Optimizing the nitrogen removal in leachate treatment during continuous-flow biological treatment (KBR)

(1)

IN

DEGREE PROJECT BIOTECHNOLOGY, SECOND CYCLE, 30 CREDITS

STOCKHOLM SWEDEN 2021,

Optimizing the nitrogen removal in leachate treatment during

continuous biological treatment (KBR)

LEANDRA ANALI DE LUCA

KTH ROYAL INSTITUTE OF TECHNOLOGY

SCHOOL OF ENGINEERING SCIENCES IN CHEMISTRY, BIOTECHNOLOGY AND HEALTH

(2)

1

(3)

2

Sammanfattning

Användandet av deponier är en av de vanligaste metoderna för avfallshantering globalt. Trots insatser som gjordes för att förbjuda hushållsavfall i deponier under millennieskiftet, deponier skapade innan restriktionerna är fortfarande en risk för miljön. Under 2014 öppnade SÖRAB en kontinuerlig biologisk reningsanläggning (KBR-anläggning) på Löt Avfallsanläggning för att hantera lakvatten från en gammal deponi som under en tid fylldes med hushållsavfall. Sedan dess har SÖRAB arbetat med att förbättra KBR-anläggningen. Målet med denna studie är att utforma en driftstrategi för KBR-anläggningen för att förbättra kvävereningen vid låga temperaturer. Ett antal laborativa försök genomfördes, såsom den mikrobiella konsortiets livsduglighet i lakvattnet och tillväxten i både rumstemperatur och vid 4°C, bioaugmentation genom att berika den mikrobiella cellkulturen som redan finns i lakvattnet och hur detta förbättrar kvävereningen i jämförelse med tillsatser av den kommersiella bakterieblandningen ClearBlu Environmental och andra externa kolkällor. Resultaten från dessa laborativa försök påvisade komplett nitrifikation i både rumstemperatur och 4°C i berikat lakvatten från KBR-anläggningens L2A bassäng efter fem dagar. Försöket visade även på syresatt denitrifikation. Dessutom påvisades komplett denitrifikation inom fem dagar, vid rumstemperatur i lakvatten från anläggningens L2B bassäng.

Under efterföljande pilotförsök påvisades möjligheten till upplivandet av den biologiska kvävereningen genom berikningen av den mikrobiella cellkulturen i lakvattnet. I ett pilotförsök då lakvatten från L2B bassängen berikades, komplett denitrifikation skedde under en anaerob fas på 16 dagar samt nitrifikation och aerob denitrifikation under ett påföljande 17 dagar lång aerob fas. Ett annat pilotförsök då lakvatten från L2A bassängen berikades påvisade både aerob och anaerob nitrifikation, då ammoniumrening skedde i både den syresatta och syrefria fasen.

Tillsatsen av nutrient broth (näringsbuljong) kan påverka KBR-anläggningen, vilket kväver vidare studier. Resultatet från detta projekt tydligt påvisar att kvävereningen i KBR-anläggningen kan förbättras genom att berika den redan närvarande mikrobiella kulturen.

Nyckelord

Microbiell anrikning

Kontinuerlig biologisk rening – KBR Deponilakvatten

Nitrifikation Denitrifikation Vattenrening

(4)

3

Abstract

Landfilling has been one of the most popular methods of handling waste globally. Despite the efforts made to stop the disposal of household waste during the turn of the millennia, the landfills formed before these restrictions are still at risk for causing harm to the environment. In 2014, SÖRAB opened a continuous-flow biological treatment (KBR) facility in Löt to treat the leachate produced in one of their older landfills, once filled with household waste. Since then, SÖRAB has been working on improving the treatment facility. The aim of this the study is to find a suitable process to enhance the nitrogen removal at low temperature. Several laboratory scale experiments were performed, such as viability of microbial consortia in the leachate and growth at room temperature and at 4°C, testing bioaugmentation by enriching the microbial cell culture in the leachate and their efficiency in removing nitrogen, compared to the commercial cell culture ClearBlu Environmental and carbon source addition. The results displayed complete nitrification at both room temperature and 4°C in bioaugmented, enriched leachate originating from the L2A basin of the KBR facility, after five days. These trials also suggested the occurrence of aerated denitrification. Complete denitrification within five days was seen at room temperature in bioaugmented, enriched leachate from the L2B basin of the same facility. The ensuing pilot scale trials proved the possibility to revive the biological nitrogen removal by microbial cell culture enrichment. In one pilot in which leachate from the L2B basin was enriched, complete denitrification in the anaerobic phase consisting of 16 days occurred, along with some nitrification and aerated denitrification in the 17 day long aerated phase that followed. Another pilot scale trail in which leachate from the L2A basin was enriched, both aerobic and anaerobic nitrification occurred, as ammonium removal occurred in both the aerated and unaerated phases. The addition of nutrient broth might influence the KBR system which needs further study. The results from this project clearly demonstrate that nitrogen removal in the KBR facility could be enhanced using a culture naturally present in the facility.

Keywords

Microbial culture enrichment

Continuous-flow biological treatment – KBR Landfill leachate

Nitrification Denitrification Bioaugmentation

(5)

4

Nomenclature and abbreviations

Landfills –Landfills are an aggregation of waste, commonly laying in a valley where rainwater falls and flows through the landfill producing landfill leachate.

KBR – Continuous-flow biological treatment

Leachate – Wastewater caused by rainwater flowing through decomposing waste.

Denitrification – The metabolic process of reducing nitrate into nitrogen gas Nitrification – The metabolic process of reducing ammonium into nitrate

EBPR – Enhanced biological phosphorus removal, a process for removing phosphorus.

TOC – Total organic carbon

NH4 or NH4-N – Shorthand for ammonium NO3 or NO3-N – Shorthand for nitrate NO2 or NO2-N – Shorthand for nitrite PO4 or PO4-P – Shorthand for phosphate

AOB – Ammonium oxidising bacteria, necessary for nitrification

NOB – Nitrite oxidising bacteria, necessary for nitrification and anammox

PAO – Phosphorus accumulating organisms, necessary for enhanced biological phosphorus removal.

