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Influence of scale on biomass growth and nutrient removal in an algal-bacterial leachate treatment system Kaitlyn D Sniffen, Jacob Ryan Price, Christopher M Sales, and Mira Olson Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b03975 • Publication Date (Web): 20 Oct 2017 Downloaded from http://pubs.acs.org on October 21, 2017

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Environmental Science & Technology

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Influence of scale on biomass growth and nutrient removal in an algal-bacterial leachate

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treatment system

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Keywords: algae, wastewater, leachate, scale-up, biomass growth, nutrient removal

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Authors: Kaitlyn D. Sniffen, Jacob R. Price, Christopher M. Sales, Mira S. Olson*

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Affiliation of all: Drexel University, 3141 Chestnut St., Philadelphia, PA, 19104

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Corresponding author*: [email protected]

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Abstract: Data collected from experiments conducted at a flask scale are regularly used as input

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data for life cycle assessments and techno-economic analyses for predicting the potential

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productivities of large scale commercial facilities. This study measures and compares nitrogen

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removal and biomass growth rates in treatment systems that utilize an algae-bacteria consortium

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to remediate landfill leachate at three scales: small (0.25L), mid (100L) and large (1000L). The

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mid- and large-scale vessels were run for 52 consecutive weeks as semi-batch reactors under

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variable environmental conditions. The small-scale experiments were conducted in flasks as

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batch experiments under controlled environmental conditions. Kolomogov-Smirnov statistical

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tests, which compare the distributions of entire data sets, were used to determine if the ammonia

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removal, total nitrogen removal, and biomass growth rates at each scale were statistically

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different. Results from the Kolmogov-Smirnov comparison indicate that there is a significant

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difference between all rates determined in the large-scale vessels compared to those in the small-

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scale vessels. These results suggest that small-scale experiments may not be appropriate as input

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data in predictive analyses of full scale algal processes. The accumulation of nitrite and nitrate

ACS Paragon Plus Environment


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within the reactor, observed midway through the experimental process, is attributed to high

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relative abundances of ammonia- and nitrite-oxidizing bacteria, identified via metagenomic

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analysis.

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Introduction: Algae are frequently investigated for use in nutrient removal from wastewater sources, as

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the presence of nutrients can cause costly economic and ecological damage to receiving water

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bodies [1, 2]. While it is most commonly suggested that algae-based nutrient removal be utilized

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in domestic wastewater treatment, the direct treatment of landfill leachate may also be favorable.

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Landfill leachate is a high strength liquid waste, containing concentrations of ammonia regularly

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exceeding 800mgN/L, and is most commonly trucked to and treated at domestic wastewater

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treatment facilities, costing landfills hundreds of thousands to millions of dollars each year.

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Treating the leachate directly may reduce these costs to landfills. Few studies have examined the

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treatment of landfill leachate by algae, and even fewer have done so on a long-term and large

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scale [3-9].

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Data from long-term, large scale studies should be used as input for predictive analyses

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such as life cycle assessments (LCAs) and techno-economic analyses (TEAs) which are used to

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evaluate if a commercial investment in a technology are environmentally favorable and

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economically feasible, respectively. However, many of these assessment tools utilize input data

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generated from small-scale studies, carried out under conditions that are significantly different

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than those that occur on a commercial scale [10-14]. Studies conducted by Moody et al. (2014)

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and Quinn et al. (2015) have shown that the wildly variable results from LCAs and TEAs are due

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to inconsistent input data and system boundaries, which define the processes included in the


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assessments. These studies emphasize that it is crucial to use input data from studies that mimic

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the environmental conditions and type of growth vessel of the proposed commercial scale

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process in these predictive analyses [15, 16].

