April 2008
Operation and evaluation of a pilot wastewater plant at GE Healthcare in Uppsala
Mats Ljung
Molecular Biotechnology Programme
Uppsala University School of Engineering
UPTEC X 08 019 Date of issue 2008-04
Author
Mats Ljung
Title (English)
Operation and evaluation of a pilot wastewater plant at GE Healthcare in Uppsala
Title (Swedish) Abstract
A lab scale pilot plant for biological wastewater treatment has been built at GE Healthcare in Uppsala. The aim was to imitate the moving bed biofilm reactors of the on-site wastewater treatment plant, creating the same environmental conditions for biological activity in the bioreactors of the pilot. Once accomplished the likeness to the main plant was evaluated using total organic carbon (TOC) reduction measurements and terminal-restriction fragment length polymorphism (T-RFLP) analyses of the biofilm communities. A correlation between the systems is obvious but unfortunately the difference is significant.
Keywords
biological wastewater treatment, biofilm, MBBR, suspended carrier, TOC, T-RFLP Supervisors
Anders Selmer
Section Manager, GE Healthcare Scientific reviewer
Sara Hallin
Associate Professor, Swedish University of Agricultural Sciences
Project name Sponsors
Language
English
Security
ISSN 1401-2138 Classification Supplementary bibliographical information Pages
38
Biology Education Centre Biomedical Center Husargatan 3 Uppsala Box 592 S-75124 Uppsala Tel +46 (0)18 4710000 Fax +46 (0)18 555217
plant at GE Healthcare in Uppsala
Mats Ljung
Sammanfattning
GE Healthcare’s fabrik i Bol¨anderna i Uppsala producerar material f¨or separation av bio- molekyler, framf¨or allt proteiner. F¨or att skydda milj¨on fr˚an det vattenburna avfallet finns ett nybyggt internt reningsverk, som renar vattnet biologiskt. Biologisk vattenrening in- neb¨ar att man l˚ater mikroorganismer, bl.a. bakterier, leva p˚a det organiska avfallet i vattnet.
Avfallet bryts p˚a s˚a s¨att ner till koldioxid och vatten samtidigt som mikroorganismerna kan f¨or¨oka sig och bli fler.
Detta examensarbete syftar till att bygga en sm˚askalig pilotanl¨aggning som ska efter- likna reningsverket. Parametrar s˚a som temperatur, syrehalt, pH och uppeh˚allstid juster- ades f¨or att skapa samma f¨oruts¨attningar f¨or biologisk rening. Pilotanl¨aggningen ¨ar t¨ankt att anv¨andas till utveckling och effektivisering av reningsverket och det ¨ar d¨arf¨or viktigt att utv¨ardera likheten mellan anl¨aggningarna. Detta gjordes p˚a tv˚a s¨att. Avfallsreduc- erande egenskaper j¨amf¨ordes genom att m¨ata halten organiskt material i inkommande och utg˚aende vatten fr˚an b˚ade pilotanl¨aggningen och reningsverket. Dessutom j¨amf¨ordes bak- teriekulturerna med hj¨alp av genetiska ”fingeravtryck”, en effektiv teknik f¨or att studera bakteriella samh¨allen.
Resultatet av studierna visar p˚a att det finns samband mellan pilotanl¨aggningens av- fallsreducerande egenskaper och reningsverkets, men att de inte ¨ar lika. Fingeravtrycken
¨ar olika men om skillnaden ¨ar stor eller liten relativt s¨att ¨ar sv˚art att uppskatta utan en referensstudie.
Examensarbete 20 p
Civilingenj¨orsprogrammet Molekyl¨ar Bioteknik Uppsala Universitet, april 2008
Contents
Abstract 1
Sammanfattning 3
Contents 5
1 Introduction 7
2 Background 8
2.1 About GE Healthcare and site Bol¨anderna . . . . 8
2.2 Wastewater Treatment . . . . 8
2.2.1 General aspects . . . . 8
2.2.2 Wastewater treatment . . . 10
2.2.3 Biological wastewater treatment and the moving bed technique . . . . 11
2.3 The on-site wastewater treatment . . . 13
2.3.1 Pre-treatment . . . 14
2.3.2 The main wastewater plant . . . 14
2.4 Molecular methods to fingerprint bacterial communities . . . 16
2.4.1 Polymerase chain reaction . . . 16
2.4.2 Terminal Restriction Fragment Length Polymorphism . . . 17
3 Materials and methods 18 3.1 LabVIEW . . . 18
3.2 The pilot plant . . . 19
3.2.1 Inlet/outlet control . . . 20
3.2.2 Aeration control . . . 20
3.2.3 Temperature control . . . 22
3.2.4 Dosage of nutrients . . . 22
3.3 Sampling . . . 22
3.3.1 Incoming water . . . 22
3.3.2 Outgoing water . . . 22
3.3.3 Sampling in the main plant . . . 23
3.3.4 Microbial biofilm community . . . 23
3.4 Experiments and analytical methods . . . 23
3.4.1 TOC . . . 23
3.4.2 Nutrients . . . 23
3.4.3 Fingerprinting the microbial biofilm community . . . 24
3.4.4 Statistical methods . . . 26
4 Result and discussion 27 4.1 Start-up phase . . . 27
4.2 TOC reduction . . . 27
4.3 Microbial community . . . 28
4.4 Nutrient study . . . 29
5 Conclusions 30
6 Acknowledgment 30
References 31
A APPENDIX: LDO and pH 33
B APPENDIX: TOC 34
C APPENDIX: T-RFLP fingerprints 35
D APPENDIX: Nutrients 38
1 Introduction
Individuals and industries produce waste, both in solid and liquid form. The treatment of the liquid portion, the wastewater, aims to remove otherwise harmful compounds from the water before it ends up in our streams, lakes and seas. Current legislation demands of industrial companies that wastewater is taken care of and that no harmful substances are released. GE Healthcare in Bol¨anderna in Uppsala manufactures media for separa- tion of biomolecules. The factory wastewater contains product remainders (e.g. dextran and agarose), lye, organic acids and salts. Even though organic solvents (e.g. ethanol and toluene) are recycled on-site, some are still present in the wastewater. The hight organic content needs to be reduced before releasing the water and this is done by a new internal wastewater treatment plant (WWTP), opened in 2007. Only wastewater from manufactur- ing steps or laboratory sinks around the site are treated. Human faeces and wastes are sent directly to the municipal WWTP.
