BIOFILTRATION OF DIMETHYL DISULPHIDE AND AMMONIA:
INVESTIGATION OF THE UNDERLYLNG MICROBIAL ACTMTIES
A Thesis
Presented to
The Faculty of Graduate Studies
of
The University of Guelph
by
MICHAEL JOHN GIBSON
In partial fulfilment of requirements
for the degree of
Doctor of Philosophy
December, 200 1
O Michael John Gibson, 200 1 Natianal Liirary Bibliiuenationale 1+1 ,cmada du Canada uisibions and Acquisitions et 3-8' wraphic Services services bibliographiques
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BIOFILTRATION OF DRWCTHYL DISULPHIDE AND AMMONIA: INVESTIGATION OF THE UNDERLYING MICROBIAL ACTMTIES
Michael John Gibson Advisor: University of Guelph, 200 1 Dr. L. Otten
Biofiltration relies on microbial degradation of the contaminants to maintain the absorptive capacity of the filter media. Biofilters are extensively used to treat gas streams with multiple contaminants with different chernical properties. This study investigates the co-removal of dimethyl disulphide (DMDS) and amrnonia fiom a gas stream using compost based biofilters. Pure cultures of Thiobacillus thioparus (ATTC 8 1 58), Nitrosornonas europaea (ATTC 25978) and Niîrobacter winogrudskyi (ATTC 2539 1) were used to inoculate the media. T~hiopurusis capable of oxiditing DMDS to sulphate, while N europaea and N. winogradskyi peerform the two stage autotrophic oxidation of ammonia to nitrate, nitrification. A quantitative polymerase chah reaction (PCR)method was developed to allow tracking of T. thiopams and N. europaea in the filter media. The PCR method developed used most probably nurnber (MPN) statistics to make population estimates fiom a senes of positivehegative tests conducted on senes dilutions of DNA extracted from the filter media. The MPN-PCR method had a lower detection limit of 100 cells per gram of dry media and an upper detection lirnit of 109 cells per gram of dry media, with a 95% confidence factor of 4.67. T.tiiioparus populations observed in the media samples ranged fiom 7.9 x 102 to 1.3 x 10' cells per gram of dry media. N europuea populations in the media samples ranged fiom 1.6 x 10) to 6.3 x 104cells per gram of dry media. In situ octivity rates for T. rhiopams change significantly, 95% confidence level, fiom 2.6 x 10-'* gram sulphur per ce11 during growth phase to 4.4 x 10'" grams sulphur per ce11 during stationary phase. T. thioparus was inhibited by ammonia levels as low as 50 ppmv in the gas phase, but if DMDS removal was established pnor to arnmonia addition chemical removal of the ammonia prevented this inhibitory effect. Autotrophic nitrifiers persisted in the biofilters treating ammonia only and arnmonia in combination with DMDS, while populations Ui the biofilters treating DMDS only dropped below detection. While they persisted there is evidence that they may not be responsible for the observed amrnonia removal. In the combined treatments the sulphate produced fiom the DMDS oxidation was suffcient to account for al1 of the observed ammonia removal. Other evidence, including preliminary denaturing gradient gel electmphoresis (DGGE) analysis, suggests that heterotrophic nitrifiers may be making a significant contribution to the biofiltration of ammonia. Acknowledgements
There are many people who have supported me during this undertaking. First, I would like to thank Dr. Otten for giving me the opportunity to conduct this work, his faith in my abilities remained even when my own faltered. Dr. Trevors accepted me, an engineer no less, into his laboratory allowing me the opportunity to experience the thrill, and the fiutration, that is microbiology lab work. I would aiso like to thank Dr. Lee for his assistance in helping me achieve my objectives with the PCR andysis. #en 1 look back on this time, some of my fondest mernories will be of the tirne spent in the labs of Drs. Trevors and Lee with the group of students and post-doctoral fellows they had assembled there. 1 would particularly like to thank pst-doctoral fellows Kam Leung and Mike Cassidy for their guidance.
1 would also like to acknowledge the support of my wife and parents as well as my extended family. When involved in something as consuming as a Ph.D. it is wonderful to have reminders that you are something more.
1 dedicate this to the memory of my dear friends Lana McLaren and Richard
Reynolds. Table of Contents
Table of Contents
List of Tables
List of Figures
Chapter 1. Introduction
1.1 Biofiltration 1.2 Odour Generation During Composting 1.3 Biofiltration of DMDS 1.4 Biofiltration of Ammonia 1.5 Indentification of Microorganisms 1.6 Polymerase-Chain-Reaction 1.7 Quantitative PCR 1.8 MPN-PCR 1.9 DNA Extraction from Soils for PCR
Chapter 2.
2.1 Biofilter Study Objectives 2.2 Microbial Analysis Objectives
Chapter 3. Methods
3.1 Laboratory Biofilter System 3.2 Airflow Measurements 3.3 Cas Analysis 3.4 Preparation of Cas Standards 3.5 Media Preparation 3.6 Cultures 3.7 Direct Counting of Liquid Cultures 3.8 Inoculum Preparation 3.9 Start-Up of Biofilter Experlments 3.10 Gas Sampling 3.1 f Sampling of Media 3.12 Moisture Determination by Microwave 3.13 Chernical Anaiysis of Media 3.14 DNA Extraction and Preparation 3.15 PCR Amplification PCR Product Purification Sequencing of PCR Products
Chapter 4 Esperimental Design
Effect of Inoculum Combined versus Individual Contaminant Treatment Long Term Combined Removal Nuhient Supplementation Effects on DMDS Removal Ceramic Media
Chapter S. Results
Development of PCR Protocol MPN-PCR Results Biofilter Results
Chapter 6.0 Discussion PCR Overall f erformance of Biofilter Studies Physical Chemical Treatment vs Biological Treatment Biological Activity Coupling of DMDS and Ammonia Removals Ammonia Inhibition of DMDS Removal DMDS Inhibition of Ammonia Removal Evidence of Heterotrophic Nitrifiers
Chapter 7.0 Preliminary DGGE Investigation
Introduction to DGGE DGGE Methodology DGGE Results Discussion of DGGE Results
Cbapter 8.0 Recommendations
Chapter 9.0 Conclusions
References Appendices
Appendix A - PCR Images Appendix B - Media Analysis Results Appendix C - Primers and Sequencing Results Appendix D - Media Compositions
List of Tables
Chapter 1
Table 1. Operating costs for air treatrnent technologies
Table 2. Removal rate constants for T. thioparus inoculated peat biofilter
Table 3. Literature reported nitrification rates
Chapter 3
Tabk 4. Identification of cultures used to inoculate biofilters
Chapter 5
Table 4. Summary of population estimates for standards
Table 5. Summary of maximum and average sulphur removal rates for al1 studies
Table 6. Summary of maximum and average ammonia removal rates for al1 studies List of Figures
Cbapter 1
Figure 1. Pathway for oxidation of DMDS by Thiobacillus thioparus.
Figure 2. Microbial nitrogen transformations and oxidation States Chapter 3
Figure 3. Schematic of laboratory biofiltration systern
Chapter 5
Figure 4. Electrophoresis gel image using psuedo-nested PCR method
Figure 5. PCR results using specific primers for Neuropaea and T.thiopums
Figure 6. Removal of DMDS and ammonia by biofilter with unsterilized-inoculated media, replicate A
Figure 7. Removal of DMDS and ammonia by biofilter with unsterilized-inoculated media, replicate B
Figure 8. Removal of DMDS and ammonia for biofilter where media was neither sterilized nor inoculated, replicate A
Figure 9. Removal of DMDS and ammonia for biofilter where media was neither sterilized nor inoculated, replicate B
Figure 10. DMDS and ammonia removal for biofilters with sterilized media
Figure 1 1. DMDS and ammonia removal for biofilter with combined loading, replicate A
Figure 12. DMDS and ammonia removal for biofilter with combined loading, replicate B Figure 13. Comparing DMDS removal between biofilters receiving both DMDS and arnmonia loading with those receiving DMDS loading only
Figure 14. Comparing ammonia removal between biofilters receiving both DMDS and ammonia and those receiving ammonia loading only
Figure 15. Ammonium, nitrate and nitrite andysis of media samples fiom biofilters treating both ammonia and DMDS
Figure 16. Ammonium, nitrate and nitrite analysis of media samples fiom biofilters treating ammonia only
Figure 17. Accumulation of inorganic and total nitrogen compared to cumulative nitrogen removal for biofilters receiving both DMDS and ammonia
Figure 18. Accumiilation of inorganic and total nitrogen compared to cumulative nitrogen removal for biofilters receiving ammonia only
Figure 19. Comparison of sulphur accumulated in filter media with cumulative removal of sulphur fiom air for biofilters receiving both DMDS and ammonia
Figure 20. Comparison of sulphur accumulated in filter media with cumulative removal of sulphur from air for biofilters receiving DMDS only
Figure 2 1. Population density of Tthioparus in filter media 95
Figure 22. Activity rates for T.thioparus in biofilters 95
Figure 23. Population density of Neuropaea in filter media 96
Figure 24. Activity rates for Neuropaea in biofilters 96
Figure 25. Amrnonia removal for biofilters loaded with media which had been stored
Figure 26. DMDS removal for biofilters loaded with media 97 which had been stored Figure 27. idet concentrations of DMDS during nutrient 98 supplementation study
Figure 28. DMDS removal during nutrient supplementation 98 StUdy
Figure 29. Ammonia and DMDS removal for biofilter packed with ceramic media
Figure 30. DMDS removal under cool lab conditions 99
Figure 3 1. DMDS inlet and outlet concentrations for biofilters 1O0 initially acclimatized to DMDS removal
Figure 32. Ammonia inlet and outlet concentrations for 1O0 biofilters initially acclimatized to DMDS removal
Chapter 6
Figure 33. Theoretical amrnonia removal based on DMDS 125 removal, unsterile inoculated biofilter, replicate A
Figure 34. Theoretical ammonia removal based on DMDS 125 removal, unsterile inoculated biofilter, replicate B
Figure 35. Theoretical ammonia removal based on DMDS 126 removal, unsterilized biofilter, replicate A
Figure 36. Theoretical arnmonia removal based on DMDS 126 removal, unsterilized biofilter, replicate B
Figure 37. Actual and theoretical ammonia removal rates for 127 long term cornbined treatment
Figure 38. Image of DGGE results 131 Biofiltration is the process by which contaminants are removed fiom an airstrearn
when brought into contact with a biologically active surface. The removal is achieved in two
steps, initially the contaminant is either adsorbed to solid sdaces in the filter or absorbed
into the water contained in the filter, secondly the contaminant is biologically degraded.
For the treatment of odours the primary alternative is wet scrubbing. Biofiltration
offers the advantages of lower costs, avoidance of chemical handling, and lower residual
odour. Activated carbon polishing of the air is often necessary after chemical scrubbing
(Ostojic et al., 1991; Pinnette et al.,1994). A fürther advantage of biofiltration, of particular
value in its application to odour treatment, is its ability to treat very divergent compounds
simultaneously. At a sludge composthg facility Hentz et al. (1992) confirmed that DMDS
was the primary odorant and that effective treatment required nearly complete removal of
organic sulphur compounds, especially DMDS. Chemical misting with sodium hypochlonte
neutraiized to a pH of 6.5 was selected for the treatment of the DMDS. In order for this to
be effective there must be no ammonia present in the air. Since ammonia is present in
composting off-gases an acid scrubbing system was required prior to the sodium hypochlode
treatment. To facilitate the removal of ketones, alkanes, aromatics and turpenes, which were
aiso present, a surfactant was added to the acid charnber. in cornparison successful treatrnent of both ammonia and organic sulphur compounds with biofilters has been widely reported
&au et al., 1994; van Durme et al., 1992; Amirhor et al., 1997). These represent studies where both ammonia and DMDS removal was determined. The treatment of composting odours by biofiltration is well established, it is also well established that ammonia is a the principle nitrogen containhg air emission and reduced organic sulphur compounds are the
most odorous, thus oxidation of organic suiphur compounds in the presence of ammonia is
implicated.
Most of the more controlled studies of biofiltration have used airstreams
contaminated with precise amounts of specific compounds. Many only consider single
contaminants, while others look at as many as four, though usually al1 within a single class
of compounds, such as BTEX compounds (Corsi and Seed, 1995), or organic sulphur
compounds (Smet et al., 1996; Smet et al. 1994; Shoda, 1993; Cho et al., 1991 ; Hirai et al..
1990; Kanagawa and Mikami, 1989).
There is a need for detailed investigation of the behaviour of biofilters loaded with
contaminants which have very distinct pathways of degradation. An understanding of the
underlying rnicrobial populations and their possible interactions is necessary to understand
the performance of biofilters treating complex airstreams such as odours fiom soiid waste
composting operations.
1.1 BIOFILTRATION
Traditionally the filter media used in biofilters have been soils, compost or peat. The
sorption capacity of biofilter beds is relatively low, the continued removal by the filter is
dependent on the biological regeneration of the sorption sites. Soils offer some advantages
over compost and peat, the higher minerai content in soils gives it a much greater buffering
capacity for acidic by-products resulting fiom the oxidation of reduced nitrogen or sulphur contaminants. Soils are also hydrophilic while compost and peat materiais tend to be hydrophobie, thus soi1 beds are more easily re-wetted after drying. Despite these advantages most biofïiters use bed material based on compost or peat due to their greater porosity, which reduces the pressure ioss as air is forced through the bed thus reducing operating costs (Bohn,
1992).
Biofilters have been used since the 1920's for the treatment of odours at wastewater treatment plants (Sakano and Kerkhof, 1998). The fint U.S. patent was awarded in 1957 for a soil biofilter. in Japan the first installation was in 1969 and by the mid 1980's biofilters were used for odour control at 80% of the night soil treatment plants and 72% of the wastewater treatment plants (Terasawa et al., 1986). By 1993 biofilters were the dominant odour treatment technology used at German wastewater treatment plants, present at 59% of the facilities (Frechen, 1995). Biofiltration has also become the most common odour treatment method at composting facilities (Goldstein, 1996). Recently biofiltration has been applied to the treatment of chemical and process industry emissions of VOCs (Ottengraf et al., 1983, Hodge et al., 199 1, Shareefdeen et al.. 1993, and van Lith et al., 1 997).
There are three principal aspects which determine the capacity to degrade a contaminant in a biofilter system. First are the physical properties of the filter, surface to volume ratio, moisture content, air flow rate and contaminant loading rate per volume of filter media. The second are the chemical properties of the contaminant, its composition, solubility and concentration. The third relate to bio logy, the biodegrability of the contaminant, the microbial density within the biofilter as well as the conditions within the bio filter to which microorganisms are sensitive. These conditions include the availability of macro and micro nutrients, pH, temperature, concentration of salts and the presence of inhibitors or toxins. It is a combination of these physical, chemical and biological aspects which determine the treatment capacity of a biofilter.
Biofilter design is media specific and balances the often confiicting requirements of large surface area and uniform air flow. Fine soi1 particles offer enonnous surface area to volume ratios, but provide very poor airflow characteristics. The high air pressures required result in a greater tendency of channeling through the bed as well as higher operating costs.
Other constraints on the media are that it mut be able to sustain a microbiai population yet be resistant to degradation itself. The media must also be self supporting, i.e. not compress under its own weight, at a practical depth. Conventional filter beds are 1 m deep. The final constraints are cost and availability. A number of materials have been used as filter media, most are natural organic materials such as peat, yard and leaf compost, bark compost, wood chips and coco fibers. Granular activated carbon (GAC) and synthetic fibers have also been explored though they are not widely used. The most common media is a mixture of compost
(typically leaf and yard waste) and wood chips. A 1 :1 ratio is cornmon, though a 4: 1 ratio of bark nuggets to compost is proving to be more resistant to degradation thus providing longer bed life (Goldstein, 1996). The general range of surface area to bed volume for biofilter media is 150 to 1 100 m2/m3,with wood chips having a ratio of 160 m'/m3 while a mixture of heather and peat has an area to volume ratio of 1100 m'lm3 (Phillips et al., 1995).
The principle design parameters for biofilters are the air loading rate and the mass loading of contaminants. Using a standard bed depth of 1 rn, the Gennan Association of
Engineers recornmend that the air loading rate not exceed 200 m3 air m" bed surface h-'
(Frechen, 1993). Typical air loading rates are in the range of 60 to 120 m3 air m" bed surface hl. The air loading rates set a practical limit to the total airflow which can be treated by biofiltration. Biofltration is deemed a suitable treatment technology for airtlow ranges of
20 to 100,000 m3 h-' (Dra1992). The loading rate of contaminants can be reported as the
total load to the filter, often reported in terms of carbon, typical ranges are 1 to 5 g C m"
media h-' (Dragt, 1992) though values as high as 160 g C mJ media h-' have been reported
for optimized systems (Mueller, 1988). Since biofilters are used for treatment of odours
loading is ofien sited in terms of odour units, the German association of Engineers
recommends that loading to biofilters not exceed 50,000 odour units mJ media h-' (Frechen,
1993). Odour units forgo the need to identi@ al1 of the odorous components and individually
quanti@ them, it also accounts for the combined effects of multiple compounds. Odour units
are determined using human subjects as an odour panel. Air sarnples are diluted with clean
air, the dilution at which 50% of the panel can no longer detect an odour, considered the
odour threshold, is deemed the odorants concentration and given units of odour units per
cubic meter (o.u. mJ). Thus a sarnple with an odour concentration of 100 O.U. m-3would
require a 100 foid dilution to be reduced to the odour threshold. The odour loading to a
biofilter is determined by taking the odour concentration of the au (o.u. m" air). multiplying
it by the airflow rate (m3 air h-') and dividing by the media volume of the biofilter (m3
media), generating a loading rate of 0.u. m-3media h-'. The odour loading rate accounts for
both the Mowrate and contaminant concentration, for constituents other than odour
concentration is typically reported in terms of gaseous concentration on a volume basis, Le.
parts per million on a volume basis (ppmv). Biofilters have been effectively used for volatile
organic compounds (VOCs) from industrial chernical industries where the VOC concentrations range fiom 50 to 2000 ppmv (Ergas et al., 1995). Since the contaminants to be treated are in the gas phase of the biofilter and the treatment takes place in the wet biomass the contaminants must first cross the gadliquid boundary. The overall transfer coefficient between these two phases is dependent on the partitionhg coefficient between air and water, defïned by Henry's law, the interface surface area per volume and the flow conditions which establish the moving phase boundary layer resistance. The latter two are properties of the filter media and airflow rate, the first is a chernical property of the contaminant. Despite the high water content in biofilters they are able to treat cornpounds with low water solubility and high Henry's Law coefficients. The
BTEX compounds benzene, toluene, ethyl-benzene and xylene are good exarnpies of compounds with high Henry's law coefficients, ranging fiom 55 7 to 887 Pa m' mol-', which have been successfùlly treated with biofilters (Corsi and Seed, 1995). This is one of the advantages that biofilters have over bioscrubbers which have a stationary media and moving phases of both water and air, bioscrubber systems are only recommended for compounds with Henry's law coefficients lower than 10 Pa m3 mol-' (van Groenestijn and Hesselink.