Stock – Abbreviation for cell culture stock, a solution of nutrient broth with respective cultivated biomass from leachate (L2A or L2B).

B+ - Abbreviation for Brenntaplus VP1 NB – Abbreviation for nutrient broth

CBE – Abbreviation for Clearblu Environmental Commercial seed mix RT – Abbreviation for room temperature

(6)

5

1 Introduction

Over the past decade the waste management company SÖRAB has collaborated with universities such as KTH in the development of their continuous-flow biological treatment (KBR) facility in Löt.

This has resulted in multiple degree projects, each reviewing different areas of a KBR process to develop an operational strategy that would enhance the KBR facility.

Goals and project overview

The goal of this project is to develop an operational strategy for SÖRAB to use in their continuous- flow biological treatment facility under low temperature conditions. This strategy needs to be scientifically grounded and applicable for the company. The initial phase of the project was understanding the previous investigations performed upon the facility regarding the microbiota present in the treatment system and of potential optimisation measures via the use of carbon sources and bioaugmentation.

The project consisted of investigating the growth of the microbial community, nitrification and denitrification of enriched cell culture at room temperature and 4ºC under laboratory conditions.

From this initial approach, the suitable culture conditions were tested in pilot scale trials.

(9)

8

2 Background

SÖRAB waste management

SÖRAB was founded in the late 1970’s to handle the waste produced in the Stockholm region.

Due to a lack of knowledge regarding the long-term effects of the produced leachate from landfills, they tended to contain nutrient-rich waste. This has changed drastically during the shift in millennia as the European Union (EU) has formed new legislation for more eco-friendly waste management (1). Since SÖRAB was founded the overall trend in using landfills for waste treatment has decreased in Sweden, from a little over 50% in year 1975 to 0,8% in 2019. However, we produce more waste than ever, between 3 to 5 million tons (2). Thus, not only do we need to handle the leachate coming from old, out-cycled landfills, but also the waste produced today. The KBR facility in Löt treats the leachate coming from a partially covered landfill marked “IFA” (icke- farligt avfall), which produces an ammonium rich leachate with heavy metals (3).

Landfill leachate treatment

The treatment of leachate produced from landfills have been an issue in Sweden since the 1970’s.

The technologies for treating landfill leachate employed by Sweden are municipal wastewater facilities or biological practises such as continuous-flow biological treatment (KBR) and sequencing batch reactors (SBR), or chemical impurity separation preformed via precipitation, etc.

(4). A major goal for any wastewater treatment facility is to use and optimize techniques that require minimum renovation to their facilities, while still being environmentally sustainable and have an increased flexibility in their treatment capacity.

An SBR system is simply put a fill-and-draw process that can contain more than a single basin that handles the leachate i.e., there can be separate basins for biological activity and sedimentation (5). There are many studies comparing KBR to SBR, which only goes so far as proof for the ideal process given a specific leachate and purification facility. In a theoretical study (model-based) aimed at comparing the two methods, this study determined that these techniques can be just as effective as the other, if optimised for parameters such as solids and hydraulic retention time (6).

In a report from Avfall Sverige published in collaboration with SÖRAB, they determined that there is no ideal solution for every kind of facility. KBR can however, provide an efficient enough nitrogen removal while still being the most economical alternative to that of SBR (7).

Continuous-flow biological treatment (KBR)

The landfill lies in a valley where rainwater falls and flows through the landfill, bringing with it the compounds being produced in the degrading waste. The leachate contains a mixture of different heavy metals and nutrients, all exiting the facility in lower than benchmark levels (3). The most notable quality in the incoming leachate is the high ammonia content in comparison to that of phosphate, bioavailable carbon and other nutrients (table 1) necessary to sustain microbial growth. This becomes evident as more carbon and phosphorus is consumed in the first step of the KBR. If left untreated, this nitrogen rich water risks causing eutrophication. Thus, the KBR facility is designed to utilize metabolisms that convert this ammonium into nitrogen gas i.e., nitrification and denitrification.

Table 1: Content and flow rate of the incoming leachate, averaged over the year.

Parameter Unit 2019 2018 2017

TOC [mg/h] 1016,68 813,03 560,63

BOD7 (ATU) [mg/h] 290,27 221,24 153,57

Total nitrogen, N [mg/h] 1053,29 881,69 676,00

Ammonium nitrogen, NH4-N [mg/h] 925,77 787,93 598,49

Nitrite nitrogen, NO2-N [mg/h] 0,01 0,01 0,32

Nitrate nitrogen, NO3-N [mg/h] 0,58 0,53 0,70

(10)

9

Total phosphorus, P [mg/h] 2,58 2,31 1,74

Phosphate phosphorus, PO4-P [mg/h] 2,11 1,39 1,26

Chlorine, Cl [mg/h] 1158,84 1037,24 707,27

Sulphate, SO4 [mg/h] 943,23 467,00 0,00

Arsenic, As [µg/h] 90,59 73,02 48,89

Chrome, Cr [µg/h] 309,51 258,76 186,43

Nickel, Ni [µg/h] 54,02 43,99 29,35

Zinc, Zn [µg/h] 55,82 32,37 11,84

Iron, Fe [mg/h] 4,42 2,13 1,00

Lead, Pb [µg/h] 1,54 1,88 0,38

Cadmium, Cd [µg/h] 0,11 0,14 0,04

Coppar, Cu [µg/h] 4,70 4,34 1,19

Flow rate [L/h] 7848,26 4394,94 2700,94

The composition of pollutants and activity of the KBR is unpredictable as weather conditions affect the microbiota present and thus reduce the efficiency of the treatment process. This is evident in Table 1, as despite being averaged throughout the year, there are still noticeable difference each year. This is also reflected in fig. 1, in that the concentrations and flow through the KBR vary almost daily which can stress the microbial consortia, and thus their efficiency in removing nutrients.

Figure 1: The flow of leachate and nutrients entering into the KBR throughout the year.

The KBR treatment facility consists of five outdoor, basins with names L2A through L2D (fig. 2).

