Effect of concentration and size of inorganic suspended...

Sustain. Environ. Res., 25(6), 315-322 (2015) 315

*Corresponding authorEmail: [email protected]

Effect of concentration and size of inorganic suspended solids on microbial-mediated nitrogen transformation in

freshwater column

Quynh Nga Le,* Chihiro Yoshimura and Manabu Fujii

Department of Civil Engineering Tokyo Institute of Technology

Tokyo 152-8552, Japan

Key Words: Inorganic suspended sediment, ammonification, nitrification, bacteria biomass

INTRODUCTION

Nitrogen (N) dynamics in freshwater systems are complex because of their various chemical states and association with microbially mediated transformation processes. Those transformations include the assimilatory and dissimilatory processes of N by bacteria. In the former process, both autotrophic and heterotrophic bacteria can obtain N for structural synthesis through the uptake and fixation of dissolved nitrogen [1]. In the latter process, bacteria catalyze electron transfer reactions, resulting in energy production, with ammonia (NH4

+) as a reducing agent (i.e., nitrification) and nitrate (NO3

-) as an oxidizing agent (i.e., denitrification) [2]. Through such bacterial processes, the N dynamics in freshwater systems are governed by factors such as pH, suspended solids (SS), temperature, dissolved oxygen (DO), and substrate availability [3].

Among these factors, the effect of SS on

microbially mediated N transformation is not well understood despite the fact that many large rivers in the world are highly turbid, with SS concentrations and sizes varying over wide ranges [4-6]. High SS concentrations, on one hand, limit phytoplankton growth by reducing light penetration in water columns resulting in reduced N uptake [7]. On the other hand, high SS concentrations increase bacterial production and rates of organic matter mineralization in water columns [8]. More specifically for N dynamics, the microbially mediated nitrification rate and biomass of ammonia-oxidizing bacteria have been found to increase as a power function of the SS concentration [9]. The effect of SS size on bacterial biomass in a freshwater column has not been well investigated. However, the river bed sediment and detrital particles in addition to biomass of bacterial communities are significantly affected by the physicochemical properties of SS, including the particle size [10-12]. Thus, a similar effect of SS size on bacterial biomass

ABSTRACT

The microbially mediated interaction between nitrogen (N) and suspended solids (SS) is not comprehensively understood despite its importance in N transport and transformation, especially in turbid freshwater columns. Herein, the biochemical interaction among inorganic SS (ISS), bacteria biomass, and aerobic N transformation (i.e., ammonification and nitrification) is examined to understand the roles of ISS in N transformation in naturally turbid rivers. Batch experiments were conducted for 7 d with an ISS concentration range of 0-1200 mg L-1 and sizes of 11-500 µm, which are typically observed in natural waters. The results indicated that bacterial biomass was higher in the system containing higher ISS concentration, and it was not significantly affected by ISS size. Furthermore, nitrogen transformation rate constants and growth and mortality rates of bacteria varied under different ISS conditions, with the highest values observed for ISS sizes in the range of 20-38 µm at concentration of 200 mg L-1. These findings indicate the substantial impact of ISS on the biological transformation of N in natural freshwaters.

Le et al., Sustain. Environ. Res., 25(6), 315-322 (2015)

carefully cleaned with a detergent, Milli-Q water, and 10% HCl acid, and finally rinsed with Milli-Q water. Just before beginning the experiments, the samples were filtered through a 38 µm-pore sieve to remove large particles, including zooplankton, and were then used for the N-transformation experiments.

Subsequently, an aliquot of the 38 µm filtered sample was used to determine the initial pH and bacterial biomass concentration. Other aliquots were further filtered with 0.45 µm membrane filters to measure the concentrations of NO2

-, NO3-, NH4

+, total nitrogen (TN), and total organic carbon (TOC). ISS was prepared by grinding and sieving silica sand (SiO2) into five size ranges: 11-20, 20-38, 38-63, 63-90, and 90-500 µm.

2. Nitrogen Transformation Experiment

Two series of experiments (series A and B) were conducted to investigate the effects of ISS size and concentration on N transformation, respectively, under the conditions summarized in Table 1. In series A, we incubated the system for 2 d prior to the main experiment.