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It has repeatedly been shown in studies using algae [15, 17], E.coli [18-21], and yeast

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[20, 22] that production efficiencies from small-scale studies using microorganisms do not scale

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proportionally to those occurring on a commercial-scale. For example, changes in vessel

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characteristics, specifically surface area to volume ratios, [20, 23, 24], mixing dynamics, such as

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types of mixing used, [20, 24, 25], and environmental conditions (e.g., temperature, light

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exposure) [26-28], can significantly affect the activity of microorganisms [20, 23, 24]. Despite

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this discrepancy between results from small-and large-scale studies, the vast majority of LCAs

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and TEAs for evaluating the overall environmental impact and technical feasibility of large-scale

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algae production systems use algal growth rates and densities from small-scale studies as input

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values for their estimation of algal growth technologies [15, 16, 29].

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The study presented here uses a system that couples algae biomass production with the

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treatment of landfill leachate. Specifically, this study compares algal growth and nitrogen

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removal using untreated leachate as a nutrient source in liquid cultures of a mixed algae/bacteria

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consortium at three different scaled systems: 0.25L, 100L, and 1000L. Algal growth and

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nitrogen removal rates measured in small-scale (0.25L) systems are statistically compared to

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rates to observed at mid- (100L) and large-scales (1000L) to determine if results from small-

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scale experiments are representative of larger-scale systems.

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The small-scale (0.25L) vessels were operated as 7-day batch experiments [30-32] in an

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environmental chamber under controlled laboratory conditions. The flask-scale and controlled

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growth conditions were deliberately chosen as the comparison scale for this study as this



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experimental set up is similar to those most frequently used to generate input data for LCAs and

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TEAs. In contrast, the mid- (100L) and large- (1000L) scale vessels were operated on a 7-day

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semi-batch cycle but under the uncontrolled environmental conditions of a greenhouse. These

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operating conditions were chosen as the environmental conditions of a full-scale outdoor system

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will not be controlled.

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Materials and Methods:

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Nutrient feed and inoculum:

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The nutrient feed used in this study was raw, untreated landfill leachate collected from the

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Sandtown Landfill in Felton, DE. Leachate was refrigerated at 2ºC from collection until use.

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Over the duration of the study, the ammonia content and pH of the leachate ranged from 320 to

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935 mgN/L and 6.48 to 7.56, respectively. A more detailed analysis of the leachate composition

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at each collection time point, data provided by the Sandtown Landfill, is described in Table S1 in

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the Supplemental Information. The algae-bacteria culture used in this study was first collected

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from a fish pond on the University of Pennsylvania campus. The inoculant was grown in the

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urban greenhouse using leachate as a nutrient feed.

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Sampling, preparation, and analysis of metagenome – The microbial population’s composition

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and structure determines a bioreactor’s kinetic behavior, which is commonly observed through

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chemical analysis. To assist in understanding these behaviors, metagenomic sequencing was

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applied to a sample collected from one of the growth vessels. Total DNA was extracted from

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four biological replicates collected prior to the beginning of the experimental period of this

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study. DNA extraction was carried out immediately after sample collection following the


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methods described in detail within Price et al 2016 [33]. The four replicate DNA extractions

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were then assessed for quality and concentration via NanoDrop 2000 and QuBit 2.0 and then

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pooled and submitted for 2x250 paired-end sequencing on an Illumina HiSeq sequencer. The

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Metagenomics RAST pipeline [34] was used to analyze the taxonomic and functional

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composition of the resulting sequences. Raw reads were uploaded to the MG-RAST server and

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paired-end reads were joined using the join-paired-ends function. Quality control measures were

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applied including the removal of artificially induced sequencing artifacts [35], the removal of

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sequences derived from H. sapiens [36], and the removal of low quality sequences (minimum

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Phred score of 15, and all sequences were trimmed such that they contained a maximum of 5 low

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quality base calls) [37]. Metagenome identification and quality control statistics are provided in

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Table S2 in the Supplemental Information.

