weftec 2013 final manuscript for booth

Novozymes A/S ∙ 5400 Corporate Circle ∙ Salem, VA 24153

HIGH-SPECIFICITY TRACKING OF BIOAUGMENTATION STRAINS IN A FULL

SCALE WASTEWATER TREATMENT FACILITY

Seth D’Imperio, Steve Leach, Matthew R. Livingston, Vaibhav P. Tale; Chrissie T. Edwards,

Justin Terra, George L. Lucas

Abstract

Wastewater treatment systems contain complex biological communities utilized to

perform a variety of functions. Operators are continuously optimizing these systems in order to stabilize biological activity, meet regulations, and improve efficiency. In order to satisfy the dynamic needs of the plant, many operations have turned to

bioaugmentation methods. While anecdotal reports of effects are often cited regarding the efficacy of bioaugmentation products, the specific role that exogenous

microbial strains play in these changes has yet to be fully understood. Questions remain regarding application strategy, product formulation optimization, and the direct link between bioaugmentation and plant performance. Compounding this

issue, most bioaugmentation products contain a mixture of microbiological strains, many of which are phylogenetically related to those found endogenously in

wastewater treatment systems. Furthermore, decisions regarding the application of these products are generally based on operational parameters that may not accurately represent the state of microbial activity in the system. The present

study utilizes two molecular techniques; quantitative polymerase chain reaction (qPCR) and Recognition of Individual Gene – Fluorescence in situ Hybridization

(RING-FISH) applied to whole genome sequences of the bioaugmentation strains. The combination of these techniques addresses the presence, quantity, and spatial distribution of several bioaugmentation strains in full scale wastewater treatment

plants.

Introduction

Advances in wastewater treatment research have emphasized the importance of the microbial populations inherent to the processes used (Wagner et al., 2002). The

total microbial community structure is responsible for both efficient processing and removal of pollutants such as COD, phosphorus, nitrate, etc, and for the maintenance of stable plant conditions. The active consortia in these systems are

strongly influenced by the influent type, treatment scheme and operational conditions (Manz et al., 1994), and their importance is reflected in the intentional

design of wastewater treatment plants specifically for the enrichment of some microbial constituents over those with detrimental effects on treatment, such as filamentous bacteria responsible causing bulking problems.


The process of adding microbial members to these complex consortia for beneficial

effects, bioaugmentation, has been practiced for years and recent findings provide considerable support for bioaugmentation methods (Bai et al., 2010). In order to better understand the mechanism of bioaugmentation products the effects of these

treatments must be closely monitored and linked to the growth and persistence of the bioaugmentation organisms. Here we applied a combination of molecular and

microscopic methods to monitor the growth and presence of several specific bioaugmentation strains in a full-scale petrochemical wastewater stream while simultaneously monitoring the performance of the plant during and after treatment

with a bioaugmentation product.

Methods

Genomic sequencing & primer/probe development Complete genome sequences of all the microbial strains present in the Novozymes

product BioRemove 5100 were sequenced via 251 cycle paired-end Illumina sequencing and assembled at Virginia Bioinformatics Institute based on the nearest-neighbor scaffolding algorithm.

The draft genomes were analyzed to identify unique regions greater than 150 base pairs that could be utilized for strain-specific qPCR and to produce Recognition of

Individual Gene Fluorescence In Situ Hybridization (RING-FISH) polynucleotide probes. These regions were compared to the NCBI non-redundant (nr) database and selected for primer and probe development when there were no significant

similarities found within the database. PCR Primers and probes with 5’ BHQ and 3’ FAM were ordered from Operon (Huntsville, AL). qPCR was performed on a

LightCycler 480 with a 96-well plate block (Roche, Indianapolis, IN).

Primer/probe sets were checked for their specificity against pure culture templates from phylogenetically related and distant organisms. Specificity was also tested against an untreated wastewater background microbial community from the field

trial site to ensure that there was no unintended amplification/fluorescence from the designed primer sets. Sensitivity was determined and qPCR standard curves

were developed through a qPCR dilution series of target strain pure cultures added to the wastewater sludge background.

Polynucleotide probes were prepared for RING-FISH visualization of specific strains within the wastewater samples according to the method previously described

(Zwirglmaier et al., 2004) Separate polynucleotide RING-FISH probes were produced for each of the strains in the BioRemove 5100 product.