1993). Bioscrubbers tend to have much more extensive biofilm growth than biofilters. this significantly reduces the available surface area as smaller pores become filled or covered with biofilm. The biofilm also covers binding sites on the support media, binding sites which facilitate contaminant removal fiom the airstrearn.
The biological nature of the treatment in biofilters requires that the contaminants be able to undergo biologicai degradation. When the source of the contaminants is itself a biological process, such as composting or animal wastes, biofiltration simply completes the biological cycle. Where the contaminants are not naturally occurring compounds biofiltration may still be a possible treatment option as long as a biologically mediated pathway exists. Treatment with biofiltration has been demonstrated for various chlorinated compounds, such as, trichloroethylene, methylene chloride and chlorobenzene, in the case of trichloroethylene a CO-metabolitein the form of toluene was required (Govind et ul.,
1997).
As a biological process biofiltration is sensitive to changes in the media which affect the microorganisms. The pnnciple parameters are moisture content, pH and ionic strength.
The oxidation of chlorinated compounds as well as reduced nitrogen and sulphur compounds results in acidification of the media as well as deposition of ions. The stationary nature of both the solid and liquid phases means these deposited ions accumulate in the media, thus media treating compounds of this nature have a nnite lifespan. Powdered calcium carbonate incorporated into the filter media has been effective in buffering pH where acid metabolites are produced (Clark and Wnorowski, 1992). The removal performance for any particular contaminant, as conditions change with time in the filter bed, will depend on the sensitivity of the particular organisms responsible for the observed activity and whether different organisms couid assume the observed activity under the new conditions. A succession of different species degrading the same compound as media conditions change is possible.
Control of moisture levels within a biofilter is critical for its proper operation. even where the incoming airstream is saturated drying occun due to the exothermic nature of the biological oxidation processes. in reality most airstreams are not fully saturated increasing the need for additional water being added to the system. It is important that the water addition maintain uniform moisture levels throughout the bed, since the media shrinks with drying, causing the void spaces to increase, creating a preferential path for airflow which promotes Merdrying. The resulting channeling through the filter bed can drastically reduce the amount of the bed actively used, reducing the contact tirne between the air and the filter media.
The main advantage biofiltration offers over other technologies is low operating costs. Two factors contribute to the lower costs. First biofiltration is a true treatment, unlike carbon adsorption, and as such does not generate a secondary waste which must either undergo Mertreatment or disposal, though periodic replacement of the filter rnatenal is required. Secondly, in biofiltration only the airstream is mobile, there are great cost savings in not having to circulate the liquid phase as occurs in scrubbing or bioscrubbing.
Dharmavaram (1991), compared operation costs for the treatment of contarninated airstrearns. Table 1 surnmarizes his findings.
Table 1: Operathg costs for air treatment technologies. Technology Costs (U.S. $ I ch)' Biofilter Scrubbing 18 -47 Inc ineration 105 - 168 Carbon absorption 179 - 210 '199 1 U.S. dollars per cubic foot per minute of air treated.
1.2 ODOURGENERATION DURINC COMPOSTCNG
The processhg of organic waste materials has associated odours. The odours result fiom a wide variety of chernical compounds, volatile fatty acids, nitrogen containing compounds, and organic and horganic sulphur compounds represent the principle groups.
Generally odours are produced during processing through biological and chemicai processes, with biological king the dominant means (Miller, 1993). Composting is an acceleration of the natural decomposition process which occurs in soils. nie accelerated decomposition results fiom proper preparation of the material including particle size reduction and mixing the different feed stocks to produce a starting material with an appropriate balance and the heat produced fiom microbiai metabolism. Typically the balance is based on the carbon to nitrogen ratio, with a target ratio of approximately 30: 1. Higher carbon ratios will result in a lower activity rate since the microorganisms will be nutrient limited, without the higher metabolic activity the compost may not reach the necessary temperatures to achieve pathogen inactivation. ifthe carbon to nitrogen ratio is too low there exists a higher chance of odour emissions. Carbon rich materials include; wood wastes, paper and paper products and autumn leaves, al1 of which are high in cellulose, hemicellulose and lignin. These compounds are resistant to degradation since they are chemically and structurally complex and nutrients are either lacking or non-available. Nitrogen nch materials include food wastes, green plant materials such as grass, animal wastes and sewage sludge. These materials contain rapidly degraded proteins, simple carbohydrates and fats. While odour generation can be minimized by mixing the proper ratio of materials and through proper management of the compost operation, they cm not be eliminated entirely.
Organic sulphur compounds are consistently identified as being the principle constituents causing offensive odows associated with the processing of organic wastes
@erikx et al. 199 1 ; Hentz et al,,1 992; Koe and Ng, 1987; Miller, 1993 ;van Dume et al., 1992; and Young and Parker, 1984). This is due to their low odour thresholds, dimethyl
sulphide @MS) and dimethyl disulphide (DMDS) are the two compounds most ofien
identified as problem odours, having odour threshold ranges of 0.6 - 40 ppb and 0. l - 3 -6
ppb, respectively (Smet et al., 1996). Despite the large number of other odorous compounds
that may be present the strength of an odour, measured as dilutions to threshold, is often
equivalent to that which would be required to treat the organic sulphur component in
isolation.
Sdphur exists in organic and inorganic forms. The sulphur containing amino acids
cysteine and methionine account for approximately 90% of the total sulphur in most plant
materials (Tabatabai, 1992). It is the degradation of organic forms of sulphur that produce
the volatile sulphur compounds associated with odour. Segal and Starkey (1 969) established that the rnicrobial degradation of methionine resulted in methyl mercaptan (MM) which readily oxidizes to DMDS, while cysteine sulphur was released as sulfate. Using monocultures under aerobic conditions Segal and Starkey (1 969) found that emissions of
MM and DMDS accounted for virtuaily 100% of the initial methionine sulphur in the system.
A wide range of soi1 microorganisms ranging fiom bacteria, actinomycetes and fungi have been identified as able to degrade organic sulphur compounds (Chattopadhyaya and Dey,
1993; Tomita et al., 1987). When organic materials were incubated aerobically with soils only 3% of the total sulphur was released as reduced organic sulphur over a 30 day period
(Banwart andBrenner, 1976). Even accounting for sulphur other than methionine these releases are much lower than would be expected. MM and DMDS are themselves biologically oxidized to sulphate, while individuai monocultures of organisms capable of degradhg methionine were not capable of oxidizing these products other agents in the soils were. Thus MM and DMDS can be seen as intermediates in the complete conversion of methionine sulphur to sulphate, the volatility of these compounds can result in their release to the environment and their low odour threshold makes them an odour nuisance when they are.
The amount of volatile organic sulphur compounds generated during composting and the amount emitted cmbe drastically different. Using the same feed stock but different set temperatures for a static pile forced air composting system resulted in varying odour ernissions. Odour emissions increased with increasing temperature, this could be attributed to higher rates of production due to the higher metabolic activity andor greater volatization due the higher temperatures (MacGregor et al., 198 1). When studying sulphur ernissions during the production of compost for mushroom production Derikx et al. (1 991) found that a 90% reduction in the emission of volatile sulphur compounds could be achieved by changing fiom an outdoor windrow system to an indoor in-vesse1 system. The total emission rate was reduced fiom 2 17 pmol S kg-'fiesh weight to 23 pmol S kg-'fiesh weight. The in- vesse1 system was indoors, which lirnited the volume of air which came in contact with the compost, and used a forced aeration system which re-circulated the air through the compost pile. The forced aeration system reduced anaerobic zones which favour production of volatile sulphur compounds, also the retirculation increased the contact time between the air and compost making the volatile sulphur compounds available for oxidation.
While volatile organic sulphur compounds rnay be the major concern for odour generation fiom composting, amrnonia production can have significant environmental impacts. Ammonia is the principal nitrogen containing compound produced during
composting with releases as high as 2 g ammonia kg" fiesh weight (Miller and Macualey,
1988). Ammonia's relatively high odour threshold, 5.2 ppm (Amoore and Hantala, l983),
means that amnionia is not a major concem for offsite odours, but its high ernission rate can
cause on-site health concems and local environrnental impacts. The threshold limit value,
a time weighted average for occupational exposure is set at 25 ppm by the American
Conference of Governmental Industrial Hygienists (Amoore and Hantala, 1983). In the
Netherlands ammonia is one of the major causes of environrnental acidification, with animal
husbandry accounting for 60% of the national amrnonia emissions (Elzing and Monteny,
1997).
1.3 BIOFILTRAT~ONOF DMDS
Smith and Kelly (1988a) isolated chemolithotrophic autotrophs fiom soils, peat,
compost, manure, marine mud and pond water. They were able to isolate a pure culture of
bactena capable of oxidizing DMDS and DMS as sole source of energy. The strain was
identified as Thiobacillus thioparus. While able to oxidize both DMDS and DMS a substantial lag time occurred in going fiom DMDS to DMS, indicating that DMS was not an intermediate in the oxidation of DMDS. In a later paper they characterize the metabolic pathway for Thiobacillus thioparus oxidation of DMDS (Smith and Kelly, 1988 b), which is shown in Figure 1.
A portion of this pathway was confhned in a study by Allen and Phatak (1993), where formaldehyde, H,S and HzOzwere shown to be the intermediates produced during the oxidation of methanethiol (CH,SH) by a Thiobacillus isolate. Sulphuric acid (H,SO,) was (CHd2S2 DMDS NADH + H+ h NAD' 2 CH,SH rnethanethiol 2 o2
2 HcOz-- 2H,O + O, 2 HCHO forrnaldehyde 2 H,S- Y ---- 2 NAD' 8 H,O -- I -- 2 NADH' + H' 2H20- 4 [~zo32-l 2 HCOOH formate
2 CO,
Figure 1: Pathway for oxidation of DMDS by ThiobaciZZus thioparus (Smith and Kelly, 1988). found to be the end product of H2S oxidation in a compost biofilter operated by Yang and
Allen (1994). inorganic S was the dominant form of sulphur (>95%) in filter beds which had been operating for long penods of time. Fifty to sixty percent of the sulphur was water soluble H$O, , though the incornplete oxidation products FeS and S0 were found in the lower regions of the bed (closest to inlet). Their presence was attributed to reduced biological activity resulting fiom exposure to high H2S concentrations.
The strong acidification which occurs with the oxidative degradation of DMDS and other volatile sulphur compounds creates long-term stability problems for biofilters. Yang and Allen (1994) found the media pH dropped fiom 8.0 to 2.5 in 32 days in a compost biofilter receiving high H,S loadiug. They found washing the filter media with deionized water or solutions of NaOH or NaHCO, maintained biological activity. Smet et al. (1 996) found that washing filter media with either tap water or potasiurn phoshpate buffers did not maintain biological activity. The sulphate was not removed as sulphuric acid but as sulphate salts with nutrient anions, leaching sulphate ions resulted in an equivalent flux of nutrient cations. They found additions of calcium carbonate (CaCO,) to the bed material provided better pH maintenance for biofilter treating dimethyl sulphide (Smet et al.. 1996).
Shoda (1 993) investigated the ability of a variety of bacteria, including Thiobacilhs thiopams, to oxidize odorous sulphur containing compounds. Bacteria were used to inoculate peat media for biofiltration studies. T.thioparzis was found to degrade al1 of the sulphur compounds tested, hydrogen suphide (H,S), methanethiol (MT), dimethy1 sulphide
@MS) and dirnethyl disulphide (DMDS). Removal rates could be desctibed using a
Michaelis-Menten type equation. Table 2 lists the removal rate constants determined for
T thiopanis. The specific uptake of DMDS by T.thioparus was 2.00 x 10 '" g S ce11 -' h -' and the population density was 5.1 x 101° cells g dry peat -' (the peat had a density of 100 kg dry peat rnJ ) (Cho et al ., 199 1). Using a Thiobacillus strain capable of oxidizing hydrogen sulphide, encapsuiated in alginate beads Chung and Huang (1997) were able to achieve a specific uptake rate of 3.26 x 10 -"g S ce11 -' h -'. Table 2: Removal rate constants for T.thioparus inoculated peat biofilter. Gas Maximum removal rate Saturation constant vm E(, g-S kg-'(dry peat) d-' PPm H2S 5.52 84.7
DMS 0.50 4.8
A pilot scale biofilter inoculated with Tthioparus treating night soi1 treatment plant air reduced arnrnonia concentrations below detection. This removal was attributed to the chemical reaction of NH, with H,SO,, the final metabolic product of the volatile sulphur compounds present, to form (NH4)$04as well as nitrification which maybe supported by the peat (Shoda, 1993).
1.4 B~OF~LTRATIONOF AMMONIA
Ammonia can be removed during biofiltration through adsorption to the media, absorption into the water phase, by means of a neutralizing chernical reaction with acidic components in the media or through biologically mediated oxidation.
Peat is a common media for biofilters, though in its natural form peat is acidic and usually requires pH adjustment before it can be used as filter material. Hartikainen et al.
(1 996) compared the chemical absorptive capacity of un-neutralized peat (pH 4.3) and peat neutralized with 170 g Ca(OH), kg-'(dry peat) and found neutralizing reduced the absorptive capacity fiom 17.9 g NH3-N kg-'to 8.6 g NH3-N kg -'. For compost at 40 % moisture the absorption capacity was determined to be 1.41 g NH, kg-' dry compost (Smet et al.. 2000).
Yani et al. (1 998) found ammonia absorption capacity of activated carbon fiber (ACF) at 75% moisture, used as biofilter media, to be 0.72 g MI, kg-' dry ACF. ln al1 cases this sorption capacity was exceeded within the £ïrst few days of loading resulting in a drop in ammonia removal, subsequent increases in removal were attributed to biologicai processes.
Liberty and Taraba (1999) conducted detailed studies into the ammonia absorption capacity of compost. Initial pH of the compost was the most significant factor in deterrnining adsorption capacity. The pH of the media increased as ammonia absorption occurred. Using compost without pH amendment at 40% moisture the absorptive capacity was determined to be 800 mg NH,-N kg-'dry compost. They concluded that for a 200 sow swine fma 400 m3 biofilter would be required to absorb 7 days worth of arnrnonia produced, this clearly demonstrates the limited amount of treatment afforded by sorption ont0 biofilter media.
The higher ammonia removal capacity for non-neutralized peat and composts is a result of the neutralizing chemical reaction occurring between the ammonia and the acidic components in the media. There are two distinct means by which ammonia can bind to the media, they relate to the nature of the binding site and affect the reversibility of the binding.
Peat, and other hurnic matenals, contain strong acidity sites as well as weak acidity sites and other oxygen-containing functional groups. The strong acidity groups, such as aromatic and aliphatic carboxylic acids as well as aromatic alchols, can trap ammonia forming ammonium salts in a neutralizing reaction. The weaker acidity sites and other oxygen containing sites, such as carbonyl and non-armatic alcohols, trap ammonia by forming hydrogen bonds with the ammonia. The ammonia trapped by hydrogen bonds is much more loosel y bound to the media than that bound through neutralizing reactions, the hydrogen bound ammonia wi Il be released easily during drying of the media (Togashi, et al., 1986). Togashi et al. (1 986) report a total amrnonia absorption capacity for a peat media at approximately 50% moistwe and initial pH of 4.1 1 of 12.73 g-N kg-' dry peat, once neutralized with Ca(OH)? to a pH of
7.25 the total absorption was reduced to 2.48 g-N kg-' dry peat, in both cases approximately half of the bound ammoaia was released when the media was dried at 80°C.
While ammonia has a higher solubility in water than most other odorous compounds considering the limited amount of water in the system removd through absorption is very smali, though as seen above water facilitated adsorption by the media can be substantial. A
Merwater mediated removal is the biologicai transformations of nitrogen which can take place in a biofilter, thus an understanding of the role and fate of nitrogen in soi1 environments is crucial for determining the ammonia treating potential of biofilters.
The transformations of nitrogen in soils are both ecologically and economically significant. Nitrogen is the limiting elernent for primary production in most ecosystems despite the fact that stable dinitrogen, which can not be used by most plants and animals, constitutes a huge portion of the atmosphere (78% by volume). Primary production in ecosystems was dependent on naturai nitrogen fixation processes until 1913 when Car1
Bosch began commercially producing ammonia using the process developed by Fritz Haber in 1909, which produced ammonia fiom nitrogen and hydrogen. By the late 1990s the chernical production of fixed nitrogen exceeded 100 million metric tons each year, comparable to the natural fixation of nitrogen which generates between 90 and 150 million metric tons per year (Homer-Dixon, 2000).
The ammonia, added as ammoniurn salts, and nitrate contained in fertilizers are only two of the stages in the continuous nitrogen cycle. Transformations. leaching of soluble nitrate, and losses in surface run off al1 reduce the ratio of nitrogen incorporated into plant
matenal to nitrogen added. This has both econornic and ecological consequences.
Transformation of added nitrogen to gaseous end products (N,O and N2)can represent
considerable losses, estimated to be as high as 40% of the applied nitrogen (Lloyd, 1993).
While biologicd transformations at the site of application are seen as undesirable, they are
critical in protecting non-target ecosystems, in particuiar surface waters. The biological
production of dinitrogen gas fiom either oxidized or reduced forms of nitrogen is the
principle rnethod by which riparian zones, the lands between terrestrial and aquatic
ecosystems, and wastewater treatment plants protect receiving waters fiom excessive
nitrogen additions (Martin et ai. 1999).
It was late in the nineteenth century that the production of nitrite (NO,-) and nitrate
(NO;) in soils was realized to be a biologically mediated process. The 1890 experiments by
Winogradsky led to the discovery of the bacteria Nitrosomonas and Nitrobacter which
oxidize ammonia to nitrite and nitrite to nitrate, respectively, the processes which are
collectively termed nitrification (Focht and Verstraete, 1981). Nicrosornonas and
Mirobacter are both gram negative, aerobic, chernoautotrophic rods, and their discovery
established the existence of chemoautotrophs, (chemo, meaning obtaining energy from
chernical sources, auto, relating to the use of CO, for carbon). Nitrifying bacteria occupy a
highly specialized niche, so the principle of cornpetitive exclusion would predict that there
would be a hi& degree of similarity among them. Isolates of Nitrosomonas and Nitrobacter fkom around the world have shown little variation (Focht and Verstraete, 198 1).