At the beginning of the incubation, 20 mg of ISS (of different sizes) and 30 mg of glucose were added to 500 mL of filtered river water, resulting in 40 mg L-1 of ISS and 21 mg L-1 of TOC in the beaker. The glucose provided labile carbon sources for bacterial growth. Thus, the incubation with glucose together with N naturally contained in river water was expected to support the bacterial adaptation to the designed growth conditions. During the two incubation days, the TOC concentration decreased to 4 mg L-1. Prior to the main experiment, other nutrients (NH 4

+, PO43-,

and NO3-) together with ISS of five different size

ranges were added equally to the samples (the final ISS concentration was fixed at 1000 mg L-1). In series B, after observing the results from series A, where the total added glucose quickly reduced within 2 d, we started the main experiment without pre-incubation. The substrate (glucose) and nutrients (NH4, NO3, and PO4) were added to the water sample, and 500 mL of the solution was put into 500 mL glass beakers with initial conditions as summarized in Table 2. Dried ISS of middle size range 38-63 µm was then added to the samples to obtain the final ISS concentrations of 0, 200, 600, 900, and 1200 mg L-1. The 38-63 µm size range was chosen because the growth of bacteria in

is expected because SS primarily originates from soil [13], and SS and bed sediment can be exchanged through suspension and deposition processes [14]. At the same time, SS has its own typical properties such as a small size, large surface area, and high mobility in aqueous environments compared to the relatively fixed state and low liquid content in soil and bed sediments. Thus, it remains largely unclear whether SS particle size has significant effects on bacterial biomass and N transformation. Furthermore, although the effect of SS concentration on N dynamics has been previously investigated by the studies noted above, the utilization of SS as a mixture of both organic and inorganic particles in experiments [9] may cause confusion in interpreting which properties of SS such as organic substrate or surface area affect bacterial biomass and enhance microbial activity and N transformation.

Given the importance of the ecological and environmental roles of N in freshwater systems, it is crucial to enhance our quantitative understanding of the biochemical N transformation processes for a range of SS concentrations and sizes. In our study, therefore, we investigated the biochemical roles of inorganic SS (ISS) on the N dynamics in freshwater systems with a specific focus on the effects of ISS concentration and size on the ammonification and nitrification processes. To this end, a series of batch experiments were conducted to investigate the effect of ISS on N dynamics and bacterial biomass. Moreover, a process-based model was employed to estimate N transformation rate constants and bacterial growth and mortality rates. This study also tested two hypotheses: 1) the presence of ISS increases bacterial biomass in the water column; and 2) the increased bacterial biomass accelerates ammonification and nitrification rates.

MATERIALS AND METHODS

1. Preparation of Water Sample and ISS

The sampling was conducted on the surface of a lower reach of the Tama River (at Futagotamagawa in Tokyo, Japan) from February to June 2012 under stable water-level conditions one day prior to the N-transformation experiment. Water samples were then transported in 500 mL high-density polyethylene bottles (74 × 74 × 146 mm) and stored in a dark refrigerator at 4 °C. Prior to sampling, the sample bottles were

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Table 1. ISS concentration and size in the two series of the experimentsExperiment ISS concentration (mg L-1) ISS size (µm)Series ASeries B

1000 0, 200, 600, 900, 1200

11-20, 20-38, 38-63, 63-90, 90-500 38-63


biomass, is a mass ratio of carbon to protein in a bacterial cell, Pr is the protein concentration (mg L-1), and is the C to N weight ratio in bacterial cells. Table 3 summarizes these parameter values. In addition, organic N (ON) concentration was calculated as the difference between TN and the sum of DIN and CNbio. Furthermore, the TOC concentration was measured with a total carbon analyzer (TOC-5000, Shimadzu, Kyoto, Japan). All samples were measured with three replicates, and their averages were taken as the measured value. The relative errors of N-species, TOC, and protein measurement were less than 5, 4, and 5%, respectively.