GE Healthcare’s internal WWTP treats the wastewater biologically, using a moving bed technique with suspended carriers. This is a biofilm process, where the microorganisms grow on the protected surface area of plastic cylindrical carriers. Organic compounds are degraded to carbondioxide and water as new biomass is produced. A common way of quantifying organic content is by measuring the total organic carbon (TOC). A present en- vironmental court decision states that GE Healthcare not is allowed to release more than 750 kg/day dissolved TOC as a monthly mean value. The average TOC reduction 2007 was 80 % and outgoing water had a dissolved TOC content of about 130 kg/day (monthly mean value).
New products are continuously being developed and from this follows new by-products in the wastewater. Before any large-scale production can take place it is important to be convinced that these compounds are either removed in the WWTP or not harmful for the recipient, Fyris˚an. This and also development and optimisation studies demand possi- bilities for small-scale testing. A pilot WWTP, a small-scale version of the main plants biological section, is to be build to fulfil this need.
The aim of this project was to build this pilot WWTP and, once up and running, evaluate its likeness to the main plant. Environmental conditions such as retention time, protected surface area per tank volume, temperature, pH and nutrient dose were set to mimic the main plant in order to achieve equal biological activity. This was evaluated using TOC re- duction measurements. Dissolved TOC content of incoming and outgoing water from both pilot and main plant were analysed on a daily basis and the results were interpreted sta- tistically. Terminal-Restriction Fragment Length Polymorphism (T-RFLP), an easy and robust way of fingerprinting microbial communities [1], was used to study the biofilm popula- tions. Biofilm samples from carriers in both pilot and main plant were taken at different occasions, meaning that both differences between pilot and main plant as well as changes over time could be studied.
For effective biomass production, sources of nitrogen and phosphorous must be present in the water. The lack of sufficient amounts in the on-site wastewater means that these must be added for optimal biological activity. Nutriol!R NP 5 (Yara), a liquid mixture of urea, am- monium nitrate and phosphoric acid is presently being used by the internal WWTP. This product was chosen before the plants reconstruction, when the biological section was a biobed. Conditions were very different then, why the optimal dosage and composition probably have changed. A study, using the pilot, was started, but due to unforeseen me- chanical problems in the main plant, had to be called off.
2 Background
2.1 About GE Healthcare and site Bol¨anderna
GE Healthcare is a unit of the American General Electric Company (GE), presently one of the three biggest companies in the world with a history stretching back to 1890 and Thomas Edison’s invention of the light bulb. GE Healthcare is a worldwide provider of instrumen- tation and knowledge in a wide range of medical applications, e.g. medical diagnostics and biopharmaceutical manufacturing technologies such as chromatography and electrophore- sis. The unit employs more than 43,000 people around the world.
The GE Healthcare site at Bol¨anderna in Uppsala was founded in 1951, at that time being a part of the Swedish, state owned, medical company Pharmacia. In 1994 Pharmacia was sold and the biotechnological part, Parmacia Biotech, became a company of its own.
Later, in 1997 Pharmacia Biotech merged with the British healthcare company Amersham and Amersham Pharmacia Biotech, later renamed Amersham Biosciences, was formed.
Since 2004 Amersham Biosciences has been a part of GE Healthcare.
The site in Bol¨anderna, an area holding about 70 buildings and 1,200 emplyees, manu- factures chromatography purification systems and media (used in production of biophar- maceuticals) and laboratory scale protein separation products (for research and drug de- velopment). It also hosts the GE Healthcare’s Life Sciences Headquarter, R&D, Operations and Marketing.
The main products produced on-site are various types of chromatography media, used for separation of biomolecules, mostly proteins. These media, also called gels, consist of small spherical particles (10-300 µm in diameter depending on type) made up of polymer networks. They are made by creating an emulsion of water dissolved dextran (polymer of glycose) or agarose (natural galactose polymer purified from seaweed) in an organic solvent (e.g. toluene). The polymer is later allowed to solidify and the small gel particles can then be removed from the suspension. The gel matrix can consist of either a single polymer or a mixture.
The gel Sephadex!R , made only of dextran, was introduced already in 1959 and the agarose gel Sepharose!R , 10 years later [2, 3]. New and modifed gels have been developed over the years and today there are many to choose from. Used without further treatment, these gels will separate biomolecules according to size, a method called gel filtration. To en- hance the separation efficiency and selectivity, functional groups can be covalently bound to the gel matrix. Ion-exchange chromatography, hydrophobic interaction chromatography and affinity chromatography are examples and various types are produced on the site.
2.2 Wastewater Treatment
2.2.1 General aspects
In order to construct effective wastewater treatment facilities it is essential to understand the nature of wastewater, with it’s physical and chemical properties. Physical characteristics include for example the colour and temperature of the water and whether pollutants are suspended particles or dissolved. Insoluble impurities, suspended or just reducing clarity of the water, are known as turbidity and can be either inorganic (e.g. clay and sand) or organic (e.g. animal or plant matter, fats and microorganisms). Particle size varies a lot from where it is considered completely dissolved1up to several centimetres in diameter.
Suspended particles that drop out of suspension is said to be sediment [5]. High turbidity
1If able to pass through a 0.45 µm pore size filter, the particles are usually considered to be dissolved [4].
values in lakes or streams will decrease light penetration and water-living photosynthesis- ing plants will not get enough light.
