73 μm size

class and BSA is to be expected. Observations suggest that the LISST device was

sensitive to the detection of small bacterial flocs and potentially planktonic bacteria,

as shown by the increase in the 3 – 10 μm range at approximately the 4-hour mark

(Figure 5). Similar results are obtained in this study with an increasing concentration

of particles in the 7.0 – 26 μm size range correlating to an increase in BSA. This

finding is consistent with the presence of bacteria-sediment composite particles in

the Lillooet River, and their detection by the LISST device (data not shown).

Although large flocculated particles may be present in situ, they are

considered fragile and subject to disturbance (Droppo et al., 1998; Hodder and

Gilbert, 2007). However, Hodder and Gilbert (2007) reported that average

flocculated particle sizes in Lillooet Lake range from 10 – 35 μm diameter. We found

no correlation between particle size and BSA in grab samples. We acknowledge that

both grab sampling and automatic sampling may alter fragile flocs (Grangeon et al.,

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2012), and that our remote field site did not lend itself to alternative sampling

techniques free of this limitation.

The addition of bacteria to the sediment slurry in this study led to changes in

particle-size distribution (Figure 5), settling velocity (Table 1) and estimated density

(Table 2). Particle-size distributions changed relative to the amount of bacterial

solution added, with a greater magnitude of change occurring at higher bacteria

concentrations. The decrease in concentration of small, primary particles is

consistent with their combining to form larger, flocculated particles; a finding

consistent with reports of flocculated particles of diameter between 10 and 35 m in

Lillooet Lake having formed from primary particles with diameter < 4 μm (Hodder and

Gilbert (2007). The results from this study indicate that bacteria can influence the

creation of flocculated particles in alpine environments. This finding contrasts with

the suggestions of Eisma (1997; cf Hodder and Gilbert 2007) who hypothesised that

a lack of organic material limited flocculation in glacial meltwater. The results from

this study and Hodder (2009) indicate that flocculated particles are present and

prevalent in this environment.

Although there is documented evidence for the presence of flocculated

particles in glacier-fed lakes (Hodder and Gilbert, 2007), the impact of these particles

on the settling velocity of sediment had yet to be quantified. The settling velocity of

flocculated particles is known to be higher than those of the constituent particles

comprising the floc; the degree of acceleration relative to the primary particle is

linked with the degree of void spaces occupied by water or lower-density organic

materials (e.g. bacteria) in the floc. This property of flocs is harnessed in water

treatment facilities (Bache and Gregory, 2010) and allows sediment, which may

otherwise not settle in slack water environments, to be more rapidly deposited on

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lake beds (Hodder, 2009). The results displayed in Figure 5 further support this

notion by showing that higher concentrations of bacteria hastened the loss of volume

concentration in the small size classes, alongside lower estimated densities (Table

2) and an increase in volume concentration in the largest size class.

Past studies have noted that flocculated particles generally settle at a rate

much lower than that suggested by Stokes’ Law (e.g. Hodder 2009). The results

displayed in Table 1 demonstrate that measured settling velocities of flocculated

sediments were lower than those estimated by Stokes’ settling velocity equation for

quartz particles (mass density of 2.65 g cm-3). These results show that, in general,

settling velocity is inversely related to bacterial concentration. Although bacteria have

previously been acknowledged to be present in flocculated particles (e.g. Droppo

2001), the impact of increasing bacterial concentrations on effective density, and

therefore hydrodynamics, of the flocs has received less attention. The measured

decrease in settling velocity for particles in the same size class requires a decrease

in effective density as more bacteria are introduced, under constant conditions. As

bacterial concentrations increase, individual bacterium may join the margins of

existing flocculated material to produce increasingly elaborate composite particles

and therefore coincide with an overall decrease in effective density (i.e. an increase

in fractal dimension; de Boer et al 2000; Hodder 2009). While the concentration of

bacteria may affect other aspects of the floc, such as shape, we expect that these

parameters would have less of an impact on settling velocity than changes in

effective density for particles within a given size class.