4. Nitrogen Transformation Model

The rates of N transformation, bacterial growth, and mortality were estimated by applying a revised process-based model of N transformation, which is a slightly modified version of the CCHE3D_WQ model [3]. In this model, the biochemical effects of ISS on N transformation are integrated by treating bacterial biomass as an additional state variable, as compared with the original version, and considering it as both a sink (i.e., bacterial N assimilation) and source (due to cell lysis) of N. Our experimental design, as described above, allowed us to neglect other processes mediated by phytoplankton (in dark conditions), denitrification (aerobic conditions), and particle settling (continuously stirring). Consequently, the governing equations used in this study were formulated as follows:

the solution containing SS in this size range in series A could be confirmed and 38-63 µm was in the middle of our tested range. All the glassware and plastic vessels were sterilized by autoclaving at 121 °C for 30 min and soaking in 10% HCl at least one day before using.

All samples in series A and B were equipped with mechanical stirrers and set at a rotation of 175 rpm to keep the particles suspended throughout the experiment. Finally, perforated aluminum foil was used to cover the beakers to prevent any photochemical processes while still allowing air exchange. Furthermore, pH, temperature, and DO were maintained at a circumneutral level (7-8), 24 °C, and approximately 7.1 mg L-1, respectively. At each time step (0, 1, 2, 3, 4, and 7 d), 20 mL water samples containing ISS were taken and divided into two aliquots (10 mL each). One was filtered through a 0.45 µm filter to determine the dissolved inorganic nitrogen (DIN), phosphorus, and TOC concentrations, and the other aliquot was placed in centrifuge vials and stored in an ultrasonic bath at 80 °C for 1 h to determine the bacterial biomass and TN concentration. These water samples were maintained at 4 °C and analyzed within three days for all parameters.

3. Chemical Analysis

The concentrations of TN, NO3-, NO2

-, and NH4+

were analyzed using a Bran+Luebbe TrAAcs 2000 Autoanalyzer (SEAL Analytical, Japan). Furthermore, TN was determined as NO3-N after digestion with K2S2O8 at 120 °C for 30 min. Bacterial biomass was determined on the basis of the protein measurement with a Pierce® BCA Protein Assay Kit (Thermo Fisher Scientific). The N of bacterial biomass (CNbio) was determined by Eq. 1:

Here, is the ratio of active to total bacteria

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Table 2. Initial concentrations of nutrients in the two series of experiments (Unit: mg L-1)

Experiment NH4-N NO3-N ON TOC PO4-PSeries ASeries B

1.52.1

2.010.6

1.91.9

4.034.1

2.02.6

(1)

Table 3. Parameter values used in model formulationParameter Symbol Value Unit Reference

Half-saturation constant for oxygen limitation of nitrification Knit 2.0 mg L-1 [15]Half-saturation constant for nitrogen KmN 0.01 mg-N L-1 [15]Fraction of dead bacteria recycled to the organic nitrogen pool fon 0.5 - [16]Temperature coefficient θ12, θ71 1.08 - [15]Ratio of active bacteria to total bacteria αab 0.5 - [17]Ratio of carbon weight in bacteria cell αCb 0.5 - [18]C:N weight ratio in bacterial cell αCN 5.6 - [19]

(2)

(3)αab

αCb

αCN


2-4 to the observed N-species concentrations at each time step using a nonlinear fitting method. Spearman’s rank correlation was applied to examine the relation between the ISS concentration and bacterial biomass. Nonparametric Kruskal-Wallis H Test was employed to test the effect of ISS size on the average bacterial biomass. The statistical analyses were conducted using R (ver. 2.15.0, the R Foundation for Statistical Computing).

RESULTS AND DISCUSSION

1. Effect of ISS on Bacterial Biomass

The average bacterial biomass concentration in series A ranged from 4.1 to 11.8 mg L-1. The highest average biomass concentration was observed in the ISS size range of 63-90 µm (Fig. 1a); however, ISS size did not significantly influence bacterial biomass (H test, p > 0.05). In contrast, in series B, a positive correlation (R = 0.68, p < 0.01) was observed between ISS concentration and bacterial biomass, which ranged from 6.37 to 25.1 mg L-1 (Fig. 1b). These results imply that the presence of ISS in the water column created favorable conditions for bacterial growth. It is likely that the availability of a solid surface area enhanced bacterial colonization and the formation of a biofilm on the ISS surface.