Chemically, the wastes are either organic or inorganic. The reduction of the total organic content in the wastewater is a very important parameter due to its environmental influence.
An increased organic content in natural waters will lead to increased growth of microor- ganisms, now having an abundance of substrate, increasing turbidity and oxygen demand.
Conditions for living organisms change and typically there is a decrease in both the organ- ism population and the number of species. There exist several ways of measuring the the organic content.
• Total Organic Carbon (TOC)
TOC is a way of quantifying the organic content in a sample. There exist two meth- ods of analyses that unfortunately do not always give the same result. Either chemical oxidation is performed at room temperature or the sample is burned at 700 - 900◦C . Both methods detect the amount of formed CO2, usually with an IR detector, giving the total carbon content. The inorganic carbon content (CO2and carbonates) in the original sample is measured by lowering the pH and then detect how much CO2that leaves the solution. Subtracting the inorganic carbon content from the total carbon content gives the TOC. The high temperature method oxidation is much more effi- cient, hence it can be used on suspensions, whereas the low temperature method is not to be used when suspended material is present in the sample [6].
• Biological Oxygen Demand (BOD)
BOD is a measurement of the easy decomposable organic content in the wastewater and is a test of how the local recipient would react on discharges. It is measured by inoculating sludge from a municipal wastewater plant to the sample. After dissolved oxygen (DO) measurement, the mixture is incubated at 20◦C for 7 days2. Then DO is measured again and the difference is the BOD, i.e. the amount of oxygen the microor- ganisms have used when degrading the substances. However, there are problems with BOD measurements. The level of uncertainty in the procedure is quite high due to many steps. First, dilution is often needed since the DO is far from enough for most wastewater samples. Second, it is impossible to standardise the type of sludge used, making comparisons difficult. Finally, the relationship between the bacterial growth, where organic matter is built into the biomass without using BOD, and the decomposition is to be known and this differ among microbial species [6].
• Chemical Oxygen Demand (COD)
COD is a measurement of the oxygen demand when organic matter is totally de- graded. The sample is boiled with a strong oxidant (dichromate) that oxidises the organic compounds. Measuring how much dichromate that was reduced makes it possible to calculate the amount of used oxygen. The reduction of dichromate can be measured spectrophotometrically. However, a few things are to be kept in mind.
Some aromatic compounds can not be oxidised by dichromate why such industrial wastewaters will give too low COD values. Industrial wastewater might as well con- tain other oxidants that will compete with the dichromate, also giving too low values.
Inorganic compounds (e.g. sulfite and nitrite) can also have a disturbing effect since they are oxidated by dichromate, resulting in too high values. Chloride is quite com- mon in wastewater and high values disturb the measurement as well, but these effects
2This is BOD7and is not to be mixed up with BOD5, where incubation for 5 days is used instead. BOD28do exist as well but is a measurement of the degradation of the more recalcitrant organic content that is harder to degrade.
are removed by adding mercury. COD used to be a very common way of measuring organic content but is nowadays more or less replaced with TOC and BOD. This is much due to the usage of mercury [6].
The main inorganic content in wastewater is dissolved salts, i.e. ions. Most common are bicarbonates, sulphates and chlorides of calcium, magnesium and sodium but also silica, iron, manganese, nitrates, nitrites and potassium can be found [5]. High amounts of inor- ganic nitrogen (nitrates, nitrites and ammonia) and phosphorus (phosphate), might cause eutrophication of the recipient [4].
Toxic compounds (both organic and inorganic) interfere with the environment in dif- ferent ways. Phenols, cyanides and some metal ions (e.g. chrome, copper and zinc) might block important enzymes within living organisms and tensides can be toxic to some algae [4]. Even compounds present at non-toxic levels might be hazardous to the environment.
Some synthetic compounds (e.g. DDT, PCB, having a hormone mimicking effect on the re- production system [7]) as well as elements (e.g. radioactive isotopes and heavy metals like mercury and cadmium) are not biodegradable. These will therefore accumulate as they move up in the food chain, sometimes leaving higher organisms such as mammals and birds with lethal doses.
Realization that synthetic chemicals can damage the environment has stimulated the development of methods for determining threat-level concentration of chemicals and doses of radiation [8]. LC50 is one example, giving the concentration of a chemical that will kill 50 % of the tested population. Toxic chemicals that can be expected to be found in wastewaters, are compounds associated with human, industrial and agricultural activities (e.g. drugs, steroids, reproductive hormones and personal care products) [9].
2.2.2 Wastewater treatment
Traditional wastewater treatment is a combination of mechanical, biological and chemical removal of pollutants, in practice often arranged in this order.
Mechanical treatment, e.g. sieves (0.5 - 2 mm [10]) or sedimentation basins, remove sus- pended material from the water (Table 1) and protects the following steps in the plants from unnecessary wear.
Biological treatment uses the metabolic pathways of microorganisms, where organic com- pounds are degraded to carbon dioxide and water, meanwhile biomass is produced. Bio- logical wastewater treatment can be acheived either by having the microorganisms in sus- pension, a technique called activated sludge, or by letting them grow on a surface, which is the case in biofilm techniques (Table 1). In an activated sludge process (Figure 1a) the
FIGURE1: Two types of biological wastewater treatment. a) an activated sludge process and b) a biobed process.
mircroorganisms are kept in suspension in big aerated basins3. The sludge can then be removed through sedimentation. In order to keep enough biomass in the process some sludge is pumped back from the sedimentation basin into the bioreactor. Problems with active sludge processes often relate to the separation characteristics of the sludge [11]. If not heavy enough, to much sludge will leave the system with outgoing water, why the amount in the system will not be enough for sufficient waste removal. In biofilm pro- cesses the microorganisms are allowed to grow on the surface of a carrier material, either attached to something (e.g. biobed or biorotor techniques) or suspended in the water (e.g.
moving bed techniques, see Section 2.2.3). In a biobed, the wastewater is sprayed over a biofilm covered surface (Figure 1b). Pollutants are removed as the water runs over the sur- face. Biofilm processes are considered to be less effective than activated sludge techniques, much because of mass transport problems, but are instead in less need of maintenance, as well as more tolerant to changes in environmental conditions (temperature, pH, nutrient concentrations, metabolic products and toxic substances) [10, 12].