Denitrification, the reductive process of converting nitrate ultimately to dinitrogen was also established as a biological process by Gayon and Dupetit in 1886 (Focth and
Verstraete, 1977). This process was observed only in the absence of oxygen and is carried out by a variety of heterotrophic bacteria.
Thus the view of the nitrogen cycle consisting of aerobic nitrification cmried out by autotrophs and anoxic denitrification performed by heterotrophs was established by the end ofthe nineteenth century and persisted for much of the twentieth century. With advances in instrumentation it has been possible to establish that aerobic denitrification actually occurs
(Lloyd et al., 1987), as does heterotrophic nitrification (C hung et al., 1997). What remains to be determined is the relative contributions of each process to the overall fluxes of nitrogen and which conditions are required for the different processes to occur.
Figure 2 illustrates the various pathways and intermediates for nitrification and denitrification. Ammonia (NH,) is the substrate for amrnonia oxidizers not ammonium
(NH,'). The concentration of arnmonia is in equilibrium with ammonium. the balance is affected by pH and temperature. There are five genera of autotrophic ammonia oxidizing bacteria. While al1 are reported to occur in soil, Nitrosococcus is pnmarily a marine nitrifier and Nitrosovibrio is relatively uncornmon in soils. The remaining three, Nitrosospira,
Niîrosolobus and Nitrosornonas are al1 widely distributed and often coexist in the same soil samples, (Schmidt and Belser, 1994; Degrange and Bardin, 1995; Kuenen and Robertson,
1988). Head et al. (1993) conducted a phylogenetic study of autotrophic ammonia-oxidizing bacteria and found Mtrosospira, Nitrosolobus and Nitrosovibrio to be very closely related and suggested they should be reclassified as a single genus. Of the four genera of autotrophic nitrite-oxidizing bacteria only Nitrobacter is well docurnented in soils, (Schmidt and Belser, 1994; Degrange and Bardin, 1995; Kuenen and Robertson, 1988).
The oxidation of ammonia by Niîrosomonus eurpaea is mediated by two enzymes, ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO). The intermediate, hydroxylarnine (NH20H) is produced fiom ammonia by AMO, and hrther oxidized to nitrite through the action of HAO. The two stage oxidation of ammonia produces 4 reducing equivalents, two of which are used in the reaction of AMO, the remaining two are available for energy generation via the respiratory chain, (Zart and Bock,
r
Figure 2: Microbial nitrogen transformations and oxidation states (du Plessis et al. 1997). 1998). It has also been demonstrated that both NO and &O are produced during the
oxidation of NH,OH even under Mly aerobic conditions, their production is between 0.01 -
2% of the nitrogen oxidized. Under oxygen stress Nitrosornonas can use nitrite as the
electron acceptor for ammonia oxidation, the NO; is reduced to NI gas though some N,O
production occurs, (Kuenen and Robertson, 1988; Zart and Bock, 1998). This alternative
pathway for ammonia oxidation by Niîrosomonas species explains their obsewed growth
under anaerobic conditions with nitrogen removal as dinitrogen (Abeliovich and Vonshak,
1992; Siegrist et al.,1998; Zart and Bock, 1998)- Autotrophic nitrifiers demonstrate a better
survival rate under anaerobic conditions in the presence of organic matter, also implicating
the ability of anaerobic denitrification (Kuenen and Robertson, 1988).
While nitrifiers have dernonstrated the ability to reduce nitrous oxide to dinitrogen
under conditions sirnilar to those required by denitrifiers, the kinetics are different (du Plessis
et al., 1998). Denitrification is a dissimilatory reduction of nitrogen oxides (nitrate and
nitrite) to gaseous oxides (NO and NO,) which may be fiuther reduced to N2 gas.
Denitrification occurs when the denitrifiers substitute the ionic nitrogen oxides for O2as the
terminal electron acceptor during respiration. Many hundreds of denitrifiers have been
isolated fiom soils and sediments, most are aerobic heterotrophs. The higher mid-point
redox potential of the 02/H20couple makes it a superior choice to the NOJN02' couple, thus denitrification is an anaerobic means of respiration for otherwise aerobic organisms.
Oxygen has been shown to inhibit tk'activities of some of the enzymes of the denitrification pathway as well as repress their synthesis. Due to the vast number of organisms capable of denitrification a wide range of sensitivity to oxygen under various conditions can be observed (Llyod, 1993). In soils denitrification rates are negligibte until oxygen levels are low (0.20 pmoVrnl) (Martin et al., 1999). While some isolated species have demonstrated aerobic denitrification, this may be a consequence of a lack of stringency in the control of the denitrification pathway. When aerobic denitrification does occur it generally results in the incomplete reduction of NO, to N,O (Lloyd, 1993).
Denitrification, the dissimilatory reduction of nitrate with N2as the ultimate product, is not the only reductive pathway for nitrate, dissimilatory reduction to ammonia and assimilatory reduction are possible. in assimilatory reduction the nitrogen is incorporated into the organism. During aerobic growth many prokaryotic microorganisms are able to reduce nitrate to ammonia which is then assimilated as organic nitrogen, this is regulated and proceeds slowly based on the nitrogen requirements for growth (Cole, 1988).
Dissimilatory nitrate reduction to nitrite is used by many anaerobic bacteria to establish a proton electrochemical gradient across the cytoplasmic membrane. This dissimilatory reduction is much more rapid than the assimilatory process, nor is it regulated by the rate of ce11 growth. The resulting nitrite can be merreduced to ammonia by a nitrite reduction pathway. The two methods of dissimilatory nitrate reduction are performed by distinct groups. Fermentative bacteria use dissimilatory reduction of nitrate and nitrite to ammonia, while non-fermentive organisms which are capable of growing anaerobically in the presence of nitrate use denitrification (Cole, 1988).
Many heterotrophs capable of denitrification under anoxic or anaerobic conditions are also capable of nitrification under aerobic conditions. He terotrophic nitrification has generally been viewed as less significant than autotrophic nitrification, due to the significantiy higher activity rates of the autotrophs (Kuenen and Roberston, 1988; Chung er
al., 1997). The role of heterotrophs in nitrification could be significant under conditions
which inhibit or restrict autotrophic activity.
Autotrophic nitrifiers are notoriously slow growers, with generation times in the
range of 8 to 24 hours or even longer depending on growth conditions. This slow growth
does not allow nitrifiers to be maintained in activated sludge of wastewater treatment
facilities unless special attention is paid to sludge age and retention. Nitrification typically
involves fixed growth systems after carbon loading is reduced to allow the establishment of
autotrophs.
Autotrophic nitrifiers are also sensitive to a variety of growth conditions, in particular
pH, temperature, and salt concentrations. Autotrophic nitrification is optimal in neutral to
slightly alkaline conditions (pH 7-8). At lower pHs the NH,MH,' equilibriurn @Ka 9.3)
shifis away fkom NH,, the true substrate of the atnmonia monooxygenase enzyme. Ammonia
oxidation by Neuropaea in liquid cultures ceases around pH 6.0 (Stein and Arp, 1998),
though others have reported activity at pH 5.0 (Focht and Verstraete, 198 1). Nirrobacfer is
also reported to have reduced activity at lower pHs not due to the absence of ammonia but
due to the toxicity of nitrous acid (HNO,), the pKa for KNOJNOi is 3.4 (Focht and
Verstraete, 1981). Hunik et al. (1 992) determined that the reduced activity of N.europaea at low pH and hi& nitrite concentrations was due to more than just the reduced ammonia and elevated salt concentrations, establishing that Neuropaea is inhibited by nitrous acid.
Nitrobacter is also sensitive to ammoniaconcentrations. Some argue that N. europaea is also inhibited by its substrate (Groeneweg et al. 1994), but others argue that such inhibitions are actually resdting from an osmotic pressure effect due to the hi& salt concentrations, no
greater inhibition was found with substrate than with other ions (Hunik et al., 1992).
Regardless of the cause Nitrobacter has a greater sensitivity to higher ammonia
concentrations at higher pHs than does Neuropaea, under these conditions nitrite
accumulation is observed (Focht and Verstraete, 198 1 ;Villaverde et al., 1997; Smith et al.,
1997). The activity rates of autotrophic nitrifiers exhibit standard temperature effects over the temperature range 15-3S°C, below this range more drastic drops in activity occur.
Autotrophic nitrifiers fail to grow above 40°C (Focht and Verstraete, 198 1).
Heterotrophic nitrifiers may be the dominant nitrifiers in environrnents where autotrophs are either inhibited or non-functioning. Environments such as acidic soils or themophilic stages of composting are likely to support significant heterotrophic nitrification
(Focht and Verstraete, 1981). Table 3 lists nitrification rates for various species under varying conditions.
Table 3: Literature reported nitrification rates. Experimental conditions Nitrification rate Source Maximum activities Ni~osomonaseuropaea Schmidt and Belser, 1994. deterrnined during 1.54 x 1 O-') g N cel1-l h-' exponential growth in liquid cultures. Nitro bacter w inogradskyi 1.68 x 10'" g N ceIl-' h-' Liquid cultures of 2.95 x 10-'5g N cell" h-' Tappa et aL, 1996. Niirosomonas europaea under no growth conditions. Chemostat mixed culture Range Laanbroek et al., 1 994. of Niîrosomonas europuea 6.16 x 1 O'" to and Nitrobacter 1.8 1 x 1 0-l3g N cell" h" hamburgensis at steady- state. Table 3: Continued Experimental conditions Nitrification rate Source . - . - .- -. -. - . - -- . -. - -- . - - Liquid cultures 5.04 x g N cell" h-' Stein and Arp, 1998b. Nitrosornonas eutropha, testing effectsof nitrite on ammonia monooxygenase activiîy. Liquid cultures No NO, addition Zart and Bock, 1998. Nitrosornonas eutropha, 1.60 x 10-'* g N cell" h-' testing effects of nitrous oxides on nitrification rates With 50 ppm NO2 9.74 x 10-16g N cell" II-' Activated carbon fiber 3.34 x 10~'~g N cell" h-' Yani, Hird and Shoda, biofilter inoculated with 1998. night soi1 sludge treating ammonia gas Nitrimng activities of 100 g N kg-' VAS*h-' Villaverde et al. 1997. submerged biofilm (using conversion of 1.47 x 10-16 kg VAS cell" fiom Tappa et al. 1996) 1.47 x 1 O-'* g N cell-' h-' Compost biofilter given Maximum removd Smet et al. 2000 high loading of ammonia achieved 0.6 1 g NH, kg-' DC" d-' Nihimng compost 0.523 mg NH,-N Liberty and Taraba, 1999 biofilter kg" DC" d-' Immobilized Nitrosornonas 5.79 x 1O-' ' g N cf&' h" Chung and Huang, 1998. europaea biofilter, cells Based on initial density of irnmobilized in alginate colony forming units per beads gram beads. Heterotrophic 5.08 x 10-'O g N cfu" h-' Chung, Huang and Tseng, Arthrobacter oxydans CH8 Based on initial density of 1997. immobilized in calcium colony forming units per alginate beads used as gram beads. packing material for ammonia treating biofilter ' VAS = volatile attached solids " DC = dry compost Biological activity in biofilters is sensitive to media conditions such as pH, ionic strength, available nutrients and the presence of inhibitors. Arnmonia impacts al1 of these, thus the biological performance of a biofilter in treating dl contaminants, including ammonia, can be affected by the amrnonia loading. The absorption of ammonia can increase pH while its oxidation acidifies, in both cases ionic strength increases. Ammonia is a source of nitrogen, which is typically limiting in compost and peat media, yet ammonia can also be an inhibitor. Thus arnmonia addition can affect the removal of other constituents both positively and negatively. This dual effect was observed in the removal of dimethyl sulphide
(DMS) by Hyphomikrobium MS3 in a biofilter also treating ammonia. At low levels of ammonia addition to the air stream (up to 14 mg NH3m-3) a slight increase in DMS removal was observed and attributed to the nutritional effects. At higher concentrations (1 00 mg NH, mJ)complete inhibition occured (Smet et al. 2000).
One biological process which is particularly sensitive to ammonia concentrations is nitrification itself. Hartikainen et al. (1 996) demonstrated that overloading a peat biofilter with ammonia (1 -78 g-N kg-' dry peat day") caused an accumulation of nitrite as the nitrite oxidizers were inhibited. Dropping the ammonia loading did not restore nitrite oxidation indicating a drastic disturbance in nitri@ing population. At a lower ammonia loading rate
(0.17 g-N kg -'dry peat day-') complete nitrification occurred. Villaverde et al. (1 997) also observed nitrite accumulation in a submerged biofilter as ammonium concentrations hcreased due to the greater sensitivity of the nitrite oxidizers to fiee ammonia inhibition. ui contrast Smet et al. 2000, found no toxic effect due to ammonia in compost biofilter even at ammonia gas concentrations of up to 550 mg NH3 mv3(778 ppm). Though they report sharp decline in ammonia removaI under various loading conditions when the cumulative removal of resulted in a combined NH,', NO; and NO,- ion concentration in the filter media of 6.6g N kg-'dry compost. This corresponds with Hunik et al. 's ( 1992) observation that salt concentrations in this range are able to inhibit Nitrosornonas europaea due to osmotic effects.
Nitrogen is an essential nutrient making up about 15% of microbial dry ce11 weight.
Through the production of Nz during denitrification, air stripping of volatile NH, and leaching of soluble NO,-, nitrogen is continually lost fiom open microbial systerns such as soils, composting and biofiltea. The depletion of available nitrogen during composting results in a reduction in microbial activity. Determined analytically as a higher carbon:nitrogen ratio and performance wise as an inability to self heat (measuring reduced thermogenic microbial activity). Both are used as mesures of compost maturity .
Biofiltration relies on the establishment of a biomass capable of utilizing the contaminants present in the airstream. The rate and extent of growth will be controlled by a variety of factors, principal among these is nutrient limitations. Biofilter media is typically a nitrogen limited environment and the effects of nitrogen supplementation on biofilter performance has been studied.
Morales et al. (1998) used gaseous ammonia as means of nitrogen supplementation to a peat biofilter treating toluene. They found there was both a short terni peak in removal and an increase in the sustained equilibrium removal of toluene. The short term peak in removal was attributed to a reversion to growth phase kinetics while the sustained long term removal was attributed to increase in toluene degrading population made possible due to the additional nitrogen. Nitrogen addition in the form of nitrate was found to enhance hexane removal in a composting biofilter (Morgenroth et al., 1996). Basing nitrogen addition on the stoichiometric utilization of nitrogen and carbon for biomass growth and assuming hexane to be the sole substrate and no nitrogen recycling, much less hexane removal than expected was observed. Thus the nitrogen was incorporated into biomass created with carbon sources other than supplied hexane. Hence, the filter media itself, compost, was being used
(Morgemoth et al. 1996). As with Mordes et al. (1998) Morgenroth et al. found that the peaks associated with growth due to nitrogen addition were short lived, but once suficient nitrogen was added a higher removal rate was sustained.
Cherry and Thompson (1997) argue that this shift from growth to nutrient-limited maintenance kinetics is the underlying principle which determines biofilter performance.
They developed a biofilter model using Monod equations for both substrate and a limiting nutrient utilization. This model was able to predict performance during acclimation and demonstrate both the short term peak and long term sustained higher removal rates observed with nutrient supplementation, events which none of the current models based on mass transfer are able to predict (Cherry and Thompson, 1997).
Removal of ammonia through nitrification is an acid forming process, Yani et ni.
(1998) found that after 20 days of activity the pH in the ACF biofilter had dropped fiom 8.4 to 5.8. While others find that pH does not &op due the neutralizing effect of absorbing ammonia which reacts in the liquid phase to form ammonium ions. Smet et al. (2000) found that only 50% of the NH,-input to the biofilter was nitrified, the remaining 50% remained in the filter as Ni&+. This 1: 1 molar ratio of NOJNH,' maintained relatively constant pH in the filter media over 72 days of operation.
1.5 ~DENTLFICATIONOF ~MICROORGANISMS
Microorganisms lack al1 of the sophisticated structures used in classification of higher
life forms such as plants and animals. Classification of microorganisms in the past has relied
on observable ce11 morphology and other phenotypic characteristics such as type and style
of colony formation. Al1 phenotypic observations are made on isolated cultures. Phenotypic
classification does not provide hereditary or relatedness information, nor does it provide
information on ecosystem function, though its greatest limitation stems £iom its reliance on
culturing of isolates. It is estirnated that ody 1% or less of cells fiom terrestrial or aquatic
environments can be isolated on agar media (Liesack and Stackebrandt, 1992).
There are several approaches to addressing the limitations of phenotypic
classification. To address the limitation resulting fiom culturing, many systems rely on the
extraction of ce11 constituents which can be used for identification. Several classes of
compounds are used to identi@ bacteria, including nucleic acids, proteins,
lipopolysaccharides, fatty acids, carbohydrates, and lipids. Of these nucleic acids and fatty
acids are the two rnost promising methods (Bouger, 1996). immunological based systems
are another method used for monitoring specific species. They aiso detect unique
components on the surface of the cells, but do so without extracting the ce11 constituents. In
an attempt to identiQ organisms based on ecosystem hction identification methods based on metabolic activity have been developed, though still culturing, these methods provide
information not given fiom classical phenotypic identification.
Which of these approaches should be used depends on the information desired, when looking at communities a measure of biodivenity is often the objective. There are three interrelated elements to biodiversity: taxonomie, genetic and functional. The first two measure biodiversity as a number of species (with species increasingly identified by their
DNA) while the third looks at the fünctional roles of the microorganisms. While variability of DNA in an environment can be measured, the ecological significance of this variability and how it affects functional diversity is poorly understood (Staddon et al., 1996). Metabolic activity is one of the functional aspects of a cornmunity which has practical applications.
There have been several automated identification systems developed which are based on metabolic activity. Many have been developed for clinical applications, some such as the
API test has been applied to terrestrial and aquatic biotypes. but with limited success
(Wunsche and Babel, 1996).