Although there are many processes that influence the physical properties of

lake sediments (Hodder and Gilbert, 2013), the presence and prevalence of bacteria,

and their impact on geomorphic processes, is often excluded. As noted by Hodder

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(2009), without flocs, the smallest primary particles may not otherwise settle to the

bottom of alpine lakes within a single season. However, in a flocculated form, such

as those formed through BSA, the smallest primary particles have a process-

pathway by which to reach the lake floor within the year. The presence of bacteria

can therefore contribute to accumulation of sediment within the lake basin that might

not otherwise occur in their absence over the same period: fine silt and clay

entrained within a floc that subsequently settles to the lake floor, for example, in

comparison with those particles that readily accumulate on the lake floor in the

absence of flocculation (sand, for example). The physical properties of the deposit

(e.g. particle-size distribution, bulk density, packing, porosity) are therefore tied to

the depositional process(es), as those depositional processes are responsible for the

population of particles that constitute the bed sediment. O’Brien and Pietraszek-

Mattner (1998) inferred the action of the flocculation process, for example, on the

basis of particle orientation within the lacustrine deposits of two Pleistocene-era

glacial lakes. Given that bacteria contribute to the formation of flocculated particles,

and that constituent grains in these particles tend to be small (e.g. ~4 m), it is likely

that bacteria modulate the hydrodynamics of at least a portion of these small grains

in the Lillooet system. As the total concentration of bacteria changes (e.g. diurnally,

seasonally) and in response to environmental conditions (e.g. nutrients,

temperature), we also expect that BSA will vary accordingly. The corresponding

geomorphic role(s) of bacteria or flocs as constituents of the freshwater sedimentary

system might therefore also shift. For example, under conditions that favour the

creation of BSA, the presence of flocs composed of small constituent particles with a

greater settling velocity may lead to a thicker sedimentary deposit in glacier-fed lakes

if those constituent grains would not otherwise settle to the basin floor. The results

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from this study illustrate the importance of flocculated particles in accelerating the

deposition of sediment when compared to individual primary particles. During

periods of high (low) bacterial concentrations, such as due to greater (lesser) nutrient

availability or warmer (colder) temperature, there may be conditions that enhance

(hinder) flocculation, and therefore a potential pathway for bacteria to contribute to

the properties of the corresponding sedimentary deposit.

Conclusions

Although noted to be present in agricultural regions, the presence of bacteria-

sediment associations in an alpine setting has gained less attention. This may be

partially due to the fact that prior studies used sample collection and analysis

methods that were both a) time sensitive and b) relied upon laboratory-proximal

techniques and sample sites. The method developed and used in this study was

successfully employed in a remote, alpine environment and this method can be

adopted for analysis in other regions thereby facilitating inter-study comparison.

A spatial transect along the Lillooet River illustrates that bacteria are

associated with sediment particles throughout the watershed, from the headwater

and throughout the channel downstream. The BSA ratio, however, varies throughout

the watershed, reaching local minimums immediately downstream of presumed

inputs of bacteria (e.g. agricultural land and wastewater treatment inflow). We infer

that bacteria are being delivered from these sources in planktonic form, and are

subsequently associating with sediment in the fluvial environment. However, it is also

noted that agricultural areas are also often sources of sediment and a) may promote

eventual BSA or b) may also be transporting bacteria in sediment-associated form.

The lowest BSA ratios found at the glacier snout, and at a major upstream

confluence. The corresponding changes in BSA measured throughout Lillooet River

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provide evidence that bacteria and sediment are associating over, for example, a

reach of river approximately 14 km in length (between sites L and M; Figure 1). BSA

ratios measured at the same site vary markedly over periods as short as 4-hours.

Results from this study confirm that bacteria aid the formation of flocculated

particles in a controlled laboratory environment. Any shift in the particle-size

distribution of suspended sediment is inherently linked to a shift in the settling

velocity of particles. Generally, increases in the concentration of bacteria coincide

with an increase in the concentration of larger, flocculated particles with a reduced

settling velocity relative to Stokes’ Law. The measured decrease in settling velocity

for particles in the same size class relative to Stokes’ Law requires a decrease in

effective density as more bacteria are introduced under constant conditions.

Understanding the geomorphic consequences for BSA, and ultimately the suite of

processes that influence the formation of lacustrine sediments, can only enhance

their utility as paleoenvironmental archives. Finally, the release of bacteria or other

potential contaminants stored within glaciers and/or bed sediment is of growing

concern due to environmental change (Blais et al., 2001; Noyes et al., 2009), and is

highlighted in the presence of flocculated particles, as flocs play a role in linking

freshwater quality with geomorphic processes.