The weak effect of ISS size on bacterial biomass concentration in series A, compared to those in river bed sediment and detrital and soil particles [10-12], was probably due to the limited ISS particle size range in our experiment and the simple inorganic composition (SiO2), as compared with the complex compositions of natural soil or bed sediments. Thus,

Here, Dp is the mortality rate of bacteria (d-1), Gp is the growth rate of bacteria (d-1), k12 is a nitrification constant (d-1), and k71 is the mineralization constant of ON (d-1). These parameters related to the N transformation processes were subjected to parameter estimation. Furthermore, (1 - ƒon) is the fraction of dead bacteria to the NH4

+ nitrogen pool. In addition, CDO, CNH4, CNO3, and CON are the concentrations (mg L-1) of DO, NH

4+-N, NO3-N, and ON, respectively. In the

above equations, CNbio is the N biomass concentration (mg L-1), Knit is the half-saturation constant for the oxygen nitrification limitation (mg L-1), and θ71 and θ12 are the temperature coefficients for mineralization and nitrification, respectively. PNH4 is the preference term for the ammonia uptake, calculated as following equation:

KmN is the half-saturation constant for N. Table 3 also provides a summary of these parameter values.

5. Statistical Analysis

The rates of N transformation, bacteria growth, and mortality were estimated by fitting model Eqs.

(4)

318

(5)

(6)

Fig. 1. The effect of ISS size (a) and concentration (b) (p indicates a probability of no significant difference among groups and no correlation, respectively) on average bacterial biomass from day 3 to 7.


concentrations in the systems with the smallest and largest ISS size ranges (11-20 and 90-500 µm) were lower than those with a middle ISS size range (20-90 µm). On the first day of the experiment (day 0-1), the NH4

+ concentration in the systems with ISS in the middle size ranges increased, indicating that ON decomposition was more dominant than nitrification. However, the NH4

+ concentration in all other size fractions decreased. The decomposition of ON, rather than the nitrification process, was probably stimulated in the ISS size range of 20 to 90 µm, where the colonization and growth of heterotrophic

further investigations of this interaction must be undertaken using ISS with different chemical qualities and structures (e.g., common clay minerals such as montmorillonite, sericite, and kaolinite). Although particles larger than 500 µm only account for a small proportion of SS [4], further tests with a wider ISS size range might be interesting.

2. Temporal Change in Concentrations of Nitrogen Species

From day 1 to 7 (Fig. 2a), in series A, the NH 4+

319

Fig. 2. N-species concentration over 7 d under various ISS concentrations (series A, Figs. 2a, 2c, and 2e) and sizes (series B, Figs. 2b, 2d, and 2f).


values. The ammonification rate k 71 ranged from 0.17 to 1.3 d-1 (Figs. 3c and 3d), which was much higher than a previous estimation (0.075 d-1) [3], and were in a wider range compared to previous results (0.18-0.69 d-1) [15]. The differences are possibly associated with the biodegradation of ON in the three studies. Nitrification rate k12 was estimated to be 0.015-0.61 d-1, which is comparable with the estimates by [3] (0.4 d-1). In both series A and B, k12 was relatively similar under different ISS sizes and concentrations, and k71, similar to Dp and Gp, was higher in the system treated with ISS in the size range of 20-63 µm (series A) and was highest with a 200 mg L-1 concentration (series B). The effect of ISS size on N transformation rates was clearly observed; however, the effect of ISS concentration was weak, except in the case of treatment with a 200 mg L-1 concentration. The result from series B, however, was specific for ISS in the size range of 38-63 µm, which is relatively large in the general ISS size range found in natural water [4]. Large particle size of ISS together with the flocculation effect in reactors may result in relatively little difference in total particle surface area among different ISS concentrations, which is one of the plausible reasons of this insignificant difference in N transformation rates in series B. Furthermore, there was no correlation among bacterial biomass and N transformation rate constants, bacterial growth, or mortality rates in either series (n = 5, R = 0.04-0.83, p > 0.05). In other words, increasing bacterial biomass did not necessarily result in higher N transformation rates. Probably, nitrification and ammonification occur mainly by autotrophic bacteria and heterotrophic bacteria, respectively, which have different specific activities. Another possible reason is that ISS characteristics affect bacterial activities and species composition.