BIOLOG is a metabolic based system designed to identiQ isolates fiom the soil and water environments. Identification is based on which sole carbon sources the isolate is capable of metabolizing. Using 95 testing wells plus one no-carbon control an identification can be made as long as three positives (ability to metabolize) are scored. A positive is indicated by a colorirnetrically determinable change of the redox dye tetrazoliurn violet into a vivid purple formazan. In applying the BIOLOG system to identiQ isolates fiom soil ecosystems Wunsche and Babel (1 996) obtained low rates of correct identification. Some of the problems cited include the limited data base used for identification, the limitations resulting fiom culturing and the inability of some isolates to grow under the BIOLOG standardized conditions, and the failure of some isolates to use three substrates. As a test of carbon use, BIOLOG is inherently for heterotrophs only, a mer restriction on characterizhg complete ecosystems.
Others have used the BIOLOG system to characterize whole communities, using direct environmental samples instead of isolates as the inocdumn. Despite its obvious limitations BIOLOG integrates the activities of a broad range of bacteria into the assessment of bctional diversity. Garland and Mills (1991) used BIOLOG for community classification by inoculating with whole cornmunity samples. They were able to distinguish significant differences between communities fiom different habitats. Using the utilization patterns fiom the BIOLOG system for soil samples, Zak et al. (1 996) were able to distinguish different soil environments and developed a measure of relatedness for the different bacterial communities. Whoie-community substrate utilization can provide a reproducible signature, but cannot determine the number of species present in the comrnunity capable of utilizing a particular substrate, nor the metabolic potential or fiction of the community. Not al1 species will grow under the conditions of the test, still only classifying culturable members.
Testing with simple communities has shown that the rate of utilization is not the surn of the individual activities and the tests are sensitive to inoculation size (Haack et al., 1995).
The challenge, and the point, of any taxonomy system is to appropriately identify, or at lest group, new species in some meaningful way. Amy et al. (1992) compared three classification systems in the identification of isolates obtained from deep subsurface rocks, isolates which are believed to be possibly 250,000 years old. Two of the systems are metabolic tests, API and BIOLOG, tests based on the ability to use specific substrates. The third system was the MIDI system, a system for analysis of fatty acid methyl esters. They found that each system had a bias towards organisms best characterized by that system. Each system identified some of the isolates though only a single isolate was identified by more
than one system, for that case the classifications did not match.
Bacteria synthesize more than 200 fatty acids and related compounds, both the
occurrence and relative arnount of the various fatty acids are characteristic of a specific
bacterial species. Since fatty acids can be extracted fiom a culture or environmental sarnple
and then analyzed by gas-liquid chromatography they have been used for characterizing and
identifjhg microbial isolates as well as populations. A particular group of fatty acids have
proven to be useful in identification of microorganisms, those are the fatty acids associated
with phospholipids. Each phospholipid contains two fatty acids in its hydrophobie tail.
Phospholipids are a major constituent of the membranes of living cells, they are rapidly degraded following ce11 death, they are not found in storage lipids or anthropogenic contaminants and have a relatively rapid turnover rate. Phospholipids are also a relatively constant proportion of the biomass of bacteria. These conditions make phospholipids a suitable ce11 constituent for monitoring biomass. The fatty acids in the phospholipids can provide even more information. Odd number and branched-chah fatty acids are produced by Gram-positive bacteria while even number straight-chah and cyclopropyl fatty acids are fiom Gram-negative bacteria. The straight-chain fatty acids are ubiquitous, thus providing little taxonomie information but making them suitable for total biomass estimations.
Quantitative phospholipid-fatty-acid (PLFA) analysis has indicated that 28 pg of PLFAs is approximately equivalent to 100 pg of biomass carbon (Zelles et ai., 1992).
Changes in fatty acid profiles during periods of starvation have been observed and often attributed to changes in membrane fluidity, thus fatty acid profiles are affected by environmental conditions (Guckert et al., 1986). For proper identification of an isolate by fatty acid analysis it mut first be grown under very specific growth conditions including media, slight variations can change fatty acid composition. Possibly related to this sensitivity to growth conditions is the difficulty in obtaining good repeatability between labs, this has resulted in many researchers maintahhg theïr own data bases. Identification of a new species is indirect since the system works on giving a similarity index to organisms present in the data base, thus the absence of an identification is how new species are identified
(Bottger, 1996).
Phospholipid fatty acid (PLFA) analysis has been used to rneasure biomass. and has been particularly successfid as a relative measure between sarnples. Often the PLFA values can be converted to a population estimate based on a reference strain, i.e. stationary phase
E. coli equivalent ceIIs (Franzmann et al., 1996). There is some suggestion that the biomass estimates made for soil and sediment samples by PLFA analysis may be too high, that as little as 10 to 20% of the lipid phosphate extracted fiom soil was denved fiom living biomass. Using direct counts in the soil, ce11 extraction followed by PLFA analysis and direct PLFA analysis on whole soil, Fostegird and BSth (1996) showed that only 26% of the cells were extracted but the PLFA analysis on the whole soil over estimated the population by approximately three fold.
Analysis of fatty acids has also been applied to total community characterization.
Haack et al. (1994) used the commercially available system fiom MicrobiaI ID Inc. (MIDI) which uses chromatography analysis of whole ce11 fatty acid methyl esters (FAME). The
MIDI system is designed to identiQ isolates, the FAME profile of the isolate is compared to a data base for identification. Haack et al. (1994) generated FAME profiles for entire communities. Applying principle characteristic analysis to the FAME profiles a measure of similarity was obtained, though the approach has limited potential for determining biomass and taxonomie composition. In looking at the profiles obtained for the same isolate grown under different environmental conditions it was found that in generai the genus-specific profiles remained clearly distinguishable fkom each other? though there were often a large nurnber of the fatty acids whose relative proportions changed. It should be noted that the
MIDI system extracts more than just the phospholipid fatty acids, including storage lipids so that it is not a suitable method of determining biomass as are more standard PLFA assay procedures
PLFA patterns fiom soils have been used to estimate population changes resulting fiom pollution deposition to soils (Baath et al., 1992), and for soils under different crop management systems (Zelles et al., 1995). One advantage of the PLFA analysis is that it allows whole community monitoring including fmgi, protozoa and actinomycetes in addition to bacteria.
Immunofluorescence methods rely on development of antibodies specific to the particular strain of bacteria. These antibodies can be either directly coupled with a fluorescence marker or have a binding site for a secondary antibody which is coupled with the fluorescence marker. Immunofluorescence staining has demonstrated comparable sensitivity to PCR,able to detect bacteria at a level of 1O3 to 1O4 cells (Hartmann er al. 1997).
The greatest challenge in using irnrnunofluorescence is the development of the antibodies with the desired degree of selectivity. Part of the problem relates to the nwnber of serotypes which might be involved. Féray et al. (1999) identified over 10 serotypes within the genus
Niiro bacter.
Another study using isolates fiom deep subsurface environments compared phylogenetic to conventional phenotypic techniques for classification. They cite that for conventional methods incorrect identifications are ofien due to well-known uncertainties of phenotypical description. These may stem fiom some traits being tested for may reside on plasmid DNA or may be a response to cultivation and not expressed in wild types.
Ribosornal DNA is always present and not likely to be af5ected by the specific ecological conditions under which the isolates were found (Boivin-Jahns et al., 1995). Thus molecular
DNA analysis has emerged as the definitive method of identification and classification.
Chernolithotrophicammonia-oxidizing bacteria are excellent examples of the failing of traditional methods of characterization. Due to the difficulty in growing them in vitro and their autotrophic physiology they cmnot be classified by traditional phenotypic methods.
Studies of their cellular lipids have yielded little information, thus, their classification has been based on morphological criteria. Phylogenetic studies have been able to provide a meanin@ classification of this group of bacteria (Head et al.. 1993). Extensive phylogenetic characterization of the P-proteobacterial arnmonia-oxidizers has been conducted. McCaig et al. 1994, have published a phylogenetic tree.
Phylogenetic classification uses the ability to exchange DNA as the definition of a species. It has been shown that bacteria with 20 % DNA difference are able to exchange
DNA, thus the molecular DNA approach classes al1 strains with 70% or more DNA-DNA relatedness as a species (O'Do~eliet al., 1994). The definitive test for identieing an isolate is genomic DNA-DNA hybndization with a known species, this can determine the degree of homology between the two DNA sequences estabiishing if they are the same species or not. This is only necessary when a complete identification of an unknown isolate is required, for most studies other techniques which work with only a fiaction of the total genomic DNA are suficient. Other DNA based detection rnethods are re-association tests, conventional hybridization as well as in situ hybridization and polymerase-chain-reaction (PCR).
Genetic diversity can be measured directly on DNA extracted fkom cornrnunities by means of re-association measurements. Re-association tests are conducted on whole genornic DNA, similar to the DNA-DNA hybridization, but in this case it is genomic DNA fiom the whole-community which is tested. Thermally denatured DNA will re-associate as the temperature is reduced. The rate of re-association will depend on the complexity of the
DNA, the greater the variability in a similar concentration of DNA, the slower the re- association process. The typical value reported for re-association studies is the Cet,, where
Co is the initial concentration of single stranded DNA, C is the concentration at any time t.
A plot of (C,C)/C, vs. Cot (initial concentration times tirne) is used to characterize the re- association, the Cotvalue at which (Co-C)/C, is equal to 0.5 is the Cot, . Re-association tests comparing the DNA obtained from cultured isolates and direct DNA extracted from the subsamples of the same soil showed that the Cot, value for the whole soi1 extract was approximately 170 times higher than for the mixture of isolates, irnplying that the isolates do not represent the total comrnunity in the soil (Torsvik et al., 1996). While this gives a measure of the complexity of a given community it does not give any specific information regarding its make-up. Free living bactena fiom soi1 and sediments have genomes ranging in size from 1S -
8 x 106 base pairs, with E. coli having 4.1 x 106 base pairs (Torsvik et al., 1996). Due to
the size of the genomic DNA and the vast number of microorganisms, knowledge of the
complete genome is not possible, instead DNA analysis has focused on specific segments,
in particular the genes for ribosomal RNA. Ribosomes are the site of protein synthesis, they
consist of a mixture of nucleic acids, ribosomal nbonucleic acids (rRNA), and proteins.
Their average size is 70s in bacteria. rRNAs are the most universally present molecule in
cellular life forms, they are functionally conserved which is reflected in the evolutionary
conservation observed in their sequences (Blackatl et al., 1998; Bottger, 1996). Ribosomes
contain three ribosomal RNAs, in bacteria they are the Ss, 16s and 23s. The three genes and
the intergenic spacer between the 16s and 23 s genes are principle targets for PCR. Most
work is done with the 16s which offers both conserved and variable regions, the intergenic
spacer offers even more variability (Nesme et al., 1995). Extensive databases of complete
and partial 16s rRNA gene sequences are accessible through the web.
Other genes of interest are the metabolic genes, those coding for specific enzymes in a particular metabolic pathway. These can be used to determine which species might be actively metabolizing a particular compound under certain conditions, different species with the same metabolic fùnction often have differences in their gene sequences. For ammonia oxidizers the ammonia mono-oxygenase (amo) genes have been used to monitor shifts in this population. Sakano and Kerkhof (1998) used amoA genes to determine that Nitrosospiru like species persisted in a biofilter treating ammonia while Nirrosomonas like amoA genes were not detectable after two weeks of operation. Conventional methods of monitoring nitrifiers would not have been able to make this distinction.
The presence of a particuiar metabolic gene in the DNA does not indicate its expression. In order for a gene to be expressed it must be transcnbed into messenger RNA which is used to produced the amino acid sequence of the protein. Muttray and Mohn (1 998) used the ratio of RNA to DNA to monitor metaboiic activity. This is possible since the amount of cellular DNA is relatively stable, while the concentration of RNA fluctuates greatly with metabolic activity. ifused with DNA-specific sequences this method could be use to target specific organisms in a mkedenvironment, allowing detennination of specific activities within a diverse community.
Hybridization is a method where two complimentary sequences of DNA are brought together; the closer their sequences match the more strongly they bïnd. In conventional hybridization methods the sample DNA is immobilized and DNA probes are applied and then washed off, only those which bind will remain with the immobilized sample DNA. The probes are designed DNA sequences with the desired specificity; they are ofien radio-labeled producing a signal where hybridization occurs. Pollard (1 998) used a reverse gene probing technique, where the radioactivity was incorporated into the DNA of the active cells in situ using methyl-3H thymidine. The specific probes were immobilized and the DNA sample was applied and washed off. Again a signal was obtained where hybridization occurred.
This method allowed for the detemination of Ni situ growth rates of specific species growing in diverse populations.
During in situ hybridization the probes are applied directly to the sample and the hybridization occurs between the probe and the DNA within the cell. The probes are usually marked with flourescent compounds, hence, flourescent in situ hybridization (FISH). FISH allows for monitoring of individual species withh complex environrnents. Hekmat et al.
(1998) used FISH to monitor specüic populations within a trickling-biofilter treating industrial air contaminated with a mixture of polyakylated benzenes, cornmon solvents in paints and adhesives. This method allowed them to rnonitor shifts in the microbial population with time. FISH has also been used for quantitative analysis of a variety of nitri fiers in activated sludge. It was found that Nitrosornonas and Nitrosocncc~.sform 1 5% of the total nitrimg population (van der Waarde et al., 1998). Another study using FISH also found Nitrosococcus mobilis to dominate in the activated sludge environment. (Wagner et al., 1998). In both studies Nitrobacter were not detected.
1.6 POLYMERASE-CHAIN-REACTION
DNA molecules consist of a long chah of deoxyribonucleotides, the backbone of the
DNA molecule is formed by phosphodiester bonds between the 3'-hydroxyl group of one deoxyribose to the 5'-hydroxyl of the adjacent sugar. Thus DNA molecules are directional, sequences are typically expressed fiom the 5' end to the 3' end. The complementary sequences in the double stranded DNA run in opposite directions. PCR is a process where selective amplification of a DNA sequence present in the sarnple is accomplished by the repeated action of a DNA polymerase enzyme. The DNA polymerase builds a double stranded DNA molecule fiom a single stranded one by moving dong the single strand and adding the complementary deoxyribonucleotide to the growing strand. DNA polymerase requires a primer to initiate replication. DNA polymerase binds to the 3' end of a double stranded DNA molecule where the complementary strand extends beyond the binding site, replication proceeds by building on the 3' end till the end of the cornplementary strand is
reached or the replication is terminated. The target, or template, DNA is first thermally
denatured so it exists in single stranded form (94 OC is the typical denaturing temperature used). Present in the PCR reaction with the template DNA are short sequences of DNA
(primers) cornplementary to specific sites on the template DNA as well as the DNA polymerase enzyme and some of each of the four deoxyribonucleotides. As the mixture is cooled the single stranded DNA will recombine to double stranded form, the primea are present in higher concentration than the template DNA, thus they are more likely to bind, forming short double stranded segments necessary for binding of the DNA polymerase. This process of the primers hding their complementary segments in the template DNA and binding with it is called anealing. The temperature used for anealing is primer specific but is typically in the range of 50 to 70 OC. DNA polymerase binds to the 3' end of the anealed primers and extends the doubled stranded segment by adding the complementary bases to the
3' end of the shorter segment. This portion of the PCR process is referred to as extension and is typically carried out at 72OC. The primers are designed in such a way that they bind one to each of the complementary strands of ternplate DNA and are spaced such that they ampli@ a common segment between them. Due to the directional nature of DNA the two primers will cause extension in opposite directions, amplifjring the segment between the two primers and the binding site of the other primer. The resulting double stranded DNA is then denatured and the cycle is repeated. What has made PCR efficient is the use of thermally stable DNA polymerases obtained fiom Thermus aquaficus (Taq) which is not denatured at the DNA denaturing temperature, other thermdly stable DNA polymerases are also available, though Tuq DNA polymerase is commonly used. Mer repeated cycles of denaturing, anealing and extension the target region of template DNA is present at a fairly high concentration. The amplified segments should al1 be of uniform length, the DNA cm be separated by size using gel electrophoresis and visualized by staining with ethidiurn bromide and illuminating with ultra violet light.
Standard gel electrophoresis separates DNA fragments based on sequence length, the longer DNA sequences migrate more slowly than shorter. If segments of the sarne length are to be separated based on differences in the actual sequence of nucleotides, a gel technique can still be applied but in this case a different principle is involved. The migration of denatured DNA is much slower than double stranded DNA. This coupled with the fact that sensitivity of a DNA segment to denahiruig forces, be they thermal or chernical, will depend on its sequence, allows for separation of same sized fragments based on differences in sequence. The development of an electrophoresis gel which maintains a denaturing gradient allows for the separation of DNA segments with very little difference in their code.
Applying this technique to PCR amplified 16s rDNA segments from complex rnicrobial populations results in distinct patterns which allow for the monitoring of changes in the population (Muyzer et al., 1993). This method is termed denaturing gradient gel electrophoresis (DGGE).
What makes PCR such an exceptionally versatile tool is the range in specificity that can be designed into the primers. PCR is extensively used for determining the presence or absence of a particula. organism in a sample by using primers which bind to sequences unique to that species. PCR can be used to generate copies of genes for cloning purposes by using prirners bracketing the gene sequence. Less selective, or "universal", primers can be used with isolated colonies, the resulting products can be sequenced, this is how much of the
16s rDNA and other databases are generated. The information obtained in this way allows for the design of more specific primers as welt as allowing phylogenetic classification.
When universal primers are used on cornmunity samples the results can be used to determine diversity. While PCR and other techniques eliminate the selectiveness introduced by culturing, they are not fiee of biases. There has been some evidence that PCR amplification fiom soil extracts using universal primers can be somewhat selective (Saano et al.. 1995).
Part of that selectivity can result fiom the non-universality of the universal primers, even whencomplementary binding sites exist they may not be equally accessible due to conditions beyond the binding site. Since PCR relies on DNA fiee from the ce11 the lysing procedure could also introduce some selectivity. While it is important to be aware of these possible biases PCR and the other DNA bases techniques have, they offer opportunities to investigate these complex ecosystems in ways which have never previously been possible.
Using PCR based diversity analysis the DNA extracted fiom a single soil sarnple corresponded to as many as 13,000 different species (Aman.et al.. 1996). Diversity analysis based on PCR arnplified segments could over estimate the diversity present due to copying error. PCR does not always result in perfect copying of the original sequence, artifacts maybe introduced. There is no proof reading function for the thermostable DNA pol ymerase hm Thermus aquaficus (the enzyme typically used in PCR amplification), an error fiequency of 1 in 1.7 x 1O4 is predicted. Studies conducted with in situ hybridization have demonstrated that the high level of diversity determined by PCR methods in activated sludge samples do exist and are not an artifact of the PCR process.