The leachate enters the system from collection well L11 into the heated, aerated basin L2A where nitrification should take place. The leachate water then enters the anaerobic L2B basin where denitrification takes place with some stirring at the bottom of the tank to avoid sedimentation.

The water then flows into the L2C basin where aeration can occur, and then into L2D where the biomass sediments and separates from the effluent water. Part of the effluent recirculates into basin L2A and the rest exits into a wetland before being discharged into the water stream. The biomass is placed in a landfill, as this type of sludge is not allowed for further use.

0 5000 10000 15000 20000 25000 30000

0,00 100,00 200,00 300,00

Flow [L/h]

Day in year

2019 2018 2017

0 1 2 3 4 5 6 7 8 9

0 100 200 300

NH4 [kg/h]

Day in year

2018 2019 2017

0 1 2 3 4 5 6 7 8 9 10

0 100 200 300

TOC [kg/h]

Day in year

2019 2018 2017

0,0 5,0 10,0 15,0 20,0 25,0

0,00 100,00 200,00 300,00

PO4 [g/h]

Day in year

2017 2019 2018

(11)

10

Figure 2: Aerial view and schematic images of the KBR treatment basins with flow of leachate. The image to the right is taken from SÖRAB’s 2019 environment report (3) and the one above is not to scale.

2.3.1 L2A – Nitrification

Nitrification is the metabolism in which ammonium is oxidized into nitrite by ammonium-oxidizing bacteria (AOB) and then into nitrate by nitrite oxidizing bacteria (NOB) with the use of oxygen, as described in eq. 1. As electron donors, ammonium and nitrite are rather weak offering a meager existence (8). Thus, the organisms are aerobic, slow growers, temperature sensitive and autotrophic. Due to these qualities the L2A tank is aerated as it accepts the incoming leachate to sustain the nitrifiers.

4 2 2 2

2 2 3

: 1,5 : 0,5

AOB NH O NO H O H

NOB NO O NO

  

 

   

  Equation 1

It has been determined that the nitrification in L2A is functional down below 10 ^◦C and not appropriately efficient (to reach benchmark levels) until the temperature reaches 16-18°C (9).

Recently, SÖRAB has explored a bioaugmentation strategy where a bacterial seed from ClearBlu Environmental (CBE, US) was tested in a pilot using L2A leachate. This experiment determined that aerated denitrification was achievable, however despite the apparent ability of the bacteria in the CBE-mix to degrade complex carbons, they demand the addition of an external carbon source (10). Adding more carbon to the basin could promote the growth of fast-growing bacteria that do not perform nitrification, this taking space and resources from the nitrifiers (9). The high concentration of ammonium can however still pose a challenge to these nitrifiers.

SÖRAB actively monitors and adjusts the facility’s functionality based on the biological activity of the basin. When there is a low nitrification activity, the leachate water is recirculated in the system.

Thus, meeting the demands according to their permits based on national regulation. In reviewing the data gained from their active monitoring (see 9.2 Content in basins between 2017 and 2019), due to the heating, the basin seems to keep a temperature allowing for an effective conversion from ammonium to nitrate. This is further proved in the case when the temperature sank below 4°C in March 2019, where a spike in ammonium exiting the basin is observed.

2.3.2 L2B – Denitrification

Denitrification is the metabolism in which suspended nitrate and nitrite is converted into nitrogen gas released into the atmosphere, as is described in eq. 2. Denitrifying organisms are often

(12)

11 facultative aerobes, meaning that in the presence of oxygen, they primarily use oxygen as an electron acceptor. Thus, denitrification is primarily an anoxic process in which the lack of oxygen allows for the use of nitrite and/or nitrate. Similarly to nitrification, this metabolism offers little energy to be conserved (8). These organisms are also temperature sensitive and slow growers.

3 2 2 2

NO^ NO^ NON ON Equation 2

Often these denitrifying organisms cannot fix oxygen and therefore demand an external carbon source. Currently SÖRAB supplies L2B with a commercial carbon mix Brenntaplus VP1 (B+), the content of which is not disclosed. This commercial carbon source has been compared to glycerol, ethanol and to combinations of the three. The results proved that a combination of glycerol and ethanol can replace if not improve upon the denitrification activity and nitrogen removal more than B+. Additionally, it was determined that phosphate addition to the L2B basin is necessary for an active denitrification (11). This finding aligns well with the findings of previous years (9, 12). However, in a different study it was determined that an excess of phosphate can inhibit denitrification and that it was likely not the concentration of phosphate limiting the denitrification (13).

2.3.3 KBR overall removal

In general, though the facility meets the requirements as per the authorities, there is still room for improvement. In reviewing the data from the last few years, the KBR facility demands recirculation as the last basin displays an increased concentration of nutrients at low temperature conditions. There is a clear trend that during the winter months, the facility is not as efficient, once the temperature surpasses 10 ˚C, the facility manages to reduce nutrients. Moreover, the revival of the microbial culture takes time which prolongs the time required to maintain a nitrification and denitrification process. The current process demands a high energy in heating the system, continuously mixing the water, long retention time and the addition of external carbon and phosphorus sources.

Anammox

Anaerobic ammonium oxidation, abbreviated as anammox, is a metabolism in which ammonium is oxidized under anoxic conditions i.e., in the absence of oxygen (as described in eq. 3). This metabolism is performed by obligately anaerobic bacteria which use nitrite as an electron acceptor to convert ammonium to nitrogen gas. Anammox bacteria are common in ammonia rich waters and co-exist well with the nitrifiers and the denitrifies, but they are inhibited by aeration and will often employ nitrification in the presence of oxygen (8). For wastewater treatment this metabolism is very beneficial as it allows for a one-pot nitrogen removal thus reducing the number of basins necessary and thus saving space. Additionally, its more energetically efficient, as aeration becomes less necessary.

4 2 2 2 2

NH^ NO^ N  H O Equation 3

An issue with anammox and wastewater treatment, is the temperature demand of this metabolism;

anammox activity has been shown to decrease below 25 ˚C and have a temperature optimum between 30-40˚C (14, 15). This becomes especially problematic as Sweden seldom consistently stays above this temperature. However, there is evidence that anammox bacteria can adapt and display a higher specific rate at lower temperatures and if aerobically grown, are less affected by temperature changes (14, 15).