Field site and sampling A petrochemical wastewater treatment plant with an average flow rate of 5700 gpm

was selected for the study. The wastewater system (shown in Appendix 1) is a modified activated sludge design. A portion of the process wastewater (“Green


stream” accounting for 80% of the BOD load) is sent to oil/water separators then to

two dissolved air floatation (DAF) units which then enter a cooling tower. Following the cooling tower the remainder of water (“Blue stream” 20% of the BOD load) is added before entering a large equalization (EQ) basin (6 million gallons volume).

The EQ basin feeds two parallel moving bed bioreactor (MBBR) tanks. MBBR tanks have attached growth treatment units with approximately 50% of the volume filled

with PVC support media. The effluent from these tanks is combined in a mix tank and fed to two parallel UNOX systems. Both the UNOX systems use pure oxygen in three different stages of treatment. The effluent from the UNOX enters the

secondary clarifiers and the return activated sludge (RAS) is recycled back to the second stage of the UNOX basins at 1,100 gpm, on average. The average wasting

rate is 4-6 gpm and averages 20,000 ppm TSS. At this wasting rate, the plant maintains a sludge retention time (SRT) of approximately 30 days.

BioRemove 5100 (Novozymes, Salem, VA) was added into the system upstream of the MBBR as indicated by the “Green stream” in figure 1.. The bioaugmentation

treatment continued for 35days. Following this period, bioaugmentation was stopped for a period of 91days to observe the washout rate of the bioaugmented strains and restarted till the conclusion of the trial. Operators on-site measured and

reported a standard set of plant performance parameters including those reported in Table 1.

Figure 1. Flow diagram of experimental field site. Green star indicates bioaugmentation product addition site. Red stars indicate position of sampling sites

15 ml samples were retrieved from each of the MBBR and UNOX outfalls daily and

stored frozen until shipped to Novozymes laboratories weekly. Upon arrival at the laboratory, samples were divided into 2 subsamples. DNA was extracted from a 2


ml subsample using the PowerLyzer PowerSoil DNA extraction kit (MoBio, Carlsbad,

CA) according to the manufacturer’s protocol. qPCR was performed for each of the 8 strains in the BioRemove 5100 product at each time point for each sampling location, indicated in figure 1.. Reactions were run in triplicate and absolute

quantification was performed based on standard curves in each wastewater type.

A second subsample was prepared for RING-FISH as previously described (Nielsen et al., 2009; Zwirglmaier et al., 2004). Briefly, the strain specific probes described above were hybridized to the sample then washed off to avoid background

fluorescence. After hybridization and washing, the samples were treated with VectaShield® mounting medium containing propidium iodide (Vector Laboratories,

Burlingame, CA) to protect against photodegredation of the fluorescein labeled probes and to provide a biological counterstain during visualization on a Nikon Eclipse80i epifluorescent microscope.

Results

Plant Performance

Plant performance data was divided into 4 discrete segments; Pre-treatment, Treatment 1 (T1), Post-Treatment, and Treatment 2 (T2). The pre-treatment period

consisted of the 70 day period (2 times the treatment period length) immediately prior to the addition of bioaugmentation product. T1 is the 35 day period (approx. 1.2 SRT) in which BioRemove 5100 was added daily. The post-treatment segment

was a 91 day period immediately following the treatment period where data was still recorded for the site and samples were retrieved though no new product was

added to the system. A final treatment period, T2 was used to repeat analysis of strain presence and growth in the system as well as performance changes due to

product addition. Plant operational conditions are presented in Table 1.

Pre-treat T1 Post-treat T2

Date 7/18-9/26 9/27-11/1 11/2-2/1 2/2-2/13

Days 70 35 91 11

Average Flow (m3/day) 22,889 23,101 25,679 27,914

Average BOD (mg/L) 515 604 499 614

Average Influent BOD

Load (kg/d) 11,786 13,770 12,712 17,071

Table 1. Plant operational characteristics for the four treatment periods of the field trial.


BOD removal efficiency at the plant improved during the treatment periods (T1 and

T2) as compared to the pre-treatment and post-treatment periods (p=0.044) (Table 2). Additionally, plant stability, with respect to BOD removal efficiency, was improved during the T1 treatment period (Figure 2). Plant stability and performance

were improved for approximately 30 days following the cessation of bioaugmentation.