PCR has also been coupled with digestion with restriction enzymes to monitor population changes in activated studge. The novel technique used universal 16s rDNA prîmers to amplie community DNA, one of the primers is labeled. The amplification products are then digested with restriction enzymes, labeled fragments of various length are made. The pattern resulting fiorn these labeled Fragments was found to be repeatable and distinctive for the various cornmunities (Liu et al.. 1998). This technique has also been used to study differences between Nitrobacter strains isolated fiom different locations. In this case the PCR amplified region was the highly variable spacer of the ribosomal operon, this region evolves rapidy thus strain specific sequences were detectable (Navarro et al., 1992).
PCR has also demonstrated advantages over imrnunofluorescence techniques which have been used for monitoring Nitrobacter populations. The advantage that PCR has demonstrated over immunofluorescence techniques when applied to monitoring members of the genus Niirobacter, is that while over 10 serotypes have been identified in the genus
Niirobacter, the primer used for PCR is effective for the entire genus.
1.7 QUANTITATIVEPCR
Ttiere are two approaches to quantimng the amount of DNA in a sample using PCR.
One involves quantimg the amount of DNA resulting fiom the PCR amplification process, then calculating the initial amount of template. The second involves diluting the initial sample and using PCR to detemine the presence or absence of template DNA at the various dilutions.
Quantification of the DNA produced by a PCR amplification can be done by direct measurement of the product DNA or indirectly by comparison with an intemal standard which has ken amplified in the same reaction. The arnount of product is dependent on the amplification efficiency, the number of cycles and the amount of initial template available.
Mathematically the amount of product should growexponentiaily with the number of cycles.
In practice the PCR will not generally depart fiom the exponential phase as long as the primer concentrations remah at least 10 times greater than the concentration of the amplification product (H&, 1994). The amplification efficiency can Vary based on the primers, the binding location, and the various constituents in the PCR reaction mixture. The amplification rates will not always be the sarne. DNA yields fiom PCR amplifications fiom pure cultures of Nih.osomonuswere found to be consistently lower than those obtained fiom cultures of Niirosospiru when species specific 16s rDNA primers were used (Hioms et al.,
1995). The use of internal standards is intended to alleviate these problems. Rongsen and
Liren (1 997) compared two different methods of using intemal standards, the first of these was the CO-amplification of the target and a heterologous standard, the second was competitive PCR where a homologous intemal standard is added to the sarnple. The heterologous standard uses different primers than the target DNA while the homologous standard is designed to use the same primers. The internal standards are added in serial dilution, the amount of template is estimated based on a comparison of the two products. Of the two internai standard methods the competitive PCR is recommended since there is no primer annealhg variability. Rongsen and Liren (1997) found the intemal standards gave better precision and repeatability than direct quantification of the PCR product. The internal standards are considered to have an advantage over other methods since any factors affecting the efficiency of the amplification are assumed to affect the amplification of the template and standard equally.
Suzuki and Giovannoni (1996) argue that even with identicai priming sites the intemal standards and the sample target sites may not necessarily undergo amplification at the same rate. The internal standard and the target DNA will differ, typicaily in length or in restriction endonuclease sites, and these differences beyond the priming site can impact the accessibility of the site. Other factors may cause extension to proceed at different rates. As amplification proceeds the concentration of products increases. Reannealing of the products occun with longer DNA molecules recognizing complementary sequences at a higher rate than shorter strands, and ones present at higher concentrations reanneal more readily as well.
Once annealed these strands are not available for amplification during that cycle. Thus if the target and internal standard are of different lengths or are present at different concentrations the reduction in amplification efficiency wiH not be uniform (Sdiand Giovannoni, 1996).
A second method for quantitative PCR is to conduct amplifications on a seriai dilution of the original DNA sample. The dilution at which no more amplification product can be obtained provides an estimate of the template concentration in the original sample.
When done with replicates this method can use the most-probable-number (MPN) statistics to estimate population. Rongsen and Liren (1997) considered dilution to extinction and cite problems of precision and reproducibility, though they did not consider how the MPN statistical approach could overcome these problems.
1.8 MPN-PCR
Most-probable-number (MPN) is based on the idea of dilution to extinction and is widely used with culturing techniques to enumerate rnicroorganisms in food, water and soils
(Woomer et al.,1990). There are two basic assurnptions critical to the application of MPN theory. First, the organisms are randomly distributed in the liquid, there is no tendency for organisms to cluster together or repel each other. Second, each sarnple which contains one or more organisms will demonstrate growth when incubated in the culture medium (Cochran,
1950). Since growth can result from one or more organisms present, MPN actually works on the probability of no growth. If al1 of the samples were taken at the sarne dilution determining the initial concentration couid be done directly fiom the nurnber of positive and negative reactions. The fact that both positive and negative results are necessary creates difficulties for using a single dilution, since a very good estimate of the density would be required to determine the dilution. Once a senes of dilutions are used the determination of the population estimate becomes much more complicated. in fact, MPN estimates are not a direct probability estimate, as are plate or direct counts, but an inverse probability theory.
That is to Say, the observations are not used directly to calculate a population estimate but instead a population estimate is chosen which can explain the observations. While a little less direct it has been proven mathematically that as the nurnber of samples becomes large. the MPN theory gives on average at Ieast as precise estimates as any other method with the same data. While the theorem can not be proven for smaller nurnber of samples, experience has indicated that even then MPN is one of the best estimation techniques (Cochran, 1950).
Scoring of traditional MPN methods has been based on growth on a selective media or the expression of a particular metabolic activity. The implementation of MPN has been facilitated through the establishment of standardized tables, and more recently computer programs, which convert positive and negative scores into a population estirnate. The MPN method has been most beneficial when a population estimate of one constituent fiom a heterogeneous microbial population is required, or when plate counting is difficult.
A culturing MPN technique monitoring for the conversion of ammonia to nitrate has been the most widely used method for enumerating nitrifiers (Schmidt and Belser, 1994).
Scoring is done by means of a colounnetric pH indicator which changes when ammonia is oxidized to nitrate. Some of the limitations with this technique are the prolonged incubations required, minimum of three weeks and often more than two months being used and the test does not distinguish between the various nitrifiers. Culturing MPN techniques have also been used for soil bacteria involved in sulphur odxidation (Lawrence and Germida, 1988).
MPN-PCR involves usïng PCR to score the different sarnples of an MPN dilution senes instead of culturing and monitoring growth or metabolic activity. The DNA sequence specified by the pnmers, instead of viable cells, is the element being monitored. The MPN score then provides an estimate of the template concentration in the initial sample.
A MPN-PCR technique has been successfùlly used to quanti@ Nitrobacter populations in soil (Degrange and Badin, 1995). MPN-PCR has also been used to monitor the fate of Mycobacterium chlorophenolicurn stain PCP-1. a PCP degrading bacteria, in various soil environments (Elsas et al.. 1997). Leung et al. (1 999) used MPN-PCR coupled with enrichment culturing to detect the presence of Shingornonas sp. UG30. After incubating the dilution series in selective medium for 7 days PCR was conducted on the various cultures. A positive PCR was used as a positive score for the MPN determinations.
Rosado et al. (1996) combined PCR and hybridization on a MPN dilution of soil DNA extracts as an effect means of quantimg Paenibacillus azotofiam, a nitrogen fixing bactena with potential as a crop innoculant in tropical soils.
As a soluble molecule in water, DNA meets the first criteria for MPN of equal distribution, possibly better than whole cells. The MPN-PCR equivalent of the second cnteria would be that oniy a single strand of template DNA would be required for a positive
PCR reaction. This is a challenge for PCR fiom soil derived DNA due to the inhibiting substances that are CO-extracted,though PCR techniques have demonstrated abilities t9 detect very low concentrations of cells in soils.
1.9 DNA EXTRACTIONFROM SOILS FOR PCR
There are two approaches to extracting DNA fiom soils, the first is to extract intact cells first and then lyse the cells, the second is to directly lyse the cells in the soil and extract the DNA. The ce11 extraction method was developed for soils with high humic acid content in an attempt to limit the amount of contarninating humic matenal in the final DNA extract.
The disadvantage to the ce11 extraction method is it can be selective, the more tightly bound cells may not be extracted, also the direct lysis method has been shown to yield 20 to 70 times more DNA than the ce11 extraction method (Steffan et ai., 1988).
Taq DNA polymerase is inhibited by humic acids. In what they claim to be the first
PCR amplification of native soil bacteria Bruce et al. (1992) used cesium chloride ultra centrifugation to separate the DNA fiom other ce11 and litter fragments. The extracted DNA band was then Mercleaned by dialysis. DNA prepared in this marner was allowed amplification by PCR.
To minimize the amount of CO-extractedcompounds, DNA can be obtained from cells isolated fiom the sample. The cells cm be rinsed and pelletized prior to lysing, resulting in a much cleaner DNA extract. Pfdler et al. (1994) found this to be necessary when tiying to obtain PCR amplification fiom compost. Compost humic acid content is 10 to 100 times greater than that of mineral soils; in general the humic acid content increases as the compost matures.
Picard et al. (1 992) recornmend direct lysis using thermal shocks (liquid nitrogen and boiling water) and sonication to extract DNA fkom soil samples. Avoid detergents which can also inhibit Taq polymerase activity. To reduce the amount of humic acids in the frnd DNA extract they used a lysing buffer containing 1% polwinylpolypyrrolidone (PVPP) which removes humic acids and other phenolic impurities by adsorption with liale or no reduction in the amount of DNA. The PVPP is non-soluble and is settled out of solution by centrifugation leaving the soluble DNA in the supernatant. Ushg this extraction process a
102dilution was required for PCR amplification of DNA fkom soils, resulting in a potential detection threshold of 103 per 100 mg of dry soil (Recorbet et ai.. 1993). Rosado et ai.
(1996) found the addition of 1% formamide and T4 gene 32 protein enhanced PCR specificity when arnplimng DNA from soi1 samples. 2.1 BIOFILTERSTUDY OBJE~~LYES
The principle objective of the biofiiter studies was to investigate the CO-removalof ammonia and DM'Sfiom an airstrearn through bio filtration. Pursuant to this objective was the design and constniction of a laboratory biofiltration system which allowed simultaneous study of biofilters treating each gas in isolation as well as in combination. Of particular interest was the possibility of inhibition of the biological removal of one constituent due to the presence of the second.
Secondary objectives included determining the effects of inoculating the filter media with the autotrophic nitrifiers Nitrosornonas europaea and Nitrobacter winogradskyi as well as the organic sulphur oxidizing bacteria Thiobucillus thioparus. Also included is monitoring the long-term deposition to the filter media to determine the combined loading effect on bed aging.
2.2 MICROB~ALANALYSIS OBJECTIVES
The principle objective of the microbial analysis was to develop a rapid and accurate method of enurnerating the three bacterial species used as inoculum in the biofilters. The intent was to monitor the population of these species over theto establish in situ growth and activity rates. An MPN-PCRmethod was explored. A secondary objective was to develop a means of monitoring the diversity of the microbial populations within the biofilters.
Denaturing gradiant gel electrophoresis @GGE) was explored as a possible technology for monitoring the diversity of the microbial populations within the biofilters. 3.1 LABORATORYBIFILTER SVSTEM
The laboratory biofilter equipment had to provide the following basic fùnctions:
allow for simultaneous biofilters to be run, deliver a variable but accurate flow of air to each
biofilter, humid@ the air, allow precise loading of ammonia and DMDS to the airstrearns,
allow some biofilters to be loaded with ammonia only some with DMDS only and others
with both at the same tirne. The biofilter and air distribution system had to be constnicted
of materials which would be non-reactive with ammonia and DMDS and their metabolites.
It was decided to constmct eight parallel biofilters, allowing for a replicate of each of the
individual loadings and the combined while leaving an additional pair for any extra
experirnents.
The air supply used was compressed air available in the building. Pressure regulation and filtering were achieved by having two filter regdators in senes. The fist filter regulator had a 10 pm filter, the second had 0.5 (rmfilter to remove any particdate matter or oïl fiom the incoming air. Once filtered the air was humidified using fine bubble diffusion. This was done using three 450 mm Long aquarium 'Bubble Curtains', a perforated flexible tubing capable of producing very fine bubbles with minimum head loss, were placed in the bottom of a 180 L plastic barre1 which was filled to a height of 60 cm with distilled water.
DMDS loading was accomplished using a programable syringe purnp (Harvard, mode1 22), equipped with 5 ml gastight syringes. The syringes used had a 1/4" by 28 UNF-
2A threaded male terminal. The syringes were connected to 1116" teflonmtubing using
'Peek' 1/16" compression fittings with a 28 W-2Athreaded female end which screwed directly ont0 the syringes. Anhydrous ammonia was supplied from a pressurized tank and
ammonia delivery was controlled via a mass flow controller (Matheson, Mode1 8270). The
mass flow controller was equipped with 114" stainless steel compression fittings and flexible
1/4" stainless steel tubing was used to deliver the ammonia to the airlines. Al1 connections
were messsteel compression fïttings (Swagelok); the airline tubing was teflon?
The distribution system varied in configuration depending on the experünental
design. Configurations were constrained by the fact that only one ammonia flow contrdler
was available. The syringe purnp could accommodate up to eight syringes. Figure 3 is a
schematic of the laboratory biofilter system showing the eight biofilters. The air distribution
system in Figure 3 is configured to have four biofilters receiving combined amrnonia and
DMDS loading, two receiving ammonia only and two DMDS only.
Flow rates were controlled at each biofilter with needle valves with stainless steel
bodies and seals made of kalrex@and viton to ensure against corrosion (Parker, 20/30 senes,
mode1 4Z(A)-NL-A-KZ-SS-V). Air was supplied to the top of each biofilter and flowed
down through the media. A down-flow configuration was used because it results in more
uniform moisture levels since drying occurs mainly at the top where additional water can
be supplied.
Each biofilter consisted of three segments: a cap, a media housing mid-section and
a bottom. Al1 were constructed fiom 150 mm schedule 40 stainless steel (SS) piping (inside diameter 154 mm). The media housing section was 400 mm in length open at both ends.
The media was supported by a SS perforated plate (3 mm diameter holes.on 5 mm centres) which was welded in place 20 mm fiom the bottom. Sample ports were dnlled 30 mm fiom the top of the media housing sections and tapped to fit 1/4" SS compression fittings
(Swagelok) which had been bored through so the air sampling probe (1/4" SS tubing) could be inserted through the fitting. The caps and bottoms were constnicted fiom 100 mm lengths of the SS piping, one end of each cap and bottom was sealed with 1/4" SS sheeting welded in place. Two ports were drilled in each cap and bottom. For the caps one port was for the inlet air supply and the second was a sampling port. For the bottoms one port was the outlet and the second a sampling port. Quarter inch (114") SS compression fittings were used for the sampling ports and the air inlet, the outlet used 1/2" SS compression fittings. The edges of the sections were smoothed and a small groove was machined to act as a seat for an O-ring used to provide a gas-tight seal between the media support section and the cap and bottom.
Small sections of angle iron were welded to the outside at the ends of the sections which were to be comected, these were used to bolt the sections tightly together. An additional port was made in the cap and media support sections for the insertion of thermocouples.
Type T thermocouples (Cole-Parmer) LOO mm long by 3 mm diarneter were used.
3.2 AIRFLOW MEASUREMENTS
The airflow rates in the biofilters were controlled with needle valves at the inlet to the biofilters, the airflow rate was measured using a bubble flow meter (Humonics Digital
Flowmeter model 730, calibrated ~2%error). Airflow was measured at the outlet of the biofilter.
3.3 GASANALYSIS
A gas chromatograph (GC) with a photo-ionization detector (PID) was used for determining DMDS levels in the gas streams (SRI instruments, model 8610). A 15 m Combined Ammonia and DMDS Loadinq
Mass Flow Controller
Air
Ammonia Only Loading4-
DMDS Only Loading Humidification Column
Figure 3: Schematic of laboratory biofiltration system, showing the eight biofiiters configured for combined and individual loading of contamùiants capillary column (Supelco, SPB-1 ) designed for separation of sulphur compounds was used.
The carrier gas was ultra pure helium and the carrier gas flow rate was set at 16 mi per minute- The column temperature was set at 60°C. . The automatic sampling system loaded a 1 ml sarnple to the column, Under these conditions the DMDS peak occurred at approximately 2.6 minutes. The solvent used to prepare standards (carbon disulphide) generated a peak which came off much earlier at approximately 1.2 minutes.
Amrnonia was also detectable with the GC-PD though arnmonia measurements were primarily taken using Matheson-Kitigawa ammonia precision detector tubes using a hand pump (Gastec Precision Gas 100 mi hand pump).
3.4 PREPARAT~ONOF CAS STANDARDS
Gas standards of DMDS in air were prepared using Tedlar bags which had been repeatedly flushed with helium and evacuated. The bags were filled with 2 L of helium and
DMDS diluted in carbon disulfide was injected into the bags. Standards were made at 50,
25, 10, 5 and 1 ppm DMDS concentrations. Standards were run in triplicate and a linear regression was preformed on the results, if no samples fell below the 10 ppm range the regression was performed on the 50,25 and 1 O ppm standard results only. If some sarnples were below the 10 ppm range, a second regression would be performed using the 10,s and
1 ppm standards results.
3.5 MEDIAPREPARATION
The filter media used was mature compost derived fiom leaf and yard waste combined with industrial paper products. The compost was provided by Growbark Inc. of
Milton, Ontario. Composting was conducted in outdoor windrows. The compost was obtained fiom the finished piles and was still warm when being
dug. After collecting the compost was screened using a shaker screen and the fraction
between the screens with the 3.2 mm diameter openings and the 4.8 mm diameter openings
was used as the filter media. Moisture holding capacity was determined by saturating compost samples (approximately 20 g dry weight) with water, placing then in a fine screened
sieve and allowing the excess water to fiee drain fiom the bottom for ten minutes. The moisture content of the sample was then determined by oven drying at 105°C for 24 hours.
3.6 CULTURES
Al1 cultures were obtained fiom the American Type Culture Collection (ATCC).