Chemical treatments aim in most cases to alter the sedimentation characteristics of com- pounds in the water, either by flocculation or precipitation. Sedimentation or filtration gener- ally follows the chemical treatment but is usually considered to be processes of their own [5]. Precipitation is obtained by adding inorganic (e.g. ammonium or iron) salts to the water and is a common technique for phosphate removal [10] (Table 1). In flocculation, smaller particles come together creating bigger ones that are easier to remove. This might be obtained by adding some kind of synthetic polymer to the water [5].
TABLE1: Different types of wastewater treatments and their expected waste removal.
For all, except for the mechanical, are some kind of mechanical pre-treatment as- sumed. [10]
Technique Reduction (%)
SSa BODb Tot-N Tot-P Pathogens
Mechanical 10-20 5 5 5 -
Active sludge 70-90 >90 20-30c 20-30 53-99.99 Biofilm (biobed) 70-80 80-90 10-20 20-30 66-99.9 Chemical (precipitation) 80-90 60-80 15 90 93-99.9 Chemical and active sludge >90 >90 20-30c >90 93-99.99
a Suspended substances b Biological oxygen demand
c 50-70 % if biological nitrogen removal is used.
2.2.3 Biological wastewater treatment and the moving bed technique
Biological wastewater treatment is a central process in most wastewater plants today. Ac- tive sludge and biofilm processes were mentioned briefly in the previous section as well as some positive and negative aspects. When new techniques have been developed fo- cus has been on increasing the biomass in the process, thus making it possible to increase the organic load. Introducing some kind of moving carrier material into the active sludge process is one way. This can be done either to enhance an already existing active sludge process and keep re-circulation of the sludge or change it into a biofilm process with no
3In cases of nitrogen removal an anaerobic step is added as well.
FIGURE2: The biocarriers used in the on-site WWTP. a) Natrix O, b) Natrix F1 and c) K3.
re-circulating sludge. The process is called moving bed and S¨arner [11] discusses a few dif- ferent types. The extreme type is the fluidised bed, where sand is used as carrier material and the wastewater is pumped with high velocity upwards through the sand, creating a very large active surface area per reactor volume for biological growth (1000-3500 m2/m3).
Biofilm growing in this type of reactor will be exposed to a lot of mechanical wear, why the huge surface is not that efficient. More common is to use larger carriers, often cylindrical, allowing water with nutrients to pass the protected surface area of the interior. Protected active surface area per tank volume can be 100-300 m2/m3[11] and here the biofilm can grow without mechanical disturbance.
The moving bed biofilm reactor (MBBRTM) was developped by the Norwegian com- pany Kaldnes (today AnoxKaldnes) in the early 1990s and is a technique that uses plastic cylindrical carriers for biofilm growth (Figure 2 and 3). The technique was a success and is
FIGURE 3: Biofilm growing on a plastic carrier model K3 from AnoxKaldnes.
now used by more than 400 large-scale wastewater treatment plants in 22 different countries all over the world [13]. Contrary to most biofilm reactors, the MBBRTM al- lows usage of almost the whole tank vol- ume for biomass growth (filling fractions4 of up to 70 % are possible) and does not, as an activated sludge process, need sludge recycling [13]. The carriers move freely in the reactor and stay there thanks to a sieve at the reactor outlet. The key pa- rameter when constructing the carriers is to obtain a large protected surface area, i.e.
the inner and protected surface where the biofilm can grow without mechanical dis- turbance (Figure 3). As mentioned earlier mass transport is often a problem in biofilm processes. Normally, the depth of full sub- strate penetration is less than 100 µm, why the ideal biofilm in the moving bed process
4The fraction of tank volume filled with carriers when no water is present.
is thin and evenly distributed over the surface of the carrier [13]. Turbulence in the reac- tor is therefore important, both for good nutrient transportation and for maintenance of the thin biofilm layer. It has been shown that turbulence caused by the air flow necessary to maintain 3 mg O2/L is more than sufficient to achieve this [13]. In processes with low organic load higher DO values might be necessary for enough turbulence.
2.3 The on-site wastewater treatment
The on-site wastewater plant was opened in 2007. The pre-existing plant was replaced to ensure a wastewater treatment capacity suitable for future production growth and tougher environmental requirements. Presently the plant is in a trial period with a temporary envi- ronmental court decision stating that this site is not allowed to release wastewater contain- ing more than 750 kg/day dissolved TOC as a monthly mean value. During this period of time data are collected, evaluating the various processes in the plant. The result will then be used when the existing court decision is renewed and up-dated, probably sometime during 2008. GE Healthcare does also have an agreement with the municipal wastewater plant, since all treated water will pass there before ending up in the recipient, Fyris˚an. This is updated once a year.
Figure 4 is a simplified drawing of the plant, showing the path of the wastewater from factory to outlet.
FIGURE4: A simplified diagram of the on-site wastewater plant.
TABLE2: The plastic carriers.
Model Tank Length Diameter Protected Fill Number Number surface fraction per m3 in tank (mm) (mm) (m2/m3) (%)
Natrix O BF1 50 60 300 33 4400 ∼3.8·105
Natrix F1 BF2 30 36 220 50 12400 ∼1.6·106
K3 V01/02 12 25 500 20 95000 61
2.3.1 Pre-treatment
Several types of amines are used during manufacturing steps, when ligands are attached to the gel matrix. A few of these are not biologically degradable and must be removed sep- arately. T-MAC (tri-metylaminechloride) is biologically degradable but due to its terrible smell it is treated separately as well. Water containing amines is sorted already at factory level and sent to either the FBA (Swedis abbreviation for pre-treatment amine, ”f¨orbehan- dling amin”) or the FBT (Swedish abbreviation for pre-treatment T-MAC, ”f¨orbehandling T-MAC”). Both FBA and FBT evaporate the incoming water and direct the amine-free vapour into the main plant, there treated as common waste water, and the sludge is sent for destruction (see Figure 4).