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Mid & large scale experimental design:

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Setup – This set of experiments used two 100L Plexiglas aquarium tanks (ATs) and two 1000L

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raceway ponds (RWPs) from MicroBio Engineering (San Luis Obispo, CA). The 100L ATs and

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the 1000L RWPs are referred to herein as mid- and large-scale vessels, respectively. The

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working volumes of the ATs and RWPs were 60L and 600L, respectively. Examples of the mid-

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and large-scale vessels used in this study can be found in Figure 1 A-B [38]. All vessels were

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inoculated with an algae-bacteria culture, at the beginning of this study in February 2016, and

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housed in a greenhouse on the roof of an academic building at Drexel University in Philadelphia,

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PA [5, 38]. These vessels were exposed to semi-ambient conditions; no additional lighting or

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heating/cooling was provided. Daylight hours ranged from 9hr:20min to 15hr:1min throughout

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the year, though the actual amount of light to reach the cultures in the greenhouse was



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substantially less due to weather and urban obstructions. Temperatures of cultures ranged from

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7.8 to 41.7°C.

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Operation – The mid and large scale vessels were operated as semi-batch reactors, on a seven –

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day batch cycle. Initial samples were taken at the beginning, and final samples were taken at the

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end of each weekly cycle, where ammonia-N, nitrate-N, nitrite-N, and biomass density were

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measured and analyzed. To prepare the system for the next weekly cycle, the entire volume of

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the mid-scale AT was pumped into the large-scale RWP. After mixing, one third of the liquid

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and biomass volume was removed and then replaced with water and leachate. A portion of this

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mixture was then transferred back into the AT and the next week’s cycle would begin. The

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volumes of the mid- and large-scale vessels were mixed together in between each week in order

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to maintain equivalent initial nutrient and biological conditions at the two scales. The volume of

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leachate added each week was increased slowly throughout the study, ranging from 5 to 40L per

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week. Exact weekly leachate additions can be found in Figure S1 of the Supplemental

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Information. These vessels were operated for 52 consecutive weeks, February 2016 – February

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2017. A more detailed description of this method can be found in Sniffen et al (2017) [38].

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Environmental Monitoring – Water temperature, pH, and dissolved oxygen within the tanks and

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raceway ponds were collected at five-minute intervals throughout the yearlong study using

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Neptune System’s APEX controller and probes (Morgan Hill, CA). Weekly maxima, minima,

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and averages were calculated for each of these parameters.

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Oxygen production analysis – The total suspended solids contained in the weekly samples was


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made up of live and dead cellular biomass as well as particulates from the leachate additions.

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The rate of oxygen production was measured to determine the oxygen production activity

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relative to the total suspended solids density [39, 40]. Oxygen production rates of the biomass

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samples were measured at the beginning and end of each week. Rates from each sample were

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measured using 450mm Unisense O2 probes (Aarhus N, Denmark) over 10 minute intervals,

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which were run in biological triplicates. Each sample was run at 100% and 25% dilution of the

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initial samples. The 25% dilution samples were found to give a better representative oxygen

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production rate than samples with high biomass densities which may have experienced

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significant self-shading (data not shown).

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Mixing dynamics – The flow rate through the raceway pond was determined using the EPA float

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method [41]. The mixing efficiency of the ponds was determined using a salt tracer test. Five

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liters of a 250 g/L sodium chloride salt tracer solution was poured into the raceway pond. Water

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samples were taken at 6 locations around the pond at 10-second intervals. The conductivity of

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these samples was measured and the concentration of salt tracer was determined using a standard

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curve.

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Small-scale experimental design:

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Flask-scale studies were performed using the same algae culture and leachate as those used in the

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mid- and large- scale vessels. The ranges of nutrient and biomass concentrations used in these

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small-scale studies mimicked those used in the mid- and large-scale studies. These small-scale

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studies used 250 mL flasks with 100mL working volume, shaken continuously in an Innova 44

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environmental chamber (Eppendorf, Hamburg, Germany), under constant 25°C temperatures and



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continuous light, as depicted in Figure 1 C. Each nutrient and biomass concentration condition

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was tested using biological triplicates. The duration of all small-scale experiments was seven

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days. Ammonia-N, nitrate-N, nitrite-N, and biomass density were measured and analyzed at the

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beginning and end of each of the experiments.