Table 4: Identification of cultures used to inoculate biofilters. ATTC nwnber Organism 8158 Thiobacillus thioparus - type strain 25978 Niirosomonas europaea - type strain 1 25391 1 Nitrobacter winogrodskyi - type strain 1
T. thioparus was maintained on plates which were incubated for 10 to 2 4 days at
30°C after which they could be stored at 4°C for up to 12 weeks, or the culture could be lyophilized and stored up to a year. When liquid culture was required, media were inoculateci fiom plate or lyophilized cultures and incubated at 30°C for approximately 7 days.
The medium used was ATCC culture medium 290 S6 (see appendix D ).
N. europaea had to be maintained in continuous liquid culture since it was not possible to maintain them on plates or lyophilized. The maintenance regime used was obtained fiom researchers at Oregon state university, see appendix D for media formula. The cultures had to be transferred every three days while being incubated at 30°C or stored at 4°C for up to 6 weeks.
N winogradskyi was maintained in liquid culture and as lyophilized cells. The culturing media was ATCC culture media 480 (see appendix D). Nitrite levels in the media had to be monitored and nitrite added in the form of sodium nitrite (NaNO,, Once the culture depleted added nitrite within 24 hours (typically after 14 days of incubation at 30°C) the culture was transferred. Niaite levels were monitored in both the N. winogradskyi and the N. europaea cultures using the colorimetric Gness-Ilosvay method, see appendix D for method.
3.7 DIRECTCOUNTING OF LIQUIDCULTURES
Liquid cultures were enumerated using direct counting under the microscope after stahing with a flourescent dye (Molecular Probes, LivelDead Baclight No. L-7012) and filtering ont0 filter paper (Corning, Nucleopore Track Etch Membrane Filters). The area of the filter paper covered with culture was 280 mm', at 1OOOX magnification the area of a field of view under the microscope was 0.01 54 mm', therefore the filtered area corresponded to
18167 views. Ce11 concentrations were determined by staining a dilution of the liquid culture, vacuum filtering the stained cells to the a filter and counting the cells which fluoresced in a number of views. Population estimates were made based on the dilution, average number of cells observed pet view and the number of views represented by the total filter area,
3.8 IN~CULUMNPREPARATION
The inoculurnn consisted of concentrations of the liquid cultures of al1 three species cultured. Al1 of the filter media was inoculated, rnixed and then divided between the various biofiltea. For the combined and individual studies approximately 3 1 of each culture was prepared to inoculate the 5 kg of mediadivided between the six biofilters. Due to difficdties in preparing 3 1 of each species and having them ail ready for tran~fe~ngat the same time, it was necessary to spin down some cells and store them at 4°C. Care was taken to ensure that the inoculumn consisted of both active culture of al1 species as well as the cultures which had been stored. Cultures were spun domand resuspended in approximately 1/ 1O the initial volume.
3.9 START-UPOF BIOFILTER EXPERIMENTS
Prior to each biofilter experiment the biofilter columns were washed and autoclaved for 20 minutes. Media was prepared by adding inoculumn (unless no inoculumn was being used) and additional autoclaved water until the media was at 60% of its water holding capacity. The media was distributed between the biofilters and loosely packed by hand.
Columns were assembled and the air supply was turned on. Air flow rates were monitored with a bubble flow meter and adjusted using the needle valves on each biofilter inlet. Once the desired airflow rate was obtained the loading of DMDS and arnmonia was initiated. Inlet concentrations were monitored and loading rates adjusted until the desired loading was achieved.
3.10 GASSAMPLING
Met and outiet concentrations of DMDS were monitored with the GC-PID daily initially (except for one day each week). Monitoring fiequency was reduced later in the experiments when activity rates were demonstrated to be consistent. Concentrations were determined fiom the average of three samples. Samples were taken by inserting the sampling probe attached to the GC directly through the sample port. The sample probe was 1/4"
stainless steel tubing which couid be inserted 150 mm into the columns and sealed with a
compression fitting nut and ferrule to the sampling port, producing an air-tight seal.
Amtnonia concentrations were monitored once every two days, daily when there were
dramatic changes apparent. The gas detector tubes used (Matheson-Kitigawa) were
approximately 130 mm long and just under 1/4" in diameter, thus could fit through the
sampling port. Sarnples were taken by inserting the tubes through the sarnple port and slowly
drawing the air sample through the tube using the sampling pump, a full stroke of the pump equal to 100 ml of air and a half stroke of 50 ml was also possible. To conserve tubes only
single samples were taken and often only one of the replicate columns was sampled.
3.11 SAMPLINCOF MEDIA
Penodically, approximately once every 2 to 4 weeks, the air and contaminant loading to the biofilters was stopped, the filters dismantled and media samples were taken for moisture, chernical and microbial analysis. Each column was dismantled and sampled individually, the media was removed and placed in a large autoclave tray where it was mixed and a grab sample taken. The sample was sub-sampled for rapid moisture determination using the microwave method allowing for determination of necessary water addition. Three sub-samples were also taken for microbial analysis and these samples were placed in sterile disposable 15 ml centrifiige tubes and irnmediately transferred to a -20°C freezer. A final sub-sample was submitted for pH, total N, total S, ammonia, nitrate and nitrite anaiysis.
Water was added to the media to bnng the moisture content up to 60% of holding capacity, the media was then placed back into the column. Each column was repacked and reassembled before the next column was opened. Once al1 columns had been sampled and reassembled the airstream and contaminant loading was reinitiated.
3.12 MOISIVREDETERMINATION BY M~CROWAVE
Sarnples for moisture determination were placed on plastic weigh dishes, weighed
(Mettler PC 2000 scale), microwaved at full power (Panasonic, mode1 NN-6449) for 1 minute then weighed again. The sample was then microwaved at hl1 power for 30 seconds, weighed again, and then microwaved for an additional 20 seconds and weighed again. If there was a significant change in weight between the last two weights (more than 1% difference) the sample was microwaved for an additional 20 seconds. The initial and final weight were used to determine initial moisture content.
3.13 CHEMICALANALYSIS OF MEDIA
Chernical analysis of the media was conducted by the Soi1 and Nutrient Laboratory
(Laboratory Services, University of Guelph). Sarnples were analyzed for pH, total N, total
S, ammonia, nitrate and nitrite analysis. The soi1 pH method used involved drying the samples, crushing and sieving then adding distilled water to form a paste, pH of the paste was determined using a pH meter. Total nitrogen was determined using a Leco FP-428
Nitrogen Andyzer following manufacturers instructions. Total sulphur was conducted using the Leco SC-444 method of sulphur determination. Ammonium, nitrite and nitrate were deterrnined using the method of Keeney and Nelson (1 982) which involves first extracting the inorganic nitrogen fiom the media sample with potassium chlonde (25 ml of 2M KCI for a 5.0g fiesh or fiozen sample). The filtrate is analyzed for ammonium colourimetrically using the Berthelot reaction. The nitrite is also determined colourimetrically using the Griess-Ilosvay reaction (see appendix D, Nitrite Analysis). Nitrate is determined by first reducing it to nitrite at pH 7.5 in a copper-cadmium reductor cell, nitrate is determined as the difference between the nitrite concentrations in the extract which was passed through the copper-cadmium reductor ce11 and that which was not.
3.14 DNA EXTRACTIONAND PREPARATION
Genomic DNA extraction fiom liquid cultures of isolates was performed using a standard chernical lysis method, see appendix D for procedure followed. The genomic DNA obtained fiom the liquid cultures was used as positive control DNA sarnples during PCR amplification.
There was extensive developrnent of the microbial analysis protocol for the environmental sarnples during this study. The extraction, purification and PCR amplification methods were al1 developed jointly to address the particular needs of this research. See section 5.1 in the results section for details regarding the development of the extraction and
PCR methodology. Listed here are the final methods used to deveIop population estimates.
The media samples for rnicrobial analysis were stored at -20°C in 15 ml disposable centrifuge tubes. Sarnples ranged fiom 3 to 5 grams wet weight. DNA was extracted using thermal shocking to physically lyse the cells. Five millilitres of TENP extraction buffer (50 rnM Tris, 20 mM EDTA, 100 mM NaCl, 1% polyvinylpolypyrrolidone) was added to the centrifuge vial, the vial was then altemated between a bath of boiling water (lOO°C) and a bath of ethanol and dry ice (-70°C) for three minutes each for three complete cycles. Three
(3) ml of chloroform/isoamyl alcohol was added to the centrifuge vial, mixed and spun. A one (1) ml aqueous sample was removed fiom the centrifuge tube and placed in a clean stenle 1.5 ml eppendorf tube to which 0.4 ml of chloroform/isoamyl alcohol was added, vortexed and centrifuged. The aqueous phase was placed in a second clean sterile 1.5 ml eppendorf tube and an equal volume of phenol in chiorofodisoamyl alcohol was added, vortexed and centrifuged. The aqueous phase placed in yet another clean sterile 1.5 ml eppendorf tube. This represented the crude DNA extract. This cmde extract was cleaned using a Sephadex spin column (see appendix D for spin column protocol). The crude DNA sample was placed in the top of the spin column, spun at 900 rpm and the cleaned DNA sample was collected in the outer 15 ml centrihige tube of the spin column. This cleaned
DNA sample was transferred to a 1.5 ml eppendorf tube. The DNA was precipitated by adding a 0.6 volume of isopropanol, the DNA was pelletized by centrifuging at 12000 rpm for 20 minutes. The supernatant was removed and the pellet was resuspended with sterile deionized water, the volume used was 1/10 the volume of cleaned DNA extract obtained from the Sephadex column.
3.15 PCR AMPLIFICATION
Nested PCR reactions, with the initial amplification using universal primers and the second reactions using primers specific to the T.thioparus and N-europaea,were performed on three replicates of 10 fold serial dilutions of the cleaned and concentrated DNA prepared as above. Positive controls were run using genomic DNA extracted from cultures of
T. llrioparus, N.europaea and Nb. winogradskyi, as weli a negative control with no template was also run with each set. A listing of the primers is given in appendix C, the development and selection of primers is reviewed in section 5.1.
The PCR reaction volumes were 25 p1 with 1.4 units of Taq DNA polymerase, 2.5 pl PCR buffer, BSA (0.1 mg/ml final concentration), formamide (2% final concentration),
dNTPs at ha1concentration of 0.3 mM each, 10 pmol of each primer, water to 24 pl and 1
pl sample. The PCR reaction used a 94OC denaturing temperature, 55°C annealing
temperature, and 72°C extension temperature. The thermal cycles were run using an
Eppendorf Mastercycler and consisted of an initial 4 minutes of denaturing followed by 30
cycles of 1 minute denaturing, 1 minute annealing 1 minute extension (the second PCR
reaction used 30 sec. extension instead of 1 min.) with a final 10 minutes of extension.
Products were run on a 1.2% agarose gel, stained with ethidium bromide and imaged.
The PCR reactions were considered successful if the appropriate bands were visible
in the positive controls and no bands were present in the negative control. PCR reactions for the dilution series of the environmental DNA were recorded as positive if the appropriate
band was present, negative if it was absent. The positive and negative scores for the various dilutions were used to estimate the population using MPN tables (Woomer, 1994).
3.16 PCR PRODUCTPURIFICATION
For band identification the PCR products were run on gels without any additional purification, but for sequencing and denaturing gradient gel electrophoresis (DGGE) analysis the PCR products were purified. A PCR purification kit fiom Qiagen (QtAquick PCR
Purification Kit @O), cat. no. 28 104) was used following the manufacturers instructions for the microcentrifuge method. This kit allowed for direct purification of double- or single- stranded PCR products between 100bp and 1Okb in length. This effectively isolates the PCR product fiom primers and genomic DNA.
3.17 SEQUENCCNCOF PCR PRODUCTS Samples were subrnitted to the Guelph Molecular Supercentre (University of Guelph) for sequencing. Sequencingwas conducted on an automatic sequencer (AB1 Prism 377 DNA
Sequencer, Applied Biosystems) using temiinator dyes (AB1 Prim BigDye Terminator, version 2).
Sequencing was conducted on the 16s rDNA fragments amplified by PCR using the universal primers Eu 49f and Eu 1070r. The PCR products were purified (as above) and concentrated to greater than 20 ng DNA pl -', a minimum sample of 80 ng DNA was required. A sample of each primer was also submitted at a concentration of 2 pmol pl-'. 4.0 EXPERIMENTALDESIGN
4.1 EFFECI-OF INOCULUMN
The initial biofilter study investigated the effect of inoculumn on the removal of
DMDS and ammonia. The airstreams to al1 eight biofilters were loaded with ammonia and
DMDS. Four different media treatments were used with a replicate of each. Two of the four treatments used fresh screened compost the other two used the same compost which was then sterilized by autoclaving for one hour on three consecutive days. One of the un-sterilized compost treatments used the compost directly. In the other unsterilized treatment, the compost was inoculated with cultures of T.thioparus, N. europaea, and N. winogradskyi.
Sixnilarly one of the sterilized treatments used sterilized compost which was inoculated while the second used sterile non-inoculated compost. To each biofilter five litres of the appropriate compost media was added. Air flow to the biofilters was set at 5.65 litres per minute, the ammonia concentration averaged 50 pprnv while the DMDS concentration was between 30 and 35 ppmv.
4.2 COMBINEDVERSUS INDIVIDUAL CONTAMINANT TREATMENT
To investigate the possible effects one contaminant might have on the treatrnent of the other, a study with some biofilters treating each gas individually as well as some treating them in combination were conducted. Six biofilters were used in this study, two treating
DMDS only, two treating ammonia only, and the final two treating both DMDS and ammonia. idet and outlet concentrations of DMDS were monitored daily by GC, the ammonia concentrations were monitored using gas detection tubes less fiequently, typically every other day. The biofilters were periodically stopped, sarnpled, repacked and put back in service.
The combined and individual study was nui three tirnes. The first experienced cold temperatures in the laboratory where column temperatures were as low as 13 OC, this study was dubbed 'The cold study7'. The second had better lab conditions, temperatures around
20°C. The term "combined and individual loading" is used to identiQ results originating fiom this shidy. The third replicate used media which had been allowed to air dry. After adjusting the moisture the media was inoculated as previously, the results from this study are identified as the "stored media"resu1ts.
43 LONGTERM COMBINED REMOVAL
At the end of the "cold study" the four columns which had been exposed to both
DMDS and ammonia (never having established removal of either), were allowed to establish
DMDS removal by turning the ammonia off. Once DMDS removal was established in al1 four columns they were stopped, the media fiom al1 four was mixed and the four columns repacked. Two of the four were loaded with DMDS only while the other two were also loaded with ammonia. The objective was to determine if ammonia loading would inhibit established DMDS removal. On day 77 of this run the two DMDS only columns were stopped to allow for the set-up of the second combined and individual study, the two columns receiving both DMDS and ammonia were continued to provide long term study of the combined removal.
4.4 NUTRIENTSUPPLEMENTATION EFFECTS ON DMDS REMOVAL
A study into the effects of various foms of nitrogen supplementation to the media was conducted using DMDS as the sole contaminant Four treatments, each with one replicate, were useci, a control of untreated compost, one supplemented with nitrate in the form of sodium nitrate, two treatments with ammonium sulphate one of which had a nitrification inhibitor added. The eight biofilters were each loaded with two litres of compost, 328 g dry compost per column and approximately 300 ml of water (to bring the compost to 60% of the water holding capacity). The nutrient additions were made to be equivalent to the ammonia holding capacity demonstrated during the start of earlier studies.
This adsorption capacity was determined based on the amount of water present in the column and was calculated to be 0.04 mol N per litre water in the column. Therefore 0.0 12 mol of
N was added to each supplemented biofilter, 0.994 g of NaNO, per column and 0.77 g
(NH4),S04 per column. The nitrification inhibitor used was sodium sulphate 2-chloro-6-
(trichloromethyl)pyridine, used to inhibit nitrification in BOD tests. Each column received
0.16 g of this inhibitor (based on the 300ml of water present in the colwnns this dose represents 50 times the recommended amount of 10 mg/L for BOD tests). The airflow rate was set to 3.5 litres per minute, the DMDS loading averaged 34 ppmv ranging fiom 32 to 38
PPrnV*
4.5 CERAMICMEDIA
A sterile media subsequently inoculated with pure cultures of T.thioparus,
N. europaea and N.winogradskyi would establish that these organisms are capable of treating the contaminants. Soils have been reported to be toxic to many organisms after autoclaving, the same is possible for compost, thus an inorganic ceramic media was used. The half inch ceramic saddles were autoclaved then aseptically transferred to 1 litre flasks with liquid media for one of the three organisms, these were then inoculated with the corresponding organism. The flasks were incubated until cultures were established. The ceramic media was transferred to biofilter columns which had been autoclaved, a mixture of al1 three cultures was added to each biofilter. Two biofilters were loaded with the inocuiated ceramic media and air, loaded with both arnmonia and DMDS, was passed üirough. The airflow and loading conditions were sirnilar to those of the combined loading expenment. 5.0 RESULTS
5.1 DEVELOPMENTOF PCR PROTOCOL
The specificity of any given PCR reaction is based on the primers. Primer sequences can be obtained from the literature, determined fiom known DNA sequences or based on sequencing the DNA of the target organisms if it is not already known. There were some specific primers for both N-europaeaand N.winograds& published in the literature. For
N-europaea there were primers for the genes for the metabolic enzyme arnmonia monooxygenase. Whiie these were obtained and tested they were abandoned in favour of primers targeting the 16s rDNA genes. The 16s rDNA genes contain non-variable sites, which can provide universal sequences for prirners, as well as highly variable regions where species specific sequences can be found. The extensive use of 16s rDNA for phlyogenetic studies of procaryotes has resulted in extensive databases of the sequences of this gene.
The 16s-rDNA sequences for T.rhiopums, N-europaeaand N. winogradskyi were obtained fiom the GenBank database. To identie unique sections of the DNA sequences small overlapping segments (40 base pairs long overlapping the previous segment by 10 base pairs) were submitted for BLAST searching using the GenBank database. BLAST searches identiq any holding in the database which matches the segment submitted, more importantly it also identifies near matches and indicates where the differences in the sequences occur.
Proceeding through the entire 16s-rDNA sequence allowed for the identification of the individual bases which have the highest degree of dissimilarity to other sequences in the database. Once the entire 16s sequences were mapped in this manner, potential pnmers were identified in those regions which had the highest concentration of dissimilar bases. The pnmers specific to the individual species were determined by selecting the segment which returned the fewest matches, ideally none. The universal primers were determined after the specific ones by identimg segments common to al1 of the sequences but which bracketed di of the specific primers identitied The finai primer sequences were based on the BLAST results and the melting temperature in degrees Celsius based on nucleotide content fo llowing the formula, valid for sequences up to 30 base pairs long;
[(Number of T and A) x 2]+ [(Number of G and C) x 41- 5 = Melting Temp. Ca(1)
The target was to have melting temperatures caiculated with equation 1 in the 50°C to 60°C range.