When the gel particles are made several types of emulsifiers are used. They are, just like the amines, not biodegradable and need to be removed separately. At factory level wa- ter containing these substances is sent to the FBE (Swedish abbreviation for pre-treatment emulsifier, ”f¨orbehandling emulgator”), where it is mixed with powder of active carbon.
The emulsifiers are adsorbed to the carbon particles, why, after filtration, the water can be sent into the main plant and the carbon sludge for incineration (see Figure 4).
2.3.2 The main wastewater plant
The plant treats the wastewater biologically, using the moving bed biofilm reactor tech- nique (MBBRTM ) described in Section 2.2.3. There are two bioreactors (BF1 and BF2) presently run in parallel, but with possibilities of connecting them in series. They are of 260 m3each with a retention time of about one day. Together they hold a total active sur- face area of about 55,000 m2(≈8 soccer fields). Due to the fact that the bioreactors were not built at the same time they hold different biocarriers. BF1 has Natrix O and BF2 has Natrix F1 (Figure 2). Tabel 2 contains information about each carrier including model K3, used in the bioreactors of the pilot.
In order to achieve the best possible waste reduction over the bioreactors the character- istics of the entering water should be as constant as possible and the water should not harm the microbial community. This and the fact that some environmental harmful substances are not removed by the microbes (discussed above in Section 2.3.1), means that the waste water needs to be processed before entering the biological section (see Figure 4).
The first section of the main wastewater plant is the AVU basin5. Here the pre-treated water from FBA, FBT and FBE and wastewater from around the site enters the main plant.
It is a basin divided into four sections. The outlet in section 1 is situated close to the bottom,
5AVU is a Swedish abbreviation for waste water equalisation (”avloppsvattenutj¨amning”). This is a relic from earlier when the basin was used for that cause.
meaning that low density solvents or particles will not be able to progress into section 2. If solvents are present gas sensors will react, redirecting the water into catastrophe tanks. In the normal case, though, the water will continue into AVU section 2, having the outlet high, why high density liquids or particles will not be able to progress. Before entering section 3 the water passes through a rotating sieve that removes big objects. Section 4 is normally not used but when needed water from any other section can be lead in there.
After the AVU basin the water is pumped into two equalisation tanks, holding 1500 m3 each. They are presently connected in series, but can be run in parallel or individually.
Since the production on-site is batchwise, flow and water composition change a lot over time, why equalisation is needed in order to get an effective biological waste reduction.
The water is mixed and aerated with mixers connected to a blower. The main purpose of the tanks is to equalise flow and water composition, but they also hold a significant spare volume6if something goes wrong.
The pH value of the incoming water is very important for the microbial community of the bioreactors, why the water leaving the equalisation tanks is pH adjusted. This is done in the pH basin just before the water enters the biological section. Incoming water is often acidic and has to be neutralised with base (NaOH).
All parts of the plant, including FBA, FBT and FBE, are controlled by an automatic computerised control system. It is programmed to control the plant, keeping important parameters where they should be and to give an alarm when something is wrong. It also visualises the plant, displaying measured parameters, such as flows, temperatures, pH values and tank water levels. Manual control is also possible if for some reason ”normal”
plant running is not desirable. The program does also save parameter values for a few weeks back in time, making analyses and studies possible.
6Only 60 % of the volume is used normally.
2.4 Molecular methods to fingerprint bacterial communities
2.4.1 Polymerase chain reaction
FIGURE 5: The polymerase chain reac- tion, where red segments are primers, blue segments are the original DNA template, green segments are synthe- sised DNA molecules and green spheres are the DNA polymerase. Illustration from Wikipedia [14].
The polymerase chain reaction (PCR) is a technique widely used in molecular biology for DNA amplifi- cation. Starting with only one or maybe a few DNA molecules, PCR makes it possible to generate mil- lions of copies of a chosen segment from the original DNA template. The method is a simplified in vitro mimic of the DNA replication machinery of the cell and is controlled by repeated cycles of temperature changes [15]. During each cycle, the amount of DNA is doubled, making the number of DNA molecules grow exponentially. Thirty cycles will theoretically yield about one billion copies. Figure 5 visualise the process, having three discrete steps [15]:
1. Denaturation
High temperature (94 - 96◦C ) melts the DNA into single-stranded molecules.
2. Annealing
The temperature is decreased (40 - 60◦C ), al- lowing the primers7 (red segments in Figure 5), provided in excess amount, to base-pair with the original DNA strands (blue in Figure 5).
3. Elongation/extension
Once the primers are on place, the tem- perature is increased (65 - 75◦C ) and the DNA polymerase can attach and start syn- thesising a new DNA strand (green in Figure 5). The polymerase keeps moving along the DNA template, combining accurate deoxynu- cleotide triphosphates to a new DNA strand, until it by accident drops off (usually after a few thousand nucleotides) or the DNA tem- plate ends.
Due to the sensitivity, selectivity, speed and ease of use, PCR has in many cases replaced other DNA
amplifying methods, e.g. bacterial cloning strategies [16]. Since the method was invented, it has been refined by many and the number of applications (e.g. DNA sequencing, in vitro mutagenesis, genetic fingerprinting and assays for the presence of specific infectious agents), has expanded enormously [17].
7Short DNA fragments, complementary to the DNA regions at the 5’ and the 3’ ends of the DNA region that is to be amplified.