Pre-treat T1 Post-treat T2

Number of readings 31 16 39 5

MBBR BOD removal (%) 50.7% 47.8% 47.6% 53.9%

UNOX BOD removal (%) 85.3% 94.5% 91.5% n/a

Total BOD removal (%) 94.8% 97.4% 96.2% 96.8%

Table 2. Average BOD reduction efficiency for treatment and non-treatment periods. n/a: data not available

Figure 2. Overall system BOD removal efficiency for the duration of the field trial. Treatent periods are denoted with dashed lines

Bioaugmentation strain monitoring

The concentrations of the eight strains that are in the BioRemove 5100 product were tracked individually via qPCR (Figure 3, A-D) at the four sampling sites twice

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weekly. The total cells per milliliter of sample from the UNOX systems in each train

(North and South) is reported in Figure 4 and compared to a theoretical total bioaugmentation strain cell count for the system. The theoretical concentration of bioaugmentation strains assumes even distribution of all strains between the North

and South trains and a constant 30-day sludge age, all bioaugmentation biomass was wasted with the waste activated sludge, and none left in the effluent over the

clarifier weir. This theoretical concentration also does not account for strain growth and serves as a reference point to assess bioaugmentation strain growth in the system.

There was immediate growth of some of the bioaugmentation strains in the system

once dosage commenced with the total concentration of bioaugmentation strains 4 logs higher than the theoretical accumulation due to strain dosage (Figure 4). Strains NZ013 and NZ094 accounted for the majority of the bioaugmentation strain

presence in the UNOX systems (Figure 3), suggesting that these strains were well suited to the treatment environment. During the post-treatment phase,

bioaugmentation strain concentration declined rapidly, though some strains, NZ012 and NZ100 remained at concentrations slightly above their theoretical accumulation levels.


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Figure 3. Individual concentrations of bioaugmentation strains in the UNOX North (A) and South (B) systems and MBBR North (C) and South (D) systems as determined via strain-specific qPCR throughout the course of the field trial. Treatment periods are denoted with dashed vertical lines.

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Figure 4. Combined bioaugmentation strain concentration in the both the North (open circles) and South (closed squares) UNOX systems of the treatment plant obtained using strain-specific qPCR. Dashed curve represents a theoretical bioaugmentation strain concentration based on dosage accumulation with a 30 day sludge age. Treatment periods are denoted with dashed vertical lines.

Bioaugmentation strains were also visualized in the UNOX mixed liquor using the RING-FISH method (Zwirglmaier et al., 2004) (Figure 5). The strains can be seen

(yellow fluorescence) incorporated in the floc (red fluorescence) suggesting good acclimation to the environment and activity within the treatment community.

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Figure 5. RING-FISH visualization of NZ012 (A) and NZ013 (B) in mixed liquor samples from the North train UNOX system taken on 31 October 2012. Arrows indicate examples of strain of interest.

Conclusions

This study was aimed at monitoring both strain presence and growth in complex wastewater treatment microbial ecosystems and improved performance as a result

of exogenous strain addition. A method for quantitatively tracking bioaugmentation strains (D’Imperio et al. 2011) was employed to track all strains in a commercially

available bioaugmentation product aimed at the petrochemical and refinery wastewater markets. Genomic information of these strains was utilized to produce strain-specific tools for the visualization and quantification of these strains in a full-

scale wastewater treatment facility. Strain concentration and persistence was dependent on the portion of the

wastewater treatment facility in which the strain is detected. In the fixed-film portion of the plant, strain presence was highly variable and was not closely tied to product dosage. Given the nature of biofilm growth, it is likely that the measured

strain concentrations reflect the proportion of surface associated to planktonic cells in the system at the time of sampling. In the UNOX systems, however, the data

show a rapid acclimation of certain strains to the wastewater ecosystem throughout the duration of product addition. At the conclusion of product addition, these strains persisted for a short time then decreased in concentration to levels below the

expected accumulation concentration. This trend suggests that there is a threshold cell density for these strains below which growth will not overcome competition

from the endogenous wastewater community. Some strains persisted in the system at low levels for several sludge ages following the cessation of product addition

A B


suggesting that they filled a niche within the community that was not filled by the

exigent population. Visualization of the strains of interest during the treatment period showed incorporation within the flocs of the mixed liquor at relatively high concentrations. This observation suggests that the added strains are involved in

high aerobic activity (Wilén et al., 2004) and are well-adapted to the environment.

Plant performance was enhanced by the addition of the bioaugmentation product despite the fact that it was running with high efficiency prior to the field trial. Two significant changes to the operational performance were perceived through the

course of the trial: 1) the average BOD removal efficiency increased by approximately 2.5% as compared to the period directly previous to the trial

(p=0.044). 2) The system ran more stably during the course of the study than it did without the bioaugmentation product added.

This information suggests that bioaugmentation could be a useful tool to improve the operations of a wastewater treatment facility. By employing highly specific

molecular techniques to analyze the presence and persistence of bioaugmentation strains and simultaneously monitoring plant performance, we are able to quantify the presence and residence time of several strains while evaluating changes in plant

performance over the same period.

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