The revised N model exhibited a reasonable simulation output, with chi-square values less than the critical value (c2

0.05,24 = 36.4). However, the coefficients of determination between the modeled and observed concentrations ranged from 0.01 (for the NO3

- model in series B) to 0.63 (for the NH4

+ model in series A). The performance of this model is still limited and must be calibrated and validated with additional experimental data. The errors are likely related to the analytical precision of our estimation of bacterial biomass and an underestimation of the total N due to the N adsorbed on the ISS surface. Furthermore, the settling process of ISS was neglected in the model formulation and could be an additional source of error.

CONCLUSIONS

This research investigated the effect of ISS concentration and size on bacterial biomass and N

bacteria are preferable (Fig. 1a). On the other hand, in experiments with ISS sizes ranging from 11 to 20 mm and from 90 to 500 mm, apparent ammonification rates remained lower than nitrification rates. As a result, the ON concentration decreased throughout the experimental period, while NO2

- + NO3- concentration

showed no significant changes for the first 3 days of the experiment but sharply increased thereafter (Figs. 2c and 2e). From day 2, there was a continuous reduction in NH4

+ concentration. These results suggest that the nitrification rate was higher than the ammonification rate. It is likely that the low ON concentration (< 1.7 mg L-1) and low TOC concentration (around 3 mg L-1) limited the ammonification processes catalyzed by heterotrophic bacteria.

Our observations from series B showed that the trends of NH4

+ over time were relatively similar among the different ISS concentrations (Fig. 2b), while the experiments with no-ISS treatment clearly exhibited the smallest NH4

+ concentration from day 2 to 4. Overall, there were sharp decreases in NH4

+ (from 2.05 to approximately 0.1 mg L-1) on day 1, while the NH4

+ concentration increased from day 2 to 4, and then reached a peak concentration on day 4 at approximately 3.5 mg L-1. The mechanism of these shifts might be explained by (i) NH4

+ assimilation by bacteria for their growth on the first day and (ii) their death after the rapid growth due to the limited substrate. The latter effect might result in the release of NH4

+ as cell-lysis product. From days 4 to 7, NH4

+ concentration slightly decreased in all cases likely due to the nitrification (the increase of NO3

-) and uptake by bacteria process. For other N species, there was a similar trend in all treatments. For example, NO3

- concentration decreased from day 1 to 3 and increased from day 3 to 7 (Fig. 2d); ON concentration decreased from the beginning to day 4 (due to mineralization) and slightly increased thereafter probably due to cell lysis and/or production from bacterial activities (Fig. 2f). No difference was found in the temporal trends of the NO3

- concentration for different ISS concentrations; however, the concentration of ON with no-ISS treatment was higher than that of the others for the duration of the experiment.

3. Nitrogen Transformation Rates

From the model application, the Dp and Gp, were estimated to be from 0.44 to 9.54 d-1 and from 0.40 to 9.93 d-1 , respectively (Figs. 3a and 3b). In series A, Dp, and Gp in the systems treated with ISS in the 20-63-µm size range were higher than those in the other systems. In series B, lower ISS concentrations (i.e., 0 and 200 mg L-1) resulted in higher Dp and Gp

320


ACKNOWLEDGEMENTS

This work was supported by the Asian Core Program and Core-to-Core Program (B. Asia-Africa Science Platforms) of the Japan Society for the Promotion of Science (JSPS).

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Fig. 3. Estimated parameters for different ISS sizes (a, c) and concentrations (b, d). Error bars indicate standard deviation of parameters estimated by nonlinear fitting of the water quality model to N species concentration.


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Discussions of this paper may appear in the discus-sion section of a future issue. All discussions should be submitted to the Editor-in-Chief within six monthsof publication.

Manuscript Received: February 21, 2015Revision Received: May 18, 2015

and Accepted: July 21, 2015

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