2.4.2 Terminal Restriction Fragment Length Polymorphism
Studies of microbial community structure and dynamics are not simple due to very com- plex assemblages of species with very diverse phylogenies and physiologies. To isolate, cultivate and study each species separately would, even if possible, still not say much about the community as a whole. Important to mention is also our inability to cultivate more than 90 % of the members of many communities [18]. Lie et al. [19] also discus the fact that any departure from the original environmental parameters during cultivation might disturb and change the community structure. An alternative way of studying microbial communi- ties is to analyse differences within them using genetic markers, either fundamental to all life forms (e.g. rRNA) or unique to populations of interest (e.g. nitrogen fixing bacteria).
Terminal Restriction Fragment Length Polymorphism (T-RFLP) is one technique where, as the name implies, the differences in fragment length after restriction enzyme digestion are studied. DNA from the whole community is extracted and the chosen marker is amplified using PCR (see Section 2.4.1), with either one or both primers labelled with a fluorescent dye. The PCR product is then digested with a restriction enzyme giving terminal frag- ments of diverse length since different species will have different gene sequences. After digestion, the product is mixed with a DNA size standard, itself labelled with a distinct fluorescent dye. The mixture of labelled terminal fragments and DNA size standard can then be separated with either gel or capillary electrophoresis and, using a laser detection system, abundance and length of the labelled fragments can be obtained. The resulting graph, a genetic fingerprint of the community (Figure 6), will look different depending on community. The number of fragments and their size will vary, giving information about the biodiversity, while the height of each peak says something about the abundance of a certain fragment.
FIGURE6: Two T-RFLP fingerprints from the 16S rRNA study of this project. Both are biofilm sam- ples, A from one of the reactors of the main plant and and B from one of the pilot reactors. Blue peaks are the sample and the small red ones are the DNA size standard.
If studying a whole community’s characteristics one needs a genetic marker present in all genomes, but the marker is also to contain well conserved sequence domains suitable for primer construction. Osborn et al. [1] states that the 16S rRNA gene is a very good
alternative. Furthermore, they mention other studies where more specific markers have been used, e.g. mercury resistance genes from organisms in polluted soils.
According to Marsh [18], T-RFLP has clear advantages over other methods where ge- netic markers are used, e.g. denaturing gradient gel electrophoresis (DGGE) [20], temper- ature gradient gel electrophoresis (TGGE) [21] and single-strand conformation polymor- phism (SSCP) [22]. One advantage is that the output is digital, making analyses much more adequate with the various statistical tools available on the market. The digital output of fragment lengths can also be compared with terminal fragments derived from sequence databases when doing phylogenetic analyses. An other advantage of T-RFLP is that it has far better resolution than other techniques. Osborn et al. [1] have evaluated T-RFLP anal- ysis for the study of microbial communities and they demonstrate that it is a robust and reproducible technique.
3 Materials and methods
3.1 LabVIEW
In this project, a LabVIEW (Laboratory Virtual Instrument Engineering Workbench, Na- tional Instruments) application was created for control of nutrient dosage and measure- ment of inlet airflow to the bioreactors of the pilot. LabVIEW is a graphical programming language that uses icons instead of lines of text to create applications. In text-based pro- gramming various types of syntax determine the order of program execution, whereas in LabVIEW dataflow programming is used. The flow of data through the nodes in a block diagram determines the execution order of the virtual instruments (VIs) or functions. There are two working environments in LabVIEW. The block diagram (Figure 7a) is where the ac- tual programming is made. Here VIs and functions are linked together, forming the back- bone of the LabVIEW application. The front panel (Figure 7b) is the graphical interface of LabVIEW, from where the application is run and controlled.
FIGURE 7: The LabVIEW application. a) shows the block diagram, the working environment in LabVIEW and b) the front panel, the graphical interface of the running application.
LabVIEW can simulate VIs but also control real instruments. To connect real instru- ments an external module is needed. A LabVIEW compatible NI DAQ (data acquisition) module that can both receive and send signals (digital or analogue) was used in this project.
The airflow was measured continuously and the nutrient dosage was controlled just by changing a number on the front panel (for further details see Section 3.2.2 and 3.2.4).
3.2 The pilot plant
A pilot-scale wastewater treatment plant was constructed as a small version of the the main biological wastewater treatment plant and placed in a ventilated hood (Figure 8, 9).
The two bioreactor vessels of the pilot (V01 and V02) were of 3.2 litres each and made of glass. They were mantled, allowing temperature control with a circulating liquid of chosen temperature in an outer volume. Plastic carriers from AnoxKaldnes (model K3) were used.
These were smaller (Table 2) than the ones in the main plant, but the number of carriers was chosen so that the protected surface area per tank volume was the same as in the main plant. This gave a total number of 61 carriers in each pilot bioreactor and a total efficient biofilm surface of 0.32 m2.
FIGURE8: A complete drawing of the pilot plant. For definitions of abbreviation see Table 3.
In order to achieve the same conditions for biological activity in the pilot as in the main plant many parameters were controlled. Incoming water flow, aeration and temperature were held at proper values manually. A LabVIEW application was made, controlling the dosage of nutrients and the collection of data from the airflow sensors, placed on the in- coming air-tubing. pH, LDO (Luminiscent Dissolved Oxygen) and temperature were mon- itored continuously using two multimeters (one per reactor) from HachLange (MM01 and MM02). They were programed to save values every five minutes during week days and
once every 15 minutes on weekends. A complete list of pilot plant parts can be found in Table 3.
FIGURE9: The pilot plant.
3.2.1 Inlet/outlet control
The wastewater entering the pilot was pumped from the the pH-basin of the main plant.
This pump was controlled, just like the inlet pumps of the big bioreactors, by the plant’s computerised control system. If the flow into the main reactors was stopped for security reasons so would the flow into the pilot.