ClearBlu Environmental (CBE) Commercial seed mix

CBE-mix (©2021 ClearBlu Environmental, US) contains a mixture of Bacillus and two Pseudomonas strains (P. fluorescens and P. putida) that together should support the removal of both organic waste and hydrocarbons. The benefits to P. fluorescens, is it will proliferate in

(13)

12 temperatures down to 5 ˚C while the P. putida is claimed to have the ability to denitrify aerobically and consume sugars (16, 17).

The Bacillus group are aerobic or facultatively aerobic and extracellularly produce hydrolytic enzymes with the ability to break down complex polymers such as polysaccharides and lipids.

Pseudomonas lie under the order Pseudomonadales which are characterized as exclusively chemoorganotrophs that respire. P. fluorescens is a commonly non-pathogenic, soil strain with the ability to form biofilms (8). P. putida is a gram-negative microorganism, commonly found in soil and in wastewater. It was found that this Pseudomonas strain removed nitrate, ammonium and phosphate in model wastewater when in a consortium with, among others, a Bacillus strain (18).

In previous investigations, this commercial seed mix was tested for its ability to support nitrogen removal in the KBR during startup. The CBE-mix seemed to adapt well in filter sterilized leachate, meaning that they can grow in the leachate and perform aerated denitrification, which depletes any available energy sources in the L2A basin and thus may demand more carbon addition. In anaerobic conditions the CBE-mix caused an increased reduction of nitrate, unaffected by the carbon source addition of B+, suggesting an ability to reduce complex carbons (10).

Carbon sources

Brenntaplus VP1 (B+) is a carbon source produced by the German chemical company Benntag for application in wastewater treatment. It is composed of a mixture of alcohols, sugars and proteins for promoting bacterial proliferation (19). Its chemical data is presented in table 2.

Glycerol is a three-carbon chain with alcoholic hydroxyl groups responsible for its miscibility in water. It is a renewable carbon source that can be used in pharmaceuticals, solvents, antifreeze and as an animal feed conditioner (20). Its chemical data is presented in table 2.

Table 2: Chemical data on carbon sources.

Brenntaplus VP1 (19) Glycerol (11)

COD (g O2/L) 1,0 1,31

Density (g/cm³) 1,27 1,26

Viscosity (mPa*s) 13,5 1412

pH 8,7 7

Freezing point (˚C) <-15 -38 (70%)

An investigation of the effect of glycerol compared to that of B+ determined that B+ promoted a more diverse growth of microorganisms, specifically gram-positives, and suggests this to be an issue as denitrifiers are often gram-negative. In comparison to glycerol, B+ contains more complex carbons and thus demand more complex metabolic reactions and thus, yielded the reduced denitrification efficiency. An economic analysis determined that in both bulk cost and in reducing potential per unit, glycerol was cheaper (11).

(14)

13

3 Materials and methods

Bacterial density analysis

The standard microbiological cultivation method on agar plates was followed to analyze the growth of microorganisms in the leachate and in pilots. The standard protocol by streaking 100 µl sample over nutrient broth (NB) agar, MacConkey or FE (phenylethyl alcohol) agar. NB agar should expose the members of the culture that grow rapidly, competing out all slower members of space. The MacConkey agar encourages the growth of gram-negative, while the FE agar the gram-positive, members of the microbial community. Growing the bacteria on these plates under different temperatures or after certain interventions, revealed an overview of the microbial community that could grow in the supplied nutrients and are viable (albeit not specific in identifying microorganisms).

To monitor the growth of microbiota during the tested interventions, optical density (OD600) was measured at 600 nm. This is done by first blanking the spectrophotometer against leachate or NB diluted as much as the sample/s in saline solution.

Media

Nutrient broth (NB), commercial mixture of 8 g/L was used. To make solid media, 15 g/L agar powder was mixed in. A commercial MacConkey agar powder 52 g/L was mixed in distilled water.

A commercial FE agar powder 42,5 g/L was mixed in distilled water. All media was autoclaved and poured onto petriplates and stored at 4ºC until use.

Saline solution with a concentration of 9 g NaCl/L used for the OD600 measurements and dilutions.

Sampling and microbial enrichment (Stock solution)

The leachate used in the laboratory scale experiments was collected from the KBR facility in Löt on the 15^th of February 2021. The facility had been shut down approximately 2 weeks prior. The leachate was stored in the cold room (4˚C) on floor 1 in Alba Nova, and some was frozen for later use.

To enrich the microbial cell culture from L2A and L2B leachate, 3 ml of leachate was mixed with 7 ml NB and placed on a shaker rotating at 70 rpm allowing the bacteria present in the leachate to grow at room temperature (RT) (ca 18 to 22 ˚C) for 4 days before storage in a 4˚C refrigerator.

After that initial inoculation, the culture was expanded upon by diluting portions of this culture into fresh NB. This expanded stock culture was used for the experiments. For aerobic growth, a 50 ml falcon tube was filled up to 10-15 ml while for anaerobic growth, a 15 ml falcon tube was filled completely for all the experiments performed in this study. This was also performed for the CBE mixture, which was used for subsequent laboratory scale experiments involving this culture.

The amount of Stock and the cell density used at the time for each experiment are mentioned throughout the methods section when relevant.

Nutrient analysis

Hach kits were used to monitor the nutrient content throughout the experiments performed both in laboratory and pilot scale. Both test strips (qualitative) and barcode labelled tubes for spectrophotometric kits (quantitative) were used. The kits used are listed in the appendix under 9.1 Hach Lange analysis.

(15)

14

Experiments

There were several experiments performed and are summarised in a flow chart as presented in fig. 3.

Figure 3: Flowchart of experiments preformed during the laboratory scale trials.

3.4.1 Nutrient analysis of the leachate

Experiment 0, the leachate collected on the 15^th of February was analysed using Hach kits for nitrate, ammonium, TOC and phosphate. Unfiltered samples diluted with MilliQ water when necessary. All the following experiment were analysed with the same parameters unless specified.