Table C 1 in appendix C contains a List of al1 the pnmers used in this study. The first primers used for PCR reactions were the universai primers Eu 23f and Eu 1070r, and the
ï3hioparu.s specific primers Ttp l94f and Ttp 833r. The PCR reactions were carried out in
50 pl volumes, with 2 units of Taq, 5 pl of PCR bufEer, dNTPs at a ha1concentration of O. 1 mM, O. lug of each primer, and water up to a volume of 49 pl. One microlitre of genomic
DNA fiom a culture of T.thioparus was used as a sample. The temperatures used in thermocycling were; denaturing 94OC, anneaiing SO°C, and extension 72°C. The thermal cycling program consisted of one cycle with 1O minutes denaturing, 2 minutes annealing, and
2 minutes extension followed by 29 cycles of 1 minute of each and a final 10 minutes of extension. Using this procedure on genomic DNA produced bands, but when T.thioparus cultures were first added to a compost sample and then the DNA extracted by the bead beating method, the above procedure failed to produce bands. Attempts to clean the DNA extracted from compost samples using the commercially available kit BI0 1O 1 had moderate success. The addition of BSA at a final concentration of 100pg/ml to the PCR reaction combined with the BI0 10 1 cleaning produced the best results, allowing amplification at a
10 fold dilution of the cleaned DNA sarnple.
While this combination of extraction, cleaning and amplification was successful, there were practical difficulties in applying it to samples fiom the biofilters. The first problem was related to the size and handling of samples in the bead beating extraction method. The bead beating vials had a total volume of 2 ml, into which beads, sample and extraction buffer were placed. The compost filter media was fibrous and some particles were too large to be placed in the bead beating vials, this resulted in either a selective and non-representative sample being used, or the addition of a grinding step to produce particles of a more suitable size. Gnnding of the samples would have to be done in a manner to prevent contamination of the samples with DNA fiom other samples as well as the lab. For these reasons the bead beating method of DNA extraction was abandoned in favour of thermal shocking, three cycles of 3 minutes at -70°Cfollowed by 3 minutes at 1 OO°C. Using this method samples could be collected in stenle disposable 15 ml centrifuge tubes sealed and stored at -20 OC until the time of extraction. The larger vials allowed non-selective sampling, sarnples ranged between 3 and 5 grams wet weight, a much more representative sample.
The basic extraction method, without a purification step, resulted in a crude DNA which was used directly for PCR analysis. With the BSA in the PCR reaction mix amplification products could be obtained from 1pl samples of 100 fold dilutions of the crude
DNA extract without additional cleaning. This method was used successfully to make population estimates from biofilter media samples, see appendix A. The problem was the high dilution required resulted in a detection limit of approximately 10' cells per dry gram compost To lower the detection limit a cleaning step was required.
The BI0 101 cleaning kit was designed for concentrated DNA extracts and small volumes (50 to 100 pl). Precipitating the crude DNA extracted as above resulted in co- precipitation of humic acids so the resulting pellet was difficult to resuspend. A cleaning method which worked on larger volumes (600 to 1O00 pl) worked directly on the crude DNA extract. Sephadex spin columns accornmodated these volumes. An additional advantage of the Sephadex columns was that they bound the impurities allowing the aqueous DNA to pass through, where as the BI0 101 columns bound the DNA allowing impurities to pass through. Thus the Sephadex columns produced cleaned DNA in one step while the BI0 101 required two, fust binding the DNA and then eluding it once the impurities were removed.
The aqueous DNA produced from the Sephadex columns was precipitated with a 0.6 volume of isopropanol and centrifiged for 20 minutes. The resulting pellet was dried and resuspended in 1/10 the original volume of water, thus making a 10 fold concentration increase. The DNA prepared in this manner could usually be arnplified by PCR without dilution, thus reducing the detection lirnit to an average of 100 cells per dry gram of compost.
inhibition due to humic acids was not the only problem encountered while developing the PCR procedure. Other problems included nonspecific amplification and failure to ampli@ anything. Nonspecific amplification can result in the presence of bands other than the ones expected or a smear of DNA without specific bands in the fuial gel. The causes can be due to the primers attaching to multiple binding sites or the conditions of the PCR reaction allowing for non-specific binding, i.e. the primer binds to a spot on the template
DNA which it does not match exactiy. The anneaiing temperature is the parameter whkh affects the specificity of the binding the most. Chernical additions to the PCR reaction mixture cmalso increase the specificity of the binding, for example formamide helps reduce non-specific priming. The failure to amplie can result from the omission or deterioration of any of the principal components in the PCR reaction mixture, the DNA polyrnerase, nucleotides, primers or template DNA, as well as the presence of any inhibitors. Isolating the specific cause can be very difncult.
In an attempt to reduce the number of PCR reactions required, a protocol based on the method presented by Bennett et al. (1996) was investigated. Bennett et al. ( 1996) used a single common fonvard primer and £ive distinct reverse primers, one for each species they were investigating. Al1 six primers were present in the PCR reaction mixture. The specific pnmers were designed so that in combination with the comrnon forward primer they each resdted in a band of a distinct length which could be distinguished from the other bands on a gel. Using Eu 49f (a slight modification of the original universal forward primer Eu 23f) as the common forward primer the three reverse primers used were; Ttp 240r (a reverse version of Ttp 194f), Nrn 459r and Nb 3 12r, specific for T.thioparus, N-europaea and
N.winogradskyi respectively. Only the T.thioparus band was consistent1y amplified. Since the primers themselves were implicated in the failure to obtain product from N.europaea and
ALwinogrudskyi new primers found in the literature were obtained; for N. winogradskyi primer FGPS 1269r fiom Degrange et al.(1995) and for N-europaea primer NmOr fiom
Pommerening-Roser et al. (1 996). This also required replacing the initiai reverse universal primer since the Nwinogradskyi primer now was beyond the Eu 1070r binding site, therefore, rP2 fiom Weisburg et al.(1 99 1) was used.
The failure to obtain consistent results caused a re-examination of the original primers Nm 459r and Nb 3 12r. Using the sequence analysis software Gene Runner it was found that both Nm 459r and Nb 312r produce thermally stable hairpin loops, they are capable of folding back on themselves binding into a hairpin loop, making them unavailable for priming. With the aid of Gene Runner and Blast searching of GeneBank these primers were modified by shifbg them a few base pairs. The resulting primers, Nm 448r and Nb
300r, were ikee of any stable hairpin loops and remained selective for Neuropaea and
Nwinogradskyi: respectively. The 2Mioparu.s primer was changed fiom Ttp 240r to Ttp
833r to produce a band more distinctive fiom the N winogradskyi.
The initial PCR products shown in Figure 4 were generated using two universal primers (Eu 49f and Eu 1070r). The secondary PCR products were obtained using one universal primer (Eu 49f) and one specific primer, (Ttp 833r for T. thiopanrs, Nm 448r for
N-europaea,Nb 300r for h! winogradskyi). The product resulting fiom EU 49f and Eu 1070r is visible in lanes 2 thru 5 (lane 5 is the negative control, indicating some contamination).
The product resulting fiom primers Eu 49f and Ttp 833r is a band approximately 785 base pairs in size, bands of this size are seen in lanes 8 and 12. The use of primers Eu 49f and Nm
448r should result in a product 400 base pairs in size, lanes 16 and 19 show bands of this length. A 250 base pair product is expected fiom an amplification using primers Eu 49f and
Nb 300r, bands of this size are seen in lanes 22 thru 26. Positive signals, in the form of appropriate sized bands, for T. thioparus and Ns. europaea were obtained using a single specific primer and a universal primer, both directly fiom genomic DNA (lanes 12 and 19) and where there had ken an initial PCR amplification of the target sequences (lanes 8 and Figure 4: Electrophoresis gel image showing first and second PCR products. First PCR products (2 to 5) using samples of T.fhioparus, N.europaea, N. winogradskyi and a blank respectively, (6) empty well. Second PCR oroducts (8 to 13) using T.thioparus specific primer, (15 to 20) using Ns. europaea specific primer and (22 to 27) using N. winogradskyi specific primer. DNA fiom the four initial PCR reactions (2 thru 5) were used, in order, as the fust four samples in each second PCR set, the fifth sample was the genomic DNA corresponding to the specific primer being used, the sixth sample was a blank. Ladders, (1)l Kb ladder, (7,14 and 21) 100 bp ladder.
16). The absence of these positive signals in the other lanes indicates that the primers do not ampli@ a corresponding sequence in the DNA present in those reactions. This includes the contaminating DNA amplified in lane 5 and transferred to reactions run in lanes 1 1 and
18, indicating that the contamination is not T. thioparus nor N europaea. The presence of the band corresponding to N winogradkyi in lane 25 suggests that N. winogradskyi could be the contaminating organism, if not, the source of contamination generates a similar size band using the Eu 49f and Nb 300r primers. While the bands in lanes 21,22, and 25 seem to be of a slightly longer base pair sequence than those in lanes 24 and 26, the lanes with
N.winogradskyi DNA present, they are not distinguishable enough to be used for indentification. The contamination persisted and the N. winogradskyi was eliminated Erom the analysis.
One variation fiom the method of Bennett and colleagues (1 996) used here was that the multiplex PCR was the second of a nested set, with Eu 49f used as the forward primer in both. One consequence of using a common primer in both the initial and second PCR reactions is that the common primer will have a binding site on al1 of the amplification products fiom the first PCR. Thus there will be continued amplification, albeit not exponential, of ail of the initial amplification products. This cm affect the amplification of the specific sequence targeted in the second PCR by using reagents, in particular reducing the amount of the common primer available for the second amplification. This reduction in reagents, especially primer, can prevent the desired amplification fiom occurring near the theoretical maximum exponential rate. A second consequence of the continued amplification of the initial PCR products is that the bands corresponding to those products will persist. A strong band corresponding to the initial PCR product can be seen in al1 secondary PCR lanes except lanes 12, 19, and 26 where the template DNA was genornic instead of resulting from an initial PCR reaction.
The non-specific bands and extensive smearing of DNA present in the gels of these
PCR products prohibit the combining of these primers in a single PCR. It would be impossible to be certain of the source of any individual band. Since individual PCR reactions would be required for each species to be identified the second PCR reactions could use two specific primers instead of one plus a universal. Additional primers specific to
T.thioparus and Meuropaea were selected to replace Eu 49f as the forward primer in the second PCR reactions. The primer Ttp 223f (a slightly modified Ttp 194) when combined with Ttp 833r amplifies a 6 10 base pair sequence in the 16s rDNA of T.thioparus. The primer NmO l9Of (reported by Pommerening-Roser et al. 1996) when combined with Nm
44th amplifies a 258 base pair sequence in the 16s rDNA of Ns-europaea. By incorporating these two new primers the double PCRs have become tmly nested. Each PCR reaction uses two distinct primers, the second sets, one set for each species, "nested" within the sequence amplified with the universai primers during the fist PCR reaction.
To confirm that the sequences of the strains being used rnatched the sequences obtained fiom GeneBank the PCR products from the initial PCR using Eu 49f and Eu1070r were subrnitted for sequencing. The sequence data obtained is included in appendix C.
Complete sequences were obtained for each of the three species, ThiobacilZus thiopams,
Nitrosornonas europaea and Nitrobacter winogradskyi. The sequence information for
T.thioparur and N-europaeaconfirmed the sequences of theirrespective specific primer pairs
Ttp SS3f and Ttp 833r and NmO WOf and Nm 448r. Sequence data for the N. winogradskyi culture demonstrated some discrepancy with the sequence of primer Nb 300r. The sequencing results indicate two additional bases within the primer binding site and a thymine substituting one of the adenines of the primer. The sequence data obtained are listed in appendix C.
Figure 5 shows the amplification products using the primer pairs NmO l9Of with Nm
448r and Ttp 223f with Ttp 833r. The gel shows the resdts of single-direct PCR where the template DNA was genomic as well as product fiom the second PCR of a nested set where the template was fiom an initial PCR reaction using universal primers Eu 49f and Eu 1O7Or.
Lanes 2 thm 6 show the products using the N.europaea specific primers, the appropriate band at approximately 250 base pairs is clear both when genomic DNA was used as template (lanes 2 and 3), and when product nom an initial PCR reaction is used as template (lanes 4 and 5). Lanes 7 thru 1 1 show the products using the T.thioparur specific primers, again both when genomic DNA (lanes 7 and 8) and product fiom an initial PCR reaction (lanes 9 and
10) are used as template the expected product of approximately 600 base pairs is observed.
The sharpest contrast between Figures 4 and 5 is the clarïty of the bands in Figure 5.
Comparing the nested results in Figure 5 to Figure 4 the bands corresponding to the initial
PCR product, which are the dominant bands in Figure 4, are absent in Fgure 5. The extensive smearing present in figure 4 is also absent in Figure 5.
This tme nested PCR method with specific primers for T.thioparus and
Nwinograh@i was used on MPN dilution series to determine their respective populations in the biofilter samples. For the population estimates the PCR reaction volumes were 25 pl with 1.4 units of Taq DNA polyrnerase, 2.5 pl PCR buffer, BSA (0.1 mg/ml final concentration), formamide (2% final concentration), dNTPs at final concentration of 0.3 mM each, 10 pmol of each primer, water to 24 pl and 1 pl sample. The PCR reaction used 94°C denaturing temperature, 55°C annealing temperature, and 72°C extension temperature. The thermal cycles were run using an Eppendorf Mastercycler and consisted of an initial 4 minutes denaturing followed by 30 cycles of 1 minute denaturing, 1 minute annealing 1 minute extension with a final 10 minutes extension. Products were run on a 1.2% (w/v) agarose gel, stained with ethidium bromide and imaged. Figure 5: PCR results using specific primers for N. europaea and T.thioparus. Lanes 2 thru 6 amplification using primers NmO 190f and Nm 448r specific for Neuropuea. (2) N-europaea genomic extract, (3) 10 fold dilution of N.europaea genomic extract, (4) product fkom an initial PCR amplification of N.europaeo, (5) a 10 fold dilution of the initial PCR product, (6) blank. Lanes 7 th11 amplification using primers Ttp 223f and Ttp 833r specific for 22hioparu.s. (7,8) T.thiopums genomic DNA at full strength and 10 fold dilution respectively, (9,l O) product of an initial amplification of T.hioparus genomic DNA using universai primers the product was used undi tuted and at a 10 fold dilution respectively, (1 1 ) blank. Ladders, (1) 100 bp plus, (1 2) 1 kb ladder. Images of ali the gels used in determining population estimates are shown in
Appendix A. Figures Al to A17 show the results fiom the true nested PCR method, where two specific primers were used in the second PCR reactions. Figures A 18 to A24 show the gels fiom the pseudo-nested PCR method, where the forward universai primer was used in both the initial and second PCR reactions. Population estimates are made using the sconng show under the gels, each lane in the gel is scored based on the presence (positive score) or absence (negative score) of the desired band. A MPN population estimate is determined fiom tables (Woomer, 1994) using the number of positive scores at each dilution level in the series. The tabular MPN value is converted to an estimate for the sample based on the media sample size, the volume of extraction baer used, the initial dilution for which positive scoring occurred and the sample volume added to the PCR vials. These values and the resulting population estirnates for each of the media sarnples are listed in tables Al through A4.
The nested PCRs were conducted on extracted DNA which had been cleaned using
Sephadex spin colurnns, while the pseudo-nested PCRs were conducted on crude DNA extracts with no Mercleaning. The detection limit using a 10 fold senes dilution with three replicates for the crude DNA extracts was 10' (log 5.0) cells per gram dry compost
(based on average sample weight), while the detection limit for the cleaned DNA was 100
(log 2.0) cells per gram dry compost (based on average sample weight). Comparing population estimates of cleaned and crude DNA sarnples fiom standards indicates that some
DNA is lost in cleaning, population estirnates for cleaned DNA are lower than those for crude extract. There was also a marked difference in the percentage of failed reactions between the pseudo-nested and true nested PCRs. A PCR is assessed as failing when the expected band is not present in the positive controls, or when bands are present in the negative control. The presence of additional bands in the environmental sarnples or any of the controls could interfere with assessing the gel if the bands are close in size to the expected band and hence not easily distinguishable. Faint and poorly defined bands could also prevent clear interpretation of the gel. During the period that the pseudo-nested technique was being used ody approxirnately 40% of the PCRs conducted resulted in successfiil population estimates.
The success rate improved to 95% when the true-nested procedure was used. While many other factors were involved, the true nested method did seem to be more robust and less sensitive to PCR conditions. For these reasons only the me-nested PCR results were used for cornparison between the different treatrnents and for the calcuiation of activity rates.