First, the incoming water entered the inlet-vessel (V03), functioning as a reservoir from where the water was pumped with pumps IN-V01 and IN-V02 to each main vessel, V01 and V02 (Figure 8). The flow into V03 was much higher then into V01 and V02 why redun- dant water was led into the sink, ending up back in the pH basin of the main plant. IN-V01 and IN-V02 maintained a flow of 2.4 ml/min or 3500 ml/day, which approximately corre- sponded to the inlet flow of the main reactors in the big plant (12 m3/h). When the water in V01 and V02 reached a certain level it left each vessel through the outlet piping, keeping the volume of about 3200 ml constant.
3.2.2 Aeration control
The aeration was controlled manually with two needle valves, one to each reactor and the airflow was measured with two microbridge mass airflow sensors, placed just after the valves on the incoming air-tubing (Figure 8). The sensors gave an output voltage propor- tional to the air mass flow through it. Connecting the sensors to a LabVIEW compatible NI DAQ (data acquisition) module that transformed the output voltage into a digital signal, readable for the computer made it possible for LabVIEW to show the real-time airflow for
TABLE3: The different parts of the pilot.
Type Name Function Description
Vessels
V01 Pilot bioreactor 1 3.2 litre glass bioreactor V02 Pilot bioreactor 2 3.2 litre glass bioreactor V03 Inlet vessel sedimentation flask, 3 outlets V04 Nutrient vessel (for V01) 15 ml falcon tube
V05 Nutrient vessel (for V02) 15 ml falcon tube S-V01 Sample from V01 10 litre plastic can S-V02 Sample from V02 10 litre plastic can S-V03 Sample from V03 10 litre plastic can
Pumps
IN-V01 Inlet pumping to V01 Peristaltic pump, Watson Marlow 520S IN-V02 Inlet pumping to V02 Peristaltic pump,
Watson Marlow 520S SP-V03 Sample pumping from V03 Peristaltic pump,
Watson Marlow 520S NUT-V01 Nutrient pumping to V01 Syringe pump,
GE Pump P500 NUT-V02 Nutrient pumping to V02 Syringe pump, GE Pump P500
TH01 Thermostat Immersion thermostat
Lauda E103
Aeration
AF01 Airflow to V01 Airflow sensor
Honeywell AWM5000
AF02 Airflow to V02 Airflow sensor
Honeywell AWM5000 AV01 Airflow valve of V01 Needle valve
AV02 Airflow valve of V02 Needle valve
Auto control
DAQ Data acquisition module Multifunction DAQ for USB NI USB-6009
LabVIEW Control and data colect. LabVIEW 8.5 (Base) National Instruments
pH, LDO & temp.
MM01 Collecting data from Multimeter
PH01 and LDO01 Hach HQ40d multi
MM02 Collecting data from Multimeter
PH02 and LDO02 Hach HQ40d multi
PH01 pH and temp. Gel-filled pH electrode measurement in V01 Hach PHC101-01 PH02 pH and temp. Gel-filled pH electrode
measurement in V02 Hach PHC101-01
LDO01 LDO and temp. LDO electrode
measurement in V01 Hach LDO101-01
LDO02 LDO and temp. LDO electrode
measurement in V02 Hach LDO101-01
each pilot reactor. A logging function was also built into the program, saving the airflow values to a text file every five minutes.
The airflow control and measurements made it possible to control the amount of dis- solved oxygen (DO) in the vessels indirectly. This was a very important parameter for aerobic biological activity and was continuously monitored as LDO with multimeter in- struments, one to each reactor (MM01 and MM02). Airflow was adjusted so that the LDO in the pilot vessels was approximately the same as in the main plants bioreactors (6-7 mg/l).
3.2.3 Temperature control
The temperature of the pilot bioreactors was held constant with an immersion thermostat (TH01), a combined pump and thermostat bath, that pumped water of a chosen tempera- ture around in the outer volume of the mantled vessels. This made it possible to hold the bioreactor temperature at a constant value close to that of the main plants reactors (Figure 8). Temperature was monitored in the reactors with MM01 and MM02. TH01 was set to maintain the same temperature as in the main plants bioreactors (31◦C ).
3.2.4 Dosage of nutrients
The dosage of nutrients (Nutriol!R NP 5) in the main plant was done just before the water entered the bioreactors, which was after the pH-basin and the connection to the pilot. This meant nutrients had to be added to V01 and V02 (Figure 8). In the plant the dosage was 7.6 l/h (supplied both reactors), giving a nutrient flow of 1 ml/day into each pilot vessel.
To start with, this dosage of nutrients was done manually with a pipette twice a day.
This was not a good solution since concentration of nutrients varied, leading to oscillations in both pH and LDO. A flow of 1 ml/day meant∼0.04 ml/h and no pumps in our pos- session could handle flows that low. Instead LabVIEW was programmed to control two syringe pumps (NUT-V01 and NUT-V02), switching them on and off a chosen number of times during a 24 h period. The user told the program how many ml/day that should be dosed and during how many pulses this was to be distributed. During a pulse the pump worked at its lowest flow, 1 ml/h. The program was also written to write to a log file each time a pulse ended. Both duration time and the volume that was pumped were saved.
3.3 Sampling
3.3.1 Incoming water
To start with, the incoming water samples, inPilot, were collected as composite samples during a 24 h period by constantly pumping water from the inlet-vessel (V03) with pump SP-V03 to a 2 litre bottle. Later, the flow through the pump was increased and the sample was collected in a 10 litre can placed in a refrigerator (Figure 8).
3.3.2 Outgoing water
In the beginning the outgoing water, denoted utV01 and utV02, was collected as spot sam- ples, 100 ml from each pilot vessel once a day. To get a more representative day sample , this was later changed to collecting all the outgoing water (∼ 3.5 l/day) from each vessel in two 10 litre cans placed in the same refrigerator as above (Figure 8).
3.3.3 Sampling in the main plant
Flow proportional samples were taken continuously at several points in the main plant.