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Analytical methods:

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Samples collected from experiments run at all scales were analyzed using the following

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analytical methods: Ammonia-N, nitrate-N, and nitrite-N were measured using Hach test

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methods 10031, 10020, and 8153, respectively, with a DR2400 spectrophotometer. Removal

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rates of each nitrogen species along with total dissolved nitrogen were calculated using

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Equations 1-2. In Eqn. 1-2, CoN and CfN are the initial and final concentrations of the nitrogen

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species [mgN/L], respectively, where t is time [days] [5], NT is the total inorganic nitrogen

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concentration [mgN/L], and Ri is the removal rate of species i [mgN/L/day] for each batch

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period. These equations were developed based on preliminary studies which monitored nitrogen

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removal and biomass growth every 1 to 2 days during week-long studies. These preliminary

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studies found that the removal and growth rates were linear throughout the week.

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(Eqn. 1)

NT= CNH3 + C NO2- + C NO3-

(Eqn. 2)

Ri= (CoN – CfN)/t

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Biomass density was measured at the beginning and end of each week by standard total

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suspended solid protocol using a 0.45µm filter and 20mL of sample [42]. Biomass density


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samples were run using biological duplicates. Weekly biomass growth rates were calculated

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using equation 3, where Co,Biomass and Cf, Biomass are the initial and final biomass concentrations

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[g/L], RB is the biomass growth rate [g/L/day], and t is time [days].

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(Eqn. 3)

RB= (Cf, Biomass – Co, Biomass)/t

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Statistical analysis – Weekly rates (nutrient removal and biomass growth) and environmental

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data were pooled for regression and correlation analysis to identify general trends. Weekly

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nutrient removal and biomass growth rates were then binned by scale for rate comparisons.

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Kolmogov-Smirnov statistical tests are used to determine if the distributions of two data sets are

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significantly different. Data binned by scale was compared using Kolmogov-Smirnov tests to

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determine if the weekly rates calculated at each scale were statistically different. Regressions and

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correlation data categories included: initial and final weekly ammonia-N, nitrate-N and nitrite-N

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concentrations and removal rates; initial and final weekly biomass concentrations and growth

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rate; initial and final weekly oxygen production rates; as well as maximum, minimum, and

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average pH, water temperature, dissolved oxygen, and number of daylight hours. SPSS 24[43]

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and Microsoft Excel were used to analyze this data.

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Results and Discussion:

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Environmental conditions – Seasonal trends of the temperature, pH, and dissolved oxygen (DO)

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in the tanks and raceway ponds, over the course of the yearlong study can be seen in Figure 2A-

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C. Over the course of a day, the pH, temperature, and dissolved oxygen in the tanks and raceway

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ponds rise and fall on a diurnal cycle. This daily trend is shown over a representative two-week



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span, depicted in Figure 3. As confirmed by observed data, the ATs and RWPs were subjected

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to identical environmental conditions due to their colocation.

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(Figure 2A-C)

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(Figure 3)

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Mid- and large-scale experimental results – The ranges in initial ammonia, total nitrogen and

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biomass concentrations over the course of the study were 0.2-161 mgN/L, 8.12- 187.8 mgN/L,

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and 40-1780 mgBiomass/L, respectively. A breakdown of initial conditions by scale is presented

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in Table S3 in the Supplemental Information. Leachate input volumes were approximately 20

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L/week during the first half of this study, while the input volumes during the second half were

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approximately 30-40L/week. During times of high nitrite concentrations in the systems, no

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leachate was added. Weekly leachate input volumes during this study can be found in Figure S1

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of the Supplemental Material.

