Golda, Rachel, Golda

Golda, Rachel

Rachel Golda is a researcher specializing in water quality investigations and assay development. She received her Ph.D. in Environmental Science and Engineering from Oregon Health & Science University in 2017.

Titles from this speaker

Genetic Analysis in Treatment Process Monitoring

Abstract

Interest in the use of genetic and genomic data for biological wastewater treatment process monitoring has risen rapidly, evidenced by the recent Water Research Foundation (WRF) request for proposals on the topic (WRF 5291). This presentation will serve as a mini 101 course on genetics and genomics for wastewater professionals, offering accessible information on relevant background, potential applications, types of tools, limitations, biomarker assay design, and study design considerations. 'Genetic tools' refers to technologies based on DNA and other nucleic acids. There are two main types, known as targeted or untargeted methods. Targeted methods typically use PCR (polymerase chain reaction) to identify a specific gene or organism of interest. Targeted genetic tools commonly used in wastewater process monitoring applications include quantitative or digital PCR (qPCR or dPCR). Nontargeted methods employ DNA sequencing, which refers to laboratory and computational methods which determine the order of the DNA building blocks; essentially spelling out the genetic code. A commonly used nontargeted method is metagenomic sequencing. (The 'metagenome' refers to all of the DNA present in a sample; see Figure 1). Nontargeted methods yield information on the composition of the microbial community and its functional potential but are not absolutely quantitative. Each type of method has its own set of advantages and limitations (Table 1). Targeted methods are highly sensitive and specific, and can provide highly accurate quantitative data, but are limited in the amount of information they can provide — typically only a single gene or organism out of thousands of relevant potential targets. Care must be exercised in choosing an appropriate target for monitoring, as well as in interpreting the results. Nontargeted methods, in contrast, provide a wealth of information; however, advanced expertise and substantial computing power is required to parse hundreds of gigabytes of data into actionable conclusions. Additionally, application of the wrong genetic technique or interpretation of genetic data without proper data context can greatly complicate or confound conclusions. Genetic tools are an attractive option for wastewater treatment process monitoring because they provide a look 'under the hood' at what is essentially a black box when it comes to biological treatment processes, particularly biological nutrient removal (BNR). These tools can offer information on the health and functioning of the microbiome, as well as insights into complex metabolic systems which can in turn inform process control. However, the field is still emerging, and many research questions remain regarding which targets are appropriate and informative; whether it's possible to develop 'universal' targets that could be widely used; and how the data should be interpreted. Clean Water Services is leading one such research effort, focused on developing biomarkers for biological phosphorus removal (BPR) process stability (WRF 5273). This presentation will describe the procedure used to develop and test these biomarkers as a case study in the application of both targeted and untargeted genetic technologies (Figure 2). Briefly, metagenomic and metatranscriptomic sequencing was performed to thoroughly characterize the aeration basin microbiomes. From these data, key genes were identified which are differentially expressed during stable versus unstable BPR conditions. These genes were used to design biomarkers which are measured with reverse transcription droplet digital PCR (RT-ddPCR). The biomarker assays were tested virtually (in silico) and validated in the laboratory prior to pilot deployment. Lastly, we will touch on the importance of robust study design when employing genetic tools. Too often, a utility will sequence a low number of samples (n<10) and draw broad conclusions from those results. Unfortunately, this can lead to spurious conclusions and a fundamental misunderstanding of the system. We will offer guidance on robust study design for genetic analyses, and suggestions for interpretation of results. This presentation will describe the different types of genetic tools available, summarize their advantages and disadvantages for biological nutrient removal applications and give an overview of the process used to develop and test biomarker targets for WRF project 5273. The goal is to improve communication between molecular biologists and process engineers to improve collaboration on the beneficial application of genetic tools to improve BNR understanding and operation.

This paper was presented at WEFTEC 2025, held September 27-October 1, 2025 in Chicago, Illinois.

Presentation time

15:30:00

16:00:00

Session time

15:30:00

17:00:00

SessionBringing Bugs into Focus: Microbial and Genetic Tools for Optimizing WRRF Performance

Session locationMcCormick Place, Chicago, Illinois, USA

TopicResearch

Author(s)

Layton, Blythe, Golda, Rachel, Coats, Erik, Menniti, Adrienne, Schauer, Peter

Author(s)B. Layton1, R. Golda1, E. Coats, A. Menniti1, P. Schauer

Author affiliation(s)Clean Water Services1, DC Water and Sewer Authority2

SourceProceedings of the Water Environment Federation

Document typeConference Paper

PublisherWater Environment Federation

Print publication date Sep 2025

DOI10.2175/193864718825160139

Volume / Issue

Content sourceWEFTEC

Word count7

Description: Genome-centric Insights into Full-scale EBPR Captured During a Biomonitoring...