3.4.2 Bioaugmentation with CBE

Experiment 1, L2A and L2B leachate was anaerobically stored in full 50 ml falcon tubes, with a lid. Additionally, a 50:50 mixture of L11:L2A was made to a final volume of 50 ml in 250 ml, baffled E-flasks. This was done twice, one duplicate was left to serve as a control, and to the second 2,46 mg CBE-mix added. The dosing was determined based on previous investigations i.e., 58 ml CBE/1000 L leachate. The CBE was measured to weigh 0,851 g/ml, yielding a dosage of 2,46 mg in 50 ml leachate and the E-flasks were left on shake table at 70 rpm, at RT. The nutrient content was then analysed on 2 and 9.

3.4.3 Bioaugmentation via enriching microbial culture

Experiment 2 was performed to test if the leachate contains any inhibiting factors. In short, resuspending/mixing centrifuged and non-centrifuged leachate with NB and incubated at RT and 4ºC. The cell density was measured using UV-Vis spectrophotometer OD600. Both aerobically grown and anaerobically grown L2B Stock was compared in their support of nutrient removal in L2B leachate. A detailed description of the experiment and results are presented in the appendix under 9.3.1 Experiment 2.

Experiment 3 consisted of three anaerobic and two aerobic enrichment experiments involving the use of cell culture stock (Stock), with their own respective control. The composition and condition in each experiment was:

 Anaerobic experiments

o 12 ml L2A leachate + 3ml L2B Stock, OD600 = 0,6, RT o 12 ml L2A leachate + 3ml L2A Stock, OD600 = 0,8, 4ºC o 12 ml L2B leachate + 3ml L2B Stock, OD⁶⁰⁰ = 0,6, 4ºC

 Aerobic experiments

o 12 ml L11 leachate + 3ml L2A Stock, OD⁶⁰⁰ = 0,8, RT, 70 rpm

(16)

15 o 12 ml L11 leachate + 3ml L2A Stock, OD⁶⁰⁰ = 0,8, 4ºC

After two days, the samples were diluted by removing half of the mixture and replacing it with fresh leachate. For more information on the thought process behind each trial, see appendix under 9.3.2 Experiment 3. The bacterial growth and nutrients were analysed as described above.

Experiment 4 was also two separate experiments, the detailed description of which can be found in the appendix under 9.3.3 Experiment 4. The Stock grown from a mixture of L2A/L2B with NB was centrifuged to remove the NB. The pellets were resuspended in L11, L2B, L2A or NB and a control of unaltered L11, L2B and L2A leachate were made as well. The trials involving L2B Stock were tested at both RT and 4ºC, while those involving L2A Stock were only tested at RT. The second experiment was designed to determine if the enrichment is useful because of the biomass or because of the NB and run for six days before analysis. This involved comparing the nutrient removal efficiency of unaltered Stock with centrifuged Stock and sterilised NB. All trials in experiment 4 were measured using OD600 spectrophotometric and Hach kits for ammonium, nitrate and phosphate. The second experiment analysed TOC as well.

3.4.4 Enrichment strategy combined with carbon addition or CBE.

Experiment 5 was designed to determine if the enrichment method can be improved, it was combined with CBE, Brenntaplus VP1 (B+) or glycerol. More details of this setup can be found in the appendix under 9.3.4 Experiment 5. Filtered sampled were analysed with Hach kits for ammonium, nitrate, phosphate, and TOC and unfiltered samples were measured using OD600

spectrophotometric analysis and blanked with L11 or L2B leachate.

Pilot scale trials

The pilot scale trials A, B and C were initiated on the 7^th of April 2021 at Löt, the leachate was taken from the KBR facility where there was no incoming leachate and the heating of L2A being off for about three weeks. Pilot scale trial D was initiated 26^th of April 2021, about a week after the startup of the KBR for the summer.. All pilot trials were run at RT (the room being consistently around 22 ˚C and the water between 16 to 18 ˚C), at a total volume of five liters and in five-liter plastic containers with no lids. The aeration of the aerobic pilot trials was provided by two Airset 540 pond-spargers. For each trial, there is a control containing only leachate sampled the same day as the trial itself. Additionally, the control is connected to the same pump as the trial to ensure identical aeration. The composition and conditions of each pilot was:

 Pilot A: L2A leachate + L2A Stock + L2B Stock (tot. OD600 0,46), aerated for 16 days, unaerated for 17 days.

 Pilot B: L2A leachate + L2A Stock (OD600 0,27), aerated for 19 days, unaerated for 14 days.

 Pilot C: L2B leachate + L2B Stock (OD600 0,25), unaerated for 16 days, aerated for 17 days.

 Pilot D: L11 leachate + L2A Stock (OD600 0,25), aerated for 7 days, + L2A Stock (OD600

0,25), aerated for 7 days.

The pilot trials are photographed in fig. 4 and illustrated in fig. 5. For more information on the sampling and microbial enrichment see appendix under, 9.5 Pilot scale trial.

(17)

16 Figure 4: Image of the pilot plant set-up.

Figure 5. Illustration of the pilot trial set-ups.

(18)

17

4 Results

Laboratory scale trials

A summary of each experiments results is depicted in fig. 6.

Figure 6: Flowchart of results produced during the laboratory scale trials.

4.1.1 Leachate analysis Experiment 0

As described in the background, the data from SÖRAB showed that the nutrient composition in the KBR vary greatly and the treatment efficiency is influenced by temperature fluctuations during the year. Hence, this results in the leachate sampled during this project period showing different nutrient concentrations than in previous investigations. Thus, it is insignificant to compare those findings within this study.

The initial value in each experiment is based on this analysis, which is presented in fig. 7. Before sampling, the facility was losing activity, resulting in ammonium exiting the system, thus the incoming stream was paused. This explains why there is some ammonium and nitrate present in the L2B basin. In terms of TOC and phosphate, their presence is explained by the decreased activity, due reducing microbial activity at low temperature, the consumption and removal has slowed down, but not completely stopped. For that reason, there is less TOC and phosphate present in the basin than there is in L11. The composition of each leachate influenced the design of subsequent experiments.