Acknowledgements

This study was completed with support from the Natural Sciences and Engineering

Research Council (NSERC) of Canada via a Discovery Grant (371779-2011) to Dr

Kyle Hodder. Infrastructure provided by a Canada Foundation for Innovation grant is

also gratefully acknowledged. The authors thank the Institute of Environmental

Change and Society at the University of Regina for access to cold-storage facilities

for conducting settling rate experiments, as well as epifluorescence microscopy

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equipment. Access to laboratory facilities provided by Dr Dena McMartin at the

University of Regina is also gratefully acknowledged. Comments from anonymous

reviewers helped improve and clarify our work; one anonymous reviewer, in

particular, is thanked for critically reading the manuscript and stimulating our

thoughts on how (or if) flocculation is linked with geomorphic processes.

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Table 1 Settling velocity estimates for each size class and Polyseed dilution expressed as a percentage of Stokes’

velocity for spheres with a density of 2.65 g cm-3 in 4°C water, excluding any run in which the concentration

increased during the observation period. Fewer than three runs with measureable settling velocity marked: two runs

(*), one run (^)

Size class (m)

Polyseed
(ml) 3.48 6.76 13.12 25.48 49.40 95.82 185.82 360.34
0 128.6 110.7 45.8 27.0* - - 0.0^ -
10 109.6 80.9 44.1 - 30.5* - -
15 99.8 70.9 25.8 1.2 - - - -
20 115.3 70.9 11.1 - - - - -
25 109.6 57.3 10.3 1.0 - - - -
30 91.6 30.7 8.2 1.2 0.3* - 0.0^ -
40 86.7 34.2 53.6 82.0 75.5* 15.6* - -

Stokes’
(cm/s) 6.9E-04 2.6E-03 9.8E-03 3.7E-02 1.4E-01 5.2E-01 2.0E+00 7.4E+00

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Table 2: Estimated density for the median-sized particle in each size class, each run, and each Polyseed dilution using

measured settling velocity, and assuming spheres in 4°C water.

Polyseed Run Size class (m)

(ml)
3.48 6.76 13.12 25.48 49.4 95.82 185.82 360.34
0 1 3.6114 2.7270 1.3954 1.8209 - - 1.0003
0 2 2.8997 2.1064 1.1708 - - - - -
0 3 3.0093 2.7270 1.5638 1.0171 - - - -
10 1 3.8228 3.1239 1.5154 - - - -
10 2 2.7130 2.2570 1.6851 - - 1.4283 - -
10 3 2.5597 2.1064 1.6851 - - 1.5205 - -
15 1 3.8228 4.1360 1.6851 2.2413 - - - -
15 2 2.5597 2.1064 1.3862 1.0171 - - - -
15 3 2.5597 2.1064 1.4013 1.0179 - - - -
20 1 3.6114 3.1239 1.8092 - - - - -
20 2 2.8016 2.1064 1.1708 - - - - -
20 3 2.8016 1.9881 1.1729 - - - - -
25 1 3.6114 2.7270 2.4034 - - - - -
25 2 2.7130 1.8926 1.1597 - - - - -
25 3 2.7130 1.8926 1.1499 - - - - -
30 1 3.4294 2.4550 2.5619 2.6420 - - 1.0002 -
30 2 2.4316 1.4774 1.1267 1.0188 1.0047 - - -
30 3 2.3229 1.4539 1.1267 1.0188 1.0047 - - -
40 1 4.0716 3.5808 2.0115 1.8632 - - - -
40 2 2.5597 1.5324 1.8325 3.7788 2.6117 1.3525 - -
40 3 2.1485 1.4539 1.4584 2.2746 1.7392 1.1330 - -

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Bacteria-sediment associations in an alpine, freshwater environment and their effects on

particle size, density and settling velocity

David C Barrett* and Kyle R Hodder

Through a suite of in-situ and experimental methods, it is found that bacteria-sediment

associations are both common in an alpine freshwater environment and that they affect the

settling velocity of sediment through the creation of composite particles (flocculation).

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