Genome-centric Insights into Full-scale EBPR Captured During a Biomonitoring Campaign

Abstract

Phosphorus removal from wastewater remains a core component of modern wastewater treatment strategies critical for protecting aquatic ecosystems and capturing valuable nutrients. Enhanced biological phosphorus removal (EBPR) is attractive since it reduces the need for chemical precipitating agents and facilitates phosphorus recovery. However, instability can limit the reliable application of EBPR for meeting stringent effluent phosphorus limits. As part of an ongoing biomonitoring campaign, samples were collected before and during an EBPR process stability episode and sequenced for paired metagenomes and metatranscriptomes. Analysis revealed that shifts in carbon and phosphorus loads coupled with changes in the population sizes of non-fermenting polyphosphate accumulating organisms (PAOs; Candidatus Accumulibacter spp.), as identified in the metagenomic data, were associated with poor P-uptake. Metatranscriptomic data from these PAO populations revealed they were indeed substrate limited, as hypothesized from process data, with key genes related to carbon storage appearing down regulated during the instability event with respect to the stable control despite having a greater cell load during the instability period. Accordingly, several gene families related to carbon uptake, VFA metabolism, and PHA synthesis were found to be sensitive to the underlying carbon-limited conditions of the process instability. Our findings illustrate how combined metagenomic and metatranscriptomic sequencing can facilitate interpretation of process data and aid in evaluating the root cause of instability. These data also indicate potential gene targets suitable for developing EBPR performance markers as part of longer term or more frequent biomonitoring efforts.

As part of an ongoing biomonitoring campaign, samples were collected before and during an EBPR process stability episode and sequenced for paired metagenomes and metatranscriptomes. Analysis revealed that shifts in carbon and phosphorus loads coupled with changes in the population sizes and gene transcription of PAOs were associated with poor P-uptake. Our findings illustrate how sequence data can facilitate interpretation of process data and aid in evaluating the root cause of instability.

SpeakerLindner, Blake

Presentation time

14:00:00

14:30:00

Session time

13:30:00

15:00:00

SessionConnecting Molecular Biology Sensors to EBPR Performance

Session locationRoom S502 - Level 5

TopicAdvanced Level, Facility Operations and Maintenance, Municipal Wastewater Treatment Design, Nutrients

Author(s)

Lindner, Blake

Author(s)B. Lindner 1; B. Layton 2 ; B. Lindner 1; B. Layton 2; R. Golda 2; K. Lesnik 3; K. Konstantinidis 4; A. Menniti 2; P. Schauer 2;

Author affiliation(s)School of Civil and Environmental Engineering, Georgia Institute of Technology 1; Clean Water Services 2 ; School of Civil and Environmental Engineering, Georgia Institute of Technology 1; Clean Water Services 2; Clean Water Services 2; Maia Analytica 3; School of Civil and Environmental Engineering, Georgia Institute of Technology 4; Clean Water Services 2; Clean Water Services 2;

SourceProceedings of the Water Environment Federation

Document typeConference Paper

PublisherWater Environment Federation

Print publication date Oct 2023

DOI10.2175/193864718825159085

Volume / Issue

Content sourceWEFTEC

Word count11

Description: Optimization Of An Ion Chromatography-Based PHA Measurement Technique To Support BPR...

Optimization Of An Ion Chromatography-Based PHA Measurement Technique To Support BPR Operation