Figure 7: The ammonium, nitrate, TOC and phosphate content of the leachate sampled on the 15/2 2021 from the KBR facility. Result of experiment 0.

0 0,2 0,4 0,6 0,8 1 1,2

0 50 100 150 200 250 300 350

L2A L2B L11

PO4 [mg/L]

NH4/TOC/NO3 [mg/L]

Leachate

NH4 NO3 TOC PO4

(19)

18 4.1.2 Bioaugmentation with CBE

Experiment 1

One of the first experiments were to test if adding CBE can support effective nutrient removal. In reviewing fig. 8, its clear that nitrification can be observed the L2A and L11 mix with or without the addition of CBE-mix, suggesting its addition having little effect. However, when there is an uptick in ammonia, CBE seems to dampen the increase. Nitrate and phosphorus removal seems to also be independent of CBE. No denitrification or phosphorus removal was observed in anaerobic experiments.

Figure 8: Ammonium, nitrate, and phosphate content of samples with the addition of CBE to L2A and L2B leachate under anaerobic conditions and to 50:50 mixed L2A and L11 leachate under aerobic conditions.

These experiments provided evidence suggesting that CBE does not support the removal of nutrients, inspiring an investigation of bioaugmentation by enriching the microbial community present in the leachate.

4.1.3 Bioaugmentation via enriching microbial culture Experiment 2

Comparisons between centrifuged and not centrifuged leachate are presented in table 3. There is little difference between the different preparation methods and thus, all cell cultures were pooled and used in subsequent experiments.

Table 3: OD600 in cell culture stock after three days

Leachate Not centrifuged Centrifuged Not Cent.:Cent.

L11 3,1 3,22 0,96

L2A 4,16 2,54 1,64

L2B 3,26 3,63 0,90

Additionally, an experiment was preformed testing if the L2B Stock should be cultivated aerobically or anaerobically. This experiment, summarized in table 4 (see more detail 9.3.1 Experiment 2) proved that both cases could perform complete denitrification but the aerobically grown L2B Stock managed to add less ammonium to the sample and consumed more phosphate and total organic carbon.

Table 4: Comparison between different methods of growing L2B Stock.

Trial NH4 addition [%] NO3 removal [%] PO4 addition [%] TOC addition [%] OD600 growth* [%]

L2B Stock, anaerobic 614 97 12280 121 222

L2B Stock, aerobic 391 97 5964 44 146

NH4 removal [%] NO3 removal [%] PO4 addition [%] TOC removal [%] OD600 growth* [%]

Control 92 0 541 8 -

*Anything less than 100% is a decrease in microbial density, the smaller the value the greater the decrease.

Experiment 3

Given the results of experiment 1, a test on enriching the already present microbial community in the L2A and L2B leachate was performed. This initial microbial enrichment experiment provided

-10 0 10 20 30 40 50 60 70

0,00 5,00 10,00

NH4 [mg/L]

Day

L2A + CBE L2A L2B + CBE L2B L2A/L11 + CBE L2A/L11

0 50 100 150 200 250

0,00 5,00 10,00

NO3 [mg/L]

Day

0 0,2 0,4 0,6 0,8 1 1,2 1,4

0,00 5,00 10,00

PO4 [mg/L]

Day

(20)

19 evidence that this method of bioaugmentation yields more nitrogen removal than that with CBE- mix.

The first half of the results are presented in fig. 9, the goal for these trials being nitrification and the effect of dilution can be observed. In every case, L2A Stock when exposed to L11 leachate at both RT and 4˚C can perform complete nitrification in five days. Denitrification occurs as well as nitrate is reduced in all cases. This suggests that the enriched culture can allow for anammox or aerobic denitrification. In comparing fig. 9.A to 9.C and 9.B to 9.D. the difference in dilution is subtle, as any detectable difference in nutrient concentration can be explained by the dilution factor. As reported in by the OD600 (fig. 9.E and 9.F), the microbial density is continuously falling, dilution making the microbial cell density fall faster.

Figure 9: Results of Experiment 3, focused on aerated trials. A: undiluted L11 leachate mixed with L2A Stock trial at RT. B: undiluted L11 leachate mixed with L2A Stock trial, at 4ºC. C: diluted L11 leachate mixed with L2A trial Stock at RT. D: diluted L11 leachate mixed with L2A Stock trial at 4ºC. E: OD600 of the RT aerated trials over the course of the experiment. F: OD600 of the 4˚C aerated trials over the course of the experiment.

The results for denitrification can be seen in fig. 10. It can be observed in fig. 10.A that some denitrification is occurring when L2A Stock is added to L2A leachate, with nitrate removal occurring 8% more in the first five days. Along with the removal of nitrate there is an increase in ammonium and phosphate, which can be observed in all anaerobic trials. The NB has ammonia as a nitrogen source and thus adds ammonium to any sample receiving Stock grown in NB. In

0 50 100 150 200 250

0 1 2 3 4 5 6

0 5

NH4 [mg/L]

NO3/PO4 [mg/L]

Day

Added stock NO3 No stock NO3 Added stock, PO4 No stock, PO4 Added stock, NH4

No stock NH4 0

50 100 150 200

0 1 2 3 4 5 6 7

0 5 14

NH4 [mg/L]

NO3/PO4 [mg/L]

Day

Added stock NO3 No stock NO3 Added stock, PO4 No stock, PO4 Added stock, NH4 No stock NH4

0 50 100 150 200 250 300

0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5

0 5

NH4 [mg/L]

NO3/PO4 [mg/L]

Day

Added stock NO3 No stock NO3 Added stock, PO4 No stock, PO4 Added stock, NH4

No stock NH4 0

50 100 150 200 250

0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5

0 5 14

NH4 [mg/L]

NO3/PO4 [mg/L]

Day

Added stock NO3 No stock NO3 Added stock, PO4 No stock, PO4 Added stock, NH4 No stock NH4

-0,15-0,1 -0,05 0 0,050,1 0,150,2 0,25 0,3 0,35

0 5 10 15

OD600

Day

L11 leac. L2A st.