Two points were of interest for this project: the incoming water to the bioreactors, inBF, taken from the pH-basin, and the outgoing water from the bioreactors, utR3, taken just before the water leaves the plant. Automated samplers were used to collect and save a few dl of water when 10 m3of water have passed the instrument. The samplers were connected to 10 litre cans, where the mixture of samples from a 24 h period was saved.
3.3.4 Microbial biofilm community
In order to compare the microbial community of the big bioreactors in the plant and the smaller ones of the pilot, samples were taken of the carrier biofilm. It was collected by placing the plastic carrier in a can with 15 ml of a storage buffer8, then using a plastic spat- ula to scratch the biofilm off the carrier. The water/biofilm mixture was then transferred into a 15 ml Falcon tube and centrifuged for 10 min at 5000 RPM. Pellet and supernatant were stored separately in a freezer for later analysis.
3.4 Experiments and analytical methods
3.4.1 TOC
The TOC content was analysed in both pilot vessels and in the incoming water on a daily basis. The reduction of TOC, redV01 and redV02, was then used as a comparative parameter when the bioreactors of the plant were compared to the ones in the pilot.
Since only the dissolved TOC content was of interest, the samples were filtered before analysis. Both in-comming and out-going water were, due to a lot of suspended material, first filtered through a rough filter and then through a 1.6 µm filter. 100 ml of the filtrate was sent for analysis of TOC. Incoming water was treated and analysed the same way.
The TOC analyses9were done by a on-site laboratory [23].
3.4.2 Nutrients
V01 was used as reference, run normally with Nutriol!R NP 5, and the experiments were done in V02. Initially the dosage of nutrients to V02 was stopped completely. TOC re- duction differences between V01 and V02 were measured as well as NH4-N and PO43+
concentrations in outgoing water. This went on for three weeks, waiting for the TOC re- duction to stabilise.
Solutions 1-7 (Table 4) were used to optimise the nutrient content. They were made by mixing accurate amounts of miliQ-water, urea, ammoniumnitrate and phosphoric acid.
Each nutrient solution was dosed to V02 for one week, starting Monday, monitoring TOC reduction and NH4-N and PO4-P content in the outgoing water. Unfortunately this study was called off due to unforeseen environmental changes in the main plant (see Section 4.4).
Ammonia-nitrogen (NH4-N) and phosphate (PO43−) contents were analysed spectropho- tometrically, using reaction kit LCK 304 (Lange) for PO43−and LCK 349 (Lange) for NH4-N.
For PO43−-analysis [24], 2 ml of the sample was added to a reaction cuvette. After addition of 0.2 ml H2SO4the cuvette is closed with a lid holding the rest of the reaction components.
8A×10 diluted di-sodium hydrogen phosphate/potassium dihydrogen phosphate solution (normally used as a pH 7.00 calibration buffer) was used since it was readily available.
9The high temperature type (see Section 2.2.1)
TABLE 4: The content of Nutriol!R NP 5 and the solutions for the nutrient study.
Product Urea-N NH4-N NO3-N PO4-P Start datea
(m%) (m%) (m%) (m%)
Nutriol!R NP 5 15.2 2.8 2.8 3.6 -
Solution 1 0 0 0 3.6 19 Oct 2007
Solution 2 7.6 1.4 1.4 1.8 26 Oct 2007
Solution 3 15.2 2.8 2.8 3.6 3 Dec 2007
Solution 4 0 2.8 2.8 0 9 Dec 2007
Solution 5 7.6 1.4 1.4 1.8 17 Dec 2007
Solution 6 15.2 0 0 0 -
Solution 7 7.6 1.4 1.4 1.8 -
a Date when solution was introduced into V02.
The cuvette is shaken and after 10 min, when the reaction has run to its end, the cuvette is placed in a spectrophotometer where the barcode on the cuvette results in measurements at the correct wavelength. The measuring range for this analyse is 0.05 - 1.50 mg/l. For the NH4-N analysis [25], 5 ml sample is added to a reaction cuvette. The cuvette is then closed with a lid holding the rest of the reaction compounds and then shaken thoroughly.
After 15 min the cuvette, having another barcode than the one for PO43−, is placed in the same spectrophotometer as above. and the NH4-N concentration in mg/l is shown on the spectrophotometer screen. The measuring range is 0.015 - 2.0 mg/l.
3.4.3 Fingerprinting the microbial biofilm community
Biofilm samples were collected at three occasions (Table 5). The first two included samples from the main plant (BF1 and BF2) and form the pilot (V01 and V02). This made it possible both to compare the main plant’s biofilm with the pilot’s biofilm at two occasions and also to see whether any changes had occurred over time. V01:3 and V02:3 were taken later when no nutrients were added to V02, investigating if its biofilm population had changed due to the new environment.
DNA extraction was done using a FastDNA!R SPIN Kit for Soil (BIO 101)10. 130-150 mg of biological material from each sample was transfered to a Multimix2 tube and 978 µl sodiumphosphate buffer and 122 µl MT buffer were added. Cells were lysed in a FastPrep instrument for 10 min. The tubes were then centrifuged for 10 min at 13000 RPM and the supernatant transferred to a new sterilised 1.5 ml ependorph tube. 250 µl PPS was added and the tubes were centrifuged for another 5 min at 13000 at 4◦C . The supernatant (>1 ml) was then mixed with 1 ml Binding Matrix Suspension for 2 min and the suspended material was then allowed to sediment for 3 min. Some of the clear solution was carefully removed not disturbing the sediment particles. The sediment was the resuspended and centrifuged through a SPIN Filter at 12300 xg for 1 min. Since all the suspension did not fit in the SPIN Filter tubes this was done twice, half the volume (∼80 µl) each time. The filtered solution was thrown away and the the filter was centrifuged until dry. 3 times
10A kit containing all material and chemicals needed for DNA extraction except for ependorph tubes. Material in kit: Multimix2 tubes and SPIN filters. Chemicals in kit: MT buffer, PPS, Binding Mix Suspension, SEWS-M and DES