Abstract

INTRODUCTION
Polyhydroxyalkanoate (PHA) is a chemical produced and used by microorganisms during biological phosphorus removal (BPR) to power uptake of phosphorus (Satoh et al., 2016; Liu et al., 2018). PHA is produced from volatile fatty acids (VFA) during the anaerobic stage of BPR. Polyhydroxybutryate (PHB) and polyhydroxyvalerate (PHV) are the two major species of PHA observed in activated sludge systems. It is useful to be able to detect this chemical because PHA has been linked to BPR stability in basin and laboratory scale studies (Schauer et al., 2018; Liu et al., 2018). Clean Water Services is an advanced wastewater treatment facility that uses BPR to achieve permit compliance with minimal use of chemical effluent polishing. We have determined biomass PHA content to be an operational parameter relevant to BPR stability, and so have chosen to develop an in-house method for PHA detection. An in-house method has the benefit of providing data quickly for use in BPR process management. The most widely favored method for PHA detection is gas chromatography (GC; Tan et al., 2014; Koller et al, 2015). While highly valued for its detection sensitivity, GC analysis of PHA is time and labor intensive, and requires the use of hazardous and volatile solvents during the digestion process. In brief, this method requires that an activated sludge sample be preserved with formaldehyde, freeze dried/lyophilized overnight, then digested at high temperature using chloroform and methanol for several hours while shaking (Tan et al, 2014). The risk of explosion during this stage is significant due to the high temperatures, volatility of the solvents, and agitation of the liquid (Koller et al, 2015). For these reasons, pursuing an alternative method for PHA detection was deemed advisable. Hesselmann et al. published an alternative to the GC method using ion chromatography (IC) for PHA preservation, digestion, and detection in 1999. While shown to be accurate and capable of quantitatively measuring and differentiating between the monomers that comprise PHB and PHV (important, since speciation of PHA is highly influenced by VFA feedstock), there have been few further developments reported in the literature since. We chose to optimize this method to develop and validate an in-house IC based protocol for PHA detection. This method is safer, faster, and requires less specialized sample handling than the currently used GC method.
METHODS
We optimized the protocol set forward by Hesselmann, et al. (1999), making alterations to further improve PHA preservation and recovery. A condensed final protocol for sample preservation is presented in Table 1. Table 2 describes the instrumentation and equipment parameters for the IC measurement of PHA, while Table 3 lists QA/QC parameters.
RESULTS
Method optimization We made alterations to the method reported by Hesselmann et al (1999) in order to improve reproducibility and reduce error between replicates. The preservation and digestion steps were optimized by measuring PHA recovery from biomass using both biological and technical replicates. The most substantial alteration to the Hesselmann method was addition of a drying step during sample preservation, as opposed to simply pipetting as dictated by the original method. We determined that decanting and drying samples to remove the supernatant resulted in better PHA recovery and less error than pipetting. The improvement to PHV recovery was particularly dramatic, increasing by 30% (Figure 1). This also improved repeatability, with error between replicates declining from approximately 10% (PHB) and 20% (PHV) to less than 1% for both PHA species (Figure 2).
PHV reference standards were not commercially available when the original method was published, requiring calculation of PHV from a PHB reference standard. However, a PHV standard is now available, allowing us to directly calibrate to PHV monomers. These alterations made to the IC method enabled us to improve the calibration range from Hesselmann's reported 1-16 mg/L to 0.05-5.0 mg/L. This gave us not only a lower detection limit but also better sensitivity, resulting in greater precision between replicates. Method validation via VFA/PHA loading The IC method to measure PHA was successful, producing high accuracy data with low variability between replicates. This was demonstrated by loading activated sludge biomass with PHA, then digesting and measuring the amount of PHA recovered. While not all VFA is expected to be recovered as PHA since we were working with a mixed microbial biomass (including glycogen accumulating organisms, as well as phosphorus accumulating organisms that do not store VFA as PHA), high PHA recovery would indicate good digestion efficiency of the method. To show this, we added precise amounts of VFA as COD to activated sludge bioreactors and allowed VFA uptake to occur under anaerobic conditions. Once uptake was complete, we measured the amount of PHA produced within the cells and compared it to original VFA added to estimate digestion efficiency and PHA recovery from biomass (Figure 3). PHA recovery was excellent, averaging 82%. Error between PHA replicates was low, highlighting the reproducibility of the IC method (Figures 4 and 2). When this experiment was repeated with varying acetate concentrations (40 mg/L and 80 mg/L), PHA recovery was doubled in the reactor with twice the amount of acetate, confirming extraction efficiency at a scale expected based on VFA input (Figure 5).
CONCLUSIONS
We have optimized and validated a safe, reliable method for PHA detection using an IC. The IC method returns PHA values that are consistent with expectations based on VFA loading. As the method is optimized to achieve maximum recovery of PHA from activated sludge mixed liquor, these data are operationally relevant to BPR stability considerations. Our results show that this method is capable of distinguishing quantitatively between PHB and PHV, with low relative error between replicates. We demonstrated high recovery of PHA from activated sludge biomass, indicating good digestion efficiency and instrumentation sensitivity. This method improves upon the originally published protocol, as demonstrated by our ability to improve the calibration range by two orders of magnitude from Hesselmann's reported 1-16 mg/L to a much more sensitive 0.05-5.0 mg/L. This allows our dilutions to generate more accurate data for high PHA samples than the previous range. The implementation of this updated protocol will make measurement of PHA and its relation to BPR stability and process control more accessible to facilities with analytical capacity. The full paper will include expanded data pertaining to method optimization and replicated experiments, as well as demonstration of successful method application to determining PHA dynamics at different stages of the BPR process, and at different stages of BPR stability.

Polyhydroxyalkanoates (PHA) are compounds that are thought to be linked to biological phosphorus removal (BPR) process stability. We optimized an ion chromatography-based method for measuring PHA in wastewater and validated it using laboratory enrichment experiments and basin profiles. This method offers a more sensitive calibration range, improved by two orders of magnitude from 1-16 mg/L to 0.05-5.0 mg/L, and will make measurement of PHA more accessible to facilities with analytical capacity.

SpeakerGolda, Rachel

Presentation time

11:30:00

11:55:00

Session time

10:30:00

12:00:00

TopicAdvanced Level, Intelligent Water, Nutrients, Research and Innovation

Author(s)

Golda, Rachel

Author(s)R. Golda1; T. Auran1; A. Menniti1; P. Schauer1; K. Williamson1; K. Chichetu1; S. Thompson1

Author affiliation(s)Clean Water Services, Hillsboro, OR1