RT undil.

undil. RT control

L11 leac. L2A st.

RT dil.

dil. RT control

-0,4 -0,2 0 0,2 0,4 0,6 0,8 1 1,2

0 5 10 15

OD600

Day

L11 leac. L2A st. 4°C undil.

undil. 4 ˚C control

L11 leac. L2A st. 4°C dil.

dil. 4 ˚C control

A B

C D

E F

Stock: L2A Stock: L2A

(21)

20 reviewing 10.B some denitrification can be seen with 13% more removal in the first five days with L2B Stock being added to L2B leachate. Again, the increase in ammonium and phosphate is evident due to the NB. After an additional seven days no more nitrate removal occurs in the L2A trial, which suggests some inhibition of the denitrification and the slight increase could be caused by error or nitrification. A potential issue could be nitrogen gas having an issue with exiting the container since the tube was closed. This, while in the L2B trial the denitrification is not stalling.

In comparing the denitrification ability of the L2A Stock and the L2B Stock to the microbial cell density of these trials visible in graph C, it can be concluded that the L2A culture needs a higher microbial density to preform denitrification.

In fig. 10.D, the results of adding L2B Stock to L2A leachate at RT can be observed. In this trial some denitrification was observed, with 23% more nitrate being removed with added Stock. After an additional seven days the nitrate concentration increases. This brings the same questions posed in the 4˚C, L2A leachate with added L2A Stock trail. There could be an issue with the microbial cell density being too low or the nitrogen gas not being able to exit. Alternatively, the cause for the increased nitrate concentration could be nitrification, as both cases may contain nitrifiers.

0 50 100 150 200 250

0 1 2 3 4 5 6 7 8

0 5 12

NO3 [mg/L]

PO4/NH4 [mg/L]

Day

Added stock, PO4 No stock, PO4 Added stock, NH4 No stock NH4 Added stock NO3

No stock NO3 0

0,5 1 1,5 2 2,5 3 3,5 4 4,5

0 20 40 60 80 100 120 140

0 5 12

PO4 [mg/L]

NO3/NH4 [mg/L]

Day

Added stock NO3 No stock NO3 Added stock, NH4 No stock NH4 Added stock, PO4 No stock, PO4

-0,3 -0,2 -0,1 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8

0 2 4 6 8 10 12 14

OD600

Day

C

L2A leach. L2A st. 4 ˚C L2A leach. control 4 ˚C L2B leach. L2B st. 4 ˚C L2B leach. control 4 ˚C

C

A Stock: L2A B Stock: L2B

(22)

21 Figure 10: Results of Experiment 3, focused on anaerobic trials. A: diluted L2A leachate mixed with L2A Stock trial at 4ºC. B: diluted L2B leachate mixed with L2B Stock trial at 4ºC. C: OD600 of the 4˚C anaerobic trials over the course of the experiment. D: diluted L2A leachate mixed with L2B Stock trial at RT. E: OD600 of the RT anaerobic trial over the course of the experiment.

Experiment 4

The results of the enrichment experiment inspired several more experiments testing if it is the NB or the biomass supporting the removal of nutrients. For more detail, see 9.3.3 Experiment 4. In tables 5 and 6, a summary of Experiment 4 is presented. In reviewing the table 5, the results for L2A Stock is summarised. In Trial AX no nitrification was achieved, likely due to the microbial density falling near zero after just four days. In Trial AY, all cases preformed complete nitrification and the microbial density stayed consistent in the case when no NB was present.

Table 5: Comparison between different L2A stock trials testing the weight of nutrient both and biomass. Leachate used for resuspension, mixing and as a control was L11.

Trial AX

Trial – After one week NH4 addition [%] NO3 removal [%] PO4 addition [%] OD600 growth* [%]

L2A biomass, no NB 935,48 39,2 4,74 30

NH4 addition [%] NO3 removal [%] PO4 removal [%] OD600 growth* [%]

Control, no bioaugmentation 935,48 43,8 85,8 10

Trial AY

Trial – After six days NH4 removal [%] NO3 removal [%] PO4 addition [%] OD600 growth* [%]

L2A biomass, no NB 90 52 36 88

L2A biomass with NB 95 36 542 278

NB, no biomass 95 42 208 -

NH4 removal [%] NO3 removal [%] PO4 removal [%] OD600 growth* [%]

Control, no bioaugmentation 0 52 85 -

In reviewing trials involving L2B Stock, summarized in table 6, there were trials proving denitrification was occurring and the microbial cell density not falling immediately like in the case of L2A Stock when NB was absent. However, as seen in Trial BX, complete denitrification could not be achieved at RT with L2A leachate or at 4ºC with L2B leachate. This changed in trial BY once the lid of the container was replaced with parafilm with holes, allowing the nitrogen gas to exit. It can be inferred that the increased denitrification could be caused by the switch in leachate from L2A to L2B, but more denitrification occurred in L2A leachate than in L2B leachate in BX.

This can however be explained by the difference in temperature.

It is evident that NB is necessary, and the microbial density suffers due to the lack of NB.

Additionally, it seems that just the addition of NB removes nitrate, but the rate of removal was not studied. A cause for the lack of denitrification when only biomass was added could be because nitrifiers took over the stock, as that was the only intervention in which ammonium was removed.

0 50 100 150 200 250

0 1 2 3 4 5 6 7 8

0 5 12

NO3 [mg/L]

PO4/NH4 [mg/L]

Day

Added stock, PO4 No stock, PO4 Added stock, NH4 No stock NH4

Added stock NO3

No stock NO3 -0,1

0 0,1 0,2 0,3 0,4 0,5 0,6

0 5 10 15

OD600

Day

L2A leach. L2B st. RT

L2A leach.

control RT

D Stock: L2B E

(23)

22 Table 6: Comparison between different L2B Stock trials testing the weight of nutrient both and biomass. Leachate used for resuspension, mixing and control was L2B for 4ºC and L2A for RT in BX while only L2B was used in BY.