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The dissolved nitrogen content of the leachate was 99% in the form of ammonia. Ammonia and

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total nitrogen removal rates along with biomass growth rates varied over the course of the study;

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the average ± standard deviation of these rates for the large-, mid- and small-scales are presented

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in Table 1. The ammonia and total nitrogen removal rates for each of the vessels are presented

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in Figure 4 A-B.

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Table 1: Average ± standard deviation of ammonia removal, total nitrogen removal, and biomass growth rates, at each scale. Large-scale Mid-scale Small-scale Total N Removal Rate 1.63 ± 2.95 1.82 ± 3.97 3.16 ± 2.73 (mgN/L/day) Ammonia Removal Rate 2.33 ± 3.05 2.68 ± 3.90 2.96 ± 2.71 (mgN/L/day) Net Biomass Growth Rate 12 ± 24 4 ± 38 18 ± 21 (mg/L/day)

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(Figure 4 A-B)

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The maximum biomass density measured over the course of this study was 2100 mg/L. Biomass

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growth rates ranged from -106 to 199 mg/L/day. A negative growth rate means that there was

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biomass loss over the course of the week. Biomass loss was seen in 70 out 205 weeks’ worth of

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data from all vessels. Negative biomass growth rates did not statistically favor any scale, season,

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or environmental condition. Detailed discussion of this point can be found in Sniffen et al. (2017,

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submitted). When accounting for only weeks with positive growth rates the average and standard

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deviation was 28 ± 32 mg/L/day. This range of growth rate is similar to rates reported for algae

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growth fed with domestic wastewater [44-47]. Weekly biomass growth rates for all vessels can

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be found in Figure S2 of the Supplemental Information.

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Metagenomics results – Alpha diversity within the metagenomic sample was estimated to be 376

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taxa. Annotations from the NCBI Reference Sequence Database [48-50] and the M5nr protein

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sequence database [51], acquired via the MG-RAST interface, were used to analyze and interpret

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the taxonomic composition of the microbial community. The vast majority of reads were

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attributed to the domains of Bacteria and Eukaryota (Table S4 in the Supplemental Information).

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Proteobacteria and Bacteroidetes were the first and second most abundant phyla respectively

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(Table S5 in Supplemental Information). Under the annotations for both reference databases,

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Chlorophyta and Cyanobacteria comprised about 2% of the annotated reads (Table S5).

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Chlamydomonas reinhardtii, Volvox carteri, and Scenedesmus obliquus accounted for the bulk

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of the reads attributed to Chlorophyta (Table S6 in the Supplemental Information). The

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distribution of reads amongst species falling within the phyla Cyanobacteria was much more



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uniform at the species level (Table S7 in the Supplemental Information); agglomerating taxa by

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Genus indicates that the genus Synechococcus accounts for roughly 30% of the reads within

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Cyanobacteria, followed by Cyanothece spp. at 19%, and Nostoc spp. at 12% within this phyla.

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AOB and NOB were observed to comprise roughly 12% and 1% of the total microbial

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population (Table S8 in Supplemental Information). The anammox bacteria Candidatus Kueneia

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was also discovered in the M5nr annotations. This indicates that anammox may be a potential

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nitrogen transformation pathway within this system (Table S8).

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Low abundances for eukaryotes, such as Chlorophyta, within this study, are common in

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metagenomic analyses, arising from their being underrepresented within reference databases,

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including those used by MG-RAST [52]. This induces bias towards the identification and

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annotation of bacterial sequences, while sequences from eukaryotic sources may fail to be

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annotated. Because of this, the abundances of Chlorophyta reported here should be viewed as a

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floor or minimum and should not be directly compared to the relative abundance of the kingdom

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Bacteria, or the taxa therein.

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Statistical analysis – A Pearson’s correlation matrix was calculated for all of the categories listed

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in the statistical methods section. Strong, statistically significant correlations were found

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between: initial ammonia concentration and ammonia removal rate (r=0.854, p

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