Trial BX– Hard lid

Trial – After one week NH4 addition [%] NO3 removal [%] PO4 addition [%] OD600 growth* [%]

L2B biomass, no NB, RT 10 888 22 348 30

L2B biomass, no NB, 4˚C 4 2 2790 10

NH4 addition [%] NO3 addition [%] PO4 removal [%] OD600 growth* [%]

Control L2A, no bioaugmentation, RT 764 6 38 -

NH4 removal [%] NO3 removal [%] PO4 addition [%] OD600 growth* [%]

Control L2B, no bioaugmentation, 4˚C 24 3 662 -

Trial BY – Punctured parafilm

Trial – After six days NH4 addition [%] NO3 removal [%] PO4 addition [%] OD600 growth* [%]

L2B biomass with NB 375 97 8275 178

NB, no biomass 439 97 6902 -

NH4 removal [%] NO3 addition [%] PO4 addition [%] OD600 growth* [%]

L2B biomass, no NB 58 43 5603 0

Control, no bioaugmentation 98 47 586 -

4.1.4 Enrichment strategy combined with carbon addition or CBE.

Experiment 5

In previous studies it was suggested that carbon addition and CBE can increase the removal of nutrients hence, another experiment involving additives was designed. The results are summarised in tables 7 and 8, see more detail in the appendix under 9.3.4 Experiment 5.

In this experiment, complete nitrification (table 7) was achieved in all cases in both RT and in 4ºC.

Aerated nitrate removal was just as efficient with and without additives. At 4ºC, NB addition removed more than just 97% of ammonium. A higher microbial density was achieved at RT and there was no big difference between carbon sources and NB.

Furthermore, it can be seen that phosphate was consumed more in the presence of B+ and glycerol. TOC is less prevalent when only Stock was added (control). The benefit of the additives was their effect on the microbial density. This accounts for both RT and 4˚C trials, the latter displaying less total organic carbon consumption and cell death due to the temperature affecting the bacterial activity.

Table 7: Summary of trials combining enrichment strategy with additives. Focused on nitrification.

RT

L11 leachate + L2A Stock +… NH4 removal [%] NO3 removal [%] PO4 addition [%] TOC addition [%] OD600 growth* [%]

CBE 97 20 1106 55 195

B+ 96 37 751 53 233

Glycerol 97 33 729 53 228

NB 97 38 1321 57 231

None 96 36 891 23 99

4˚C

L11 leachate + L2A Stock +… NH4 removal [%] NO3 removal [%] PO4 addition [%] TOC addition [%] OD600 growth* [%]

CBE 98 20 1461 56 38

B+ 97 37 729 56 199

Glycerol 97 27 698 56 199

NB 100 35 1518 55 171

None 97 36 891 56 148

Complete denitrification also occurs in this experiment at RT with and without additives (Table 8).

Additionally, the amount of ammonium, phosphate and total organic carbon is at its lowest when only Stock is added. The microbial cell density was stable but does increase further with the addition of carbon sources and CBE. At 4˚C denitrification is more effective with additives, the best being NB, as the control at 4ºC on only removed 33% of nitrate after 5 days. This implies

(24)

23 that the addition of NB with microbial cell enrichment influences the microbial cell density and nitrogen removal at 4ºC as well. Similarly to the nitrification experiment at 4˚C, total organic carbon is consumed less and B+ and glycerol does not contribute to the increase of phosphate or ammonium.

Table 8 Summary of trials combining enrichment strategy with additives. Focused on denitrification.

RT

L2B leachate + L2B Stock +… NH4 addition [%] NO3 removal [%] PO4 addition [%] TOC addition [%] OD600 growth* [%]

CBE 913 97 26902 37 157

B+ 422 98 9511 27 220

Glycerol 431 98 8907 29 188

NB 1333 98 31845 37 295

None 498 97 5621 12 152

4˚C

L2B leachate + L2B Stock +… NH4 addition [%] NO3 removal [%] PO4 addition [%] TOC addition [%] OD600 growth* [%]

CBE 521 75 29831 37 103

B+ 409 50 15278 36 107

Glycerol 413 44 15918 35 92

NB 650 92 39717 37 125

None 467 33 16101 34 95

4.1.5 Microbial density and responses

Throughout the laboratory scale experiments, microbial growth was observed by plating the leachate samples on agar plates to determine the presence of viable cells.

The microbial cell viability was analysed in the leachate samples to determine on which agar and what temperatures the culture grows best in. The results of this are summarised in fig. 11 and pictures are available in the appendix under 9.4 Petridish images.

The culture in each leachate, seems to consist mainly of gram-negative bacteria given that most growth can be observed in the MacConkey agar in comparison to the growth visible in FE agar.

Additionally, it can be observed that, each culture in all leachate’s grew the fastest at 25 °C, with RT being second. In comparing the culture present in the L2B and L2A leachates, it can be observed that there are more gram-positive bacteria in the L2B community than in L2A. It can also be observed that the L2B community grew faster overall. All this growth on the agar proves that there is a microbial community present in the leachate that can be enriched in NB.

0 50 100 150 200 250 300 350 400 450 500

4 °C 25 °C RT 37 °C

Number of colonies

Temperature

L2A L2B L11

0 20 40 60 80 100 120 140

4 °C 25 °C RT 37 °C

Number of colonies

Temperature

L2A L2B L11

0 50 100 150 200 250 300 350 400 450 500

4 °C 25 °C RT 37 °C

Number of colonies

Temperature

L2A L2B L11

A1 A2 A3

Optimizing the nitrogen removal in leachate treatment during continuous-flow biological treatment (KBR)

Optimizing the nitrogen removal in leachate treatment during

continuous biological treatment (KBR)

LEANDRA ANALI DE LUCA

Sammanfattning

Abstract

Nomenclature and abbreviations

Table of contents

1 Introduction

Goals and project overview

2 Background

SÖRAB waste management

Landfill leachate treatment

Continuous-flow biological treatment (KBR)

Anammox

ClearBlu Environmental (CBE) Commercial seed mix

Carbon sources

3 Materials and methods

Bacterial density analysis

Sampling and microbial enrichment (Stock solution)

Nutrient analysis

Experiments

Pilot scale trials

4 Results

Laboratory scale trials