Standards for population estimation were conducted b y spiking compost samples with some liquid culture of the various isolates. The concentration of the culture was previously determined by direct counting under the light microscope. The inoculurn strength was calculated based on the estimate of the number of ce[ls added and the dry weight of the compost sample. Table 4 summarizes population estimates for the controls. The inoculum size was calculated by direct counts on the liquid cultures prior to inoculation. Table 4: Siimmary of population estimates for standards. T.thioparus Population Estirnates Using True-Nested PCR, [log (cells/g-dry media)] Cnide Extract Cleaned Extract Inocuiumn - Species (log celldg) Pop. 95% Con. ht. Pop. 95% Con. Int. Estimate Estimate & 95% confidence Lower Upper Lower Upper
Ttp (320.1) Not m 3 -8' 3.5 4.2 Blank ~o~ulationestimate performed ushg two fold dilution series instead of standard ten fold dilution series. hoculating the filter media shortened the time before biological rernoval was established in biofilters receiving both DMDS and ammonia loading. Comparing the removal rates for the inoculated columns to the rernoval rates for the non-inoculated columns, Figures 6 through 9, the inoculated columns established removal sooner than the non-inoculated. The inoculated column A, which was re-inoculated on day 6, attained 60% removal of DMDS by day 15. Inoculated column B, which only received the initial inoculation, attained 60% removal of DMDS on day 27. The non-inoculated columns A and B attained 60% removal on days 29 and 31 respectively. The stenlized media failed to establish biological removal even after inoculation, the peaks in ammonia removal evident in Figure 10 can be attnbuted to water additions on days 7,21 and 43. 82 During the combined and individual loading experiment al1 of the columw were loaded with media which had been inoculated. Al1 columns commenced biological removal about day ten. The ammonia and DMDS removal efficiencies for the combined loading columns are showin Figures 1I and 12. Cornparison of the DMDS removal for the DMDS only and combined colurnns is shown in Figure 13. More DMDS is removed by the biofilters receiving ammonia loading as well. The average cumulative removal for the columns receiving ammonia is 37.7 g-S per colurnn after 99 days (Figure 19), compared to 24.5 g-S per column &er 99 days for the columns receiving DMDS only (Figure 20), thus the ammonia addition resulted in a 54% increase in DMDS removal. For most of the study ammonia removal was 100% though an initial dip in removal is observed for the columns also loaded with DMDS, as show in Figure 14. While the arnrnonia removal was similar for the combined and arnmonia only treatrnents the speciation of the inorganic nitrogen accumulating the filter media was drastically different. Figures 15 and 2 6 show the nitrogen accurnuIation in the media for the combined and ammonia only biofilters, respectively. In the biofilters receiving both DMDS and ammonia, amnonia accounted for al1 of the inorganic nitrogen found in the media. For the filters treating arnmonia only both nitrate and nitrite were observed to accumulate in the filter media as well as ammonia. The cumulative amount of nitrogen and sulphur removed fiom the air was calculated using the air flow rate and the inlet and outlet concentrations. These cumulative removals were compared to the nitrogen and sulphur determined to be accumulating in the media based on the media analysis. For nitrogen, both the inorganic (ammonia, nitrate and nitrite combined) and the total nitrogen (Kjedhal nitrogen plus nitrate) were compared to the nitrogen removed from the air. These results are show in Figures 17 and 18 for the combined and ammonia only treatments respectively. The total sulphur accumulating in the media is compared to the sulphur removed nom the airstreams for the combined and DMDS only treatments, Figures 19 and 20 also demonstrate the greater removal of DMDS when the air is also loaded with arnrnonia. Media analysis results are given in Appendix B. In al1 columns the pH of the media dropped fiom the onginal value of 8.3. The greatest drop in pH occurred in the biofilters treating DMDS alone, with fuial pH values of 4.0 and 3.9 for the two replicates. Despite removing a greater amount of DMDS the combined columns experienced a smaller drop in pH, final pH values for the combined columns were 6.1 and 5.5 for the two replicates. The rnost modest drop in pH occurred in the columns treating ammonia only, with final pH values of 6.7 and 6.5 for the two replicates. The MPN population estimates are plotted against time for the combined and individual treatments in Figures 21 and 23, for T.thioparus and Neuropaea respectively. Activity rates based on these population estimates and removal rates at the tirne of sampling are shown in figures 22 and 24, for T.thioparus and Meuropaea respectively. The removal efficiencies for cornbined and individual loadings of DMDS and arnmonia for biofilters whose media had been stored air dry prior to use are shown in Figures 25 and 26. For both amrnonia, Figure 25, and DMDS, Figure 26, removal was only established for the individual loading condition, the observed spike in ammonia removal for the combined treatment corresponds to the addition of water on day 33. The effect of nutrient supplementation on DMDS removal is reported in Figure 28. The initial erratic results, including some negative removds, were caused by leaks in the delivery and distribution system. This is demonstrated by the variable inlet concentrations show in Figure 27. The system was shut down for a day while repairs were made, higher and more consistent inlet concentrations were maintained for the remainder of the study. For the ceramic media which had been sterilized and then inoculated some removal of amnonia was observed, though not sustained, and very Little DMDS removal occurred, Figure 29. Under cold laboratory conditions only DMDS removal was observed, and only when DMDS was the sole contaminant. No ammonia removal was observed during the coId study for either the ammonia only or the combined loading condition. Once the ammonia was tumed off DMDS removal was established in those columns which had previously been receiving both, Figure 30. Figures 3 1 and 32 show the continued performance of the four biofilters which had established DMDS removai at the end of the cold study. Two of the four continued receiving only DMDS while the other two received both ammonia and DMDS. There was no obvious effect of ammonia on the established DMDS removal. DMDS removal showed drastic fluctuations in both the DMDS only biofilters and those treating both DMDS and ammonia. On day 77 of this study the DMDS only biofilters were terminated to allow for the set-up of the second cornbined and individual loading study. Table B5 in appendix B shows the media analysis for this long term combined removal study, again amrnonia accounts for al1 of the inorganic nitrogen accumulating in the filter media of the biofilters treating both ammonia and DMDS. The final ammonium concentration in the media was 23 g-NH,-N kg-' dry media, this represents the highest inorganic nitrogen accumulation in the media observed during this research. A sununary of the experimental conditions and removal rates attained for al1 of the experiments are contained in tables 5 and 6. A maximum and an average removal rate is reported for each experimental condition of each study. Maximum removal rates represent the highest removal rate observed during the study while the average removal rate is the average of ail the individual removai rates determined during the study. Removal rates for DMDS are listed in table 5 while removal rates for ammonia are listed in table 6. The maximum removal rate of 1.7 g S-' kg-' (dry media) for DMDS was obtained with sodium nitrate addition during the nutrient supplementation experiment. For the ammonia loading al1 of the expenments achieved 100% removal due to initial absorption of ammonia, so the maximum removal rates represent the loading rate. Differences in maximum removal rates is principally due to differences in the amount of media present in the columns. The overall maximum removai rate for ammonia was 0.30 g-N d-' kg-'(dry media), obtained during the stored media study. lnoculumn Effects on Removal Unsterile inoculated A O 10 20 30 40 50 60 70 Time (days) + DMDS +Ammonia k Figure 6: Removai of DMDS and ammonia by biofilter with unsterilized-inoculated media, replicate A. Column was re-inoculated with T.thioparus on day 6. - lnoculumn Effects on Removal Unsterile inoculated 6 O IO 20 30 40 50 60 70 Time (days + DMDS -Ammonia - Figure 7: Removal of DMDS and ammonia for biofilter with unsterile-inoculated media, replicate B. Inoculumn Effects on Removal Unsterilized A 30 40 Tirne (days) + DMDS - Ammonia Fipre 8: Removal of DMDS and ammonia for biofilter where media was neither stenlized nor inoculated, replicate A. Inoculumn Effects on Removal Unsterilized B 30 40 Time (days) +DMDS + Ammonia Fipre 9: Removal of DMDS and amrnonia by biofilter with media which was neither sterilized nor inoculated, replicate B. Inoculumn Effects on Removal Sterilized Media 20 30 40 Tirne (days) + DMDS Inoculated +Ammonia Inoculated -a- DMDS No Inoculation * Ammonia No Inoculation Figure 10: DMDS and ammonia removal for biofilters with sterilized media, comparing performance between those which were inoculated after sterilization and those which were not. Each treatment was run in duplicate, results are average of replicate biofilters. Combined and Individual Loading Combined Loading, replicate A O 20 40 60 80 100 120 Time (days) - DMDS * Ammonia I Figure 11: DMDS and ammonia removal for biofilter with combined loading, replicate A. - Combined and Individual Loading Combined Loading, replicate B O 20 40 60 80 100 120 Time (days) - DMDS * Ammonia - Figure 12: DMDS and ammonia removal for biofilter with combined loading, replicate B. Combined and Individual Loading DMDS Removal O 20 40 60 80 1O0 120 140 Time (days) o Combined (A) + Combined (B) DMDS (A) - DMDS (B) - Figure 13: Cornparing DMDS removal between biofilters receiving both DMDS and ammonia loading and those receivhg DMDS loading only. Combined and Individual Loading Ammonia Removal O 20 40 60 80 1O0 120 Time (days) -0- Combined (A) -+- Combined (B) * Ammonia (A) + Ammonia (B) Figure 14: Comparing ammonia removal between biofilters receiving both DMDS and ammonia Ioading and those receiving ammonia loading only. Media Analysis for Inorganic Nitrogen Combined Loading Time (days) Figure 15: Ammonium, nitrate and nitrite anaiysis of media sarnples from biofilters treating both ammonia and DMDS, results are average fiom two repiicate Media Analysis for Inorganic Nitrogen Arnmonia Only Loading 13 41 Time (days) Figure 16: Ammonium, nitrate and nitrite analysis of media samples fiom biofilters treating ammonia only, results are average of two replicate columns. Nitrogen Accumulation For Combined Treatrnent 13 41 72 99 Tirne (days) Inorganic N Total N N Removed Fipre 17: Accumulation of inorganic and total nitrogen compared with cumulative nitrogen removal for bio filters receiving both DMDS and ammonia loading, results are average fiom two replicate columns. Nitrogen Accumulation Ammonia Only Treatment 13 41 Time (days) Inorganic N Total N N Removed Figure 18: Accumulation of inorganic and total nitrogen compared with cumulative nitrogen removd for biofilters receiving only ammonia loading, results are average from two replicate columns. Sulphur Accumulation For Combined Loading 13 41 Tirne (days) Total S S Removed Figure 19: Comparison of sulphur accumulated in filter media with cumulative removal of sulphur fiom air for biofilten receiving both DMDS and ammonia loading, results are average for two replicate columns. Sulphur Accumulation DMDS Only Loading 13 41 The(days) S Removed Figure 20: Comparison of sulphur accumulated in filter media and sulphur removed from air for biofilters treating air loaded widi DMDS only, results are average for two replicate columns. Population density Thiobacillus thiopams 40 Time (days) 0 DMDS and Ammonia DMDS only - - Figure 21: population density of T.thiopams in compost biofilters treating DMDS either alone or in combination with ammonia, 95% confidence intervais are indicated. Activity Rates for T.thioparus in Compost Biofilters 40 Time (days) DMDS and Ammonia DMDS only Figure 22: Activity rate for Zthioparus in compost biofilters loaded with DMDS and arnmonia or DMDS alone, activity based on DMDS removal fiom air, 95% conficence intervals indicated. Population density Nitrosornonas europaea 40 The (days) DMDS and Ammonia Ammonia only Figure 23: Population estimates for N-europaea in compost biofilters treating ammonia either done or in combination with DMDS, 95% confidence intervals are indicated. Activity Rates for N.europaea in Compost Biofilters 40 Time (days) DMDS and Ammonia Ammonia only Figure 24: Activity rates of N-europaea in compost biofilters treating ammonia either alone or in combination with DMDS, activity is measured as removal of ammonia fiom airstrearn, 95% confidence intervals are indicated. Combined and Individual Treatment Stored Media, Ammonia Removal 30 40 Time (days) --Combined (a) - -- Combined (b) -Ammonia (a) - Ammonia (b) Figure 25: Ammonia removal for inoculated compost biofilters receiving either DMDS and amrnonia (combined) of ammonia only (ammonia). Media had been stored air dry prior to use. DMDS loading was stopped on day 44 to promote ammonia removal in combined treatements. Combined and Individual Treatment Stored Media, DMDS Removal 30 40 Time (days) -c- Combined (a) - -- Combined (b) - DMDS (a) a- DMDS (b Figure 26: DMDS removal for inoculated compost biofilters receiving either DMDS and arnmonia (combined) or DMDS only (DMDS). Media was stored air dry prior to use. DMDS was turned off on day 44 to promote amrnonia removal in combined treatments. Nutrient Supplementation Study DMDS Inlet Concentrations 40 35 30 25 20 15 10 5 O O 15 30 45 Time (days) -COI. 1 - 4 - Col. 5 - 6 - - Col. 7 - 8 i Figure 27: Met concentrations of DMDS for nutrient supplementation study. Columns 1-4 were supplied by a cornmon air line, columns 5 and 6 a second and a third supplied columns 7 and 8. Nutrient Supplementation Study DMDS Removal 15 30 Tirne (days) -untreated --(NH4)2S04 Figure 28: DMDS removal as percentage of inlet concentration for various nutrient supplementation. Each series is the average of two columns, untreated controls (columns 1 and 2), sodium nitrate (columns 3 and 4), ammonium sulphate (columns 5 and 6) and ammonium sulphate with inhibitor (columns 7 and 8). Ceramic Media Sterilized and Inoculated O 5 10 15 20 25 Time (days) * Amrnonia (a) * Ammonia (b) DMDS (a) -DMDS (b) Figure 29: Ammonia and DMDS removaÏfor biofilter packed with ceramic saddle packing which had ken sterilized then inoculated with Tthiopa~s,N etrropaea and M.winogradskyi. DMDS Removal Under Cold conditions O 20 40 60 80 Time (days) + Combined (A) -s+ Combined (B) -+- Combined (C) + Corn bined (D) + DMDS Only (A) -E)- DMDS Only (b) -Tempertaure (Celcius) Figure 30: DMDS removal when sole gas (DMDS only) and when combined with arnmonia (combined) under cool lab conditions. Ammonia was tumed off on day 60. Combined A and B had not been inoculated, while combined C and D had. Long Term Combined Removal Inlet and Outlet DMDS Conc. 60 80 100 120 140 160 180 Time (days) -Outlet DMDS and arnmonia - Outlet DMDS only + Inlet DMDS and ammonia -c- Inlet DMDS only Figure 31: DMDS iniet and outlet concentrations for biofilters initially acclimatized to DMDS loading only. On day 77 DMDS only biofilters were discontinued. Long Term Combined Removai Inlet and Outlet Ammonia Conc. 20 40 60 80 100 120 140 160 180 Tirne (days) - Inlet ammonia --Outlet ammonia Figure 32: Met and outlet ammonia concentrations for biofilters initially acclimatized to DMDS loading only. Ammonia loading was initiated on day 0. Table 5: Summary of maximum and average sulphw removal rates for al1 studies. Treatment Experimental Conditions DMDS Removal Rates g-S 6' kg*'(dry media) 1 Effect of Inoculumn Study ------Inoculuated media Media per col. = 1O00 g Max removal = 0.42 I Air flow rate = 5.6 1 min-' Ave removal = 0.19 Met Conc- Non-inoculated media DMDS = 32 pprn Max removal = 0.56 Ave removal = 0.19 1 1 Amrnonia = 50 pprn Autoclaved media Max removal = 0.1 1 Ave removal = 0.0039 I - Autoclaved then inoculated Max removal = 0.068 Imedia Ave removal = 0.0048 1 Combined and Individuil Loading Study DMDS and Amrnonia Media per col. = 789 g Max removal = 0.95 ' Loading Air flow rate = 4.3 1 min-' Ave removal = 0.44 hiet Conc. DMDS = 42 pprn Ammonia = 43 pprn DMDS Loading Only Media per col. = 944 g Max removal = 0.5 14 Air flow rate = 4.3 1 min" Ave removal = 0.24 inlet Conc. = 42 pprn Untreated Media per col. = 257 g Max removal = 1.2 Air flow rate = 3.5 1 min-' Ave removal = 0.63 I Inlet Conc. = 34 pprn Sodium Nitrate Addition Media per col. = 280 g Max removal = 1.7 Air flow rate = 3.5 1 min-' Ave removal = 0.94 Met Conc. = 34 pprn Ammonium Sulphate Media per col. = 272 g Max removal = 1.2 Addition Air flow rate = 3.5 1 minc' Ave removal = 0.72 I Met Conc. = 34 pprn Table 5 cont.: 1 Expcrimeital- Conditions 1 DMDS Removnl Rates 1 g-S d " kg -'(dry media) r I 1 Stored Media Shidy DMDS and Ammonia Media per col. = 458 g Max removal = 0.14 Loading Air flow rate = 3.5 1 min" Ave removal = 0.070 Inlet Conc. DMDS = 5 1 pprn Ammonia = 54 pprn -- r Media per col. = 453 g Max removal = 0.83 Air flow rate = 3.5 1 min-' Ave removal = 0.40 Idet Conc. = 44 pprn DMDS Loading Only Media per col. = 994 g Max removal = 0.27 Air flow rate = 5.5 1 min-' Ave removal = 0.096 I Inlet Conc. = 29 pprn DMDS and Ammonia Media per col. = 994 g Max removal = 0.10 Loading Air flow rate = 5.5 1 min-' Ave removal = 0.0097 Inlet Conc. DPvlDS = 29 pprn After arnmonia turned off Ammonia = 35 pprn Max removal = 0.5 1 Ave removal = 0.1 0 1 Long Term Combined Removal Study -- - DMDS and Ammonia Media per col. = 861 g Max removal = 0.72 Loading Air flow rate = 4.5 1 min'' Ave removal = 0.26 Inlet Conc. DMDS = 41 pprn Amrnonia = 35 pprn -- DMDS Loading Only Media per col. = 861 g Max removal = 0.52 Air flow rate = 4.5 1 min" Ave removal = 0.22 I Met Conc. = 39 pprn Table 6: Summary of maximum and average ammonia removal rates for al1 studies, in al1 cases maximum removal represents maximum loading. Treatment Experimental Conditions Ammonia Removal Rates g-N d-' kg-'(dry media) / Effect of Inoculation Study .- - - -- Inoculated media Media per col. = 1000 g Max removai = 0.23 I Air flow rate = 5.6 1 min" Ave removal = 0.19 Met Conc. = 0.23 Non-hoculated media DMDS = 32 pprn Max removal = Ammonia = Ave removal 0.15 I - .. - - - - 50 ppm Autoclaved media Max removal = 0.23 I Ave removal = 0.067 Autoclaved then inoculated Max removal = 0.23 Imedia Ave removal = 0.063 1 Combined and Individual Loading Study Media per col. = 789 g Max removal = 0.22 Air flow rate = 4.3 1 min" Ave removal = 0.17 Met Conc. DMDS = 42 pprn Ammonia = 43 pprn Ammonia Loading Only Media per col. = 808 g Max removal = 0.22 I 1 Air flow rate = 4.3 1 min-' Ave removal = 0.1 8 Inlet Conc. = 43 pprn I 1 Stored Media Study .- .-- - .. DMDS and Amrnonia Media per col. = 458 g Max removal = 0.28 Loading Air flow rate = 3.5 1 min-' Ave removal = 0.079 Inlet Conc. After DMDS turned off DMDS = 5 1 pprn Max removal = 0.14 Ammonia = 54 pprn Ave removal = 0.049 Ammonia Loading Only Media per col. = 474 g Max removal = 0.30 Air flow rate = 3-5 1 min" Ave removal = 0.24 Inlet Conc. = 42 pprn Table 6 cont.: Treatment Experimental Conditions Ammonia Removal Rates g-N d -'kg -'(dry media) Long Term Combined Removal Study DMDS and Ammonia Media per col. = 86 1 g Max removal = 0.21 Loading Air flow rate = 4.5 1 min" Ave removal = 0.13 inlet Conc. DMDS = 4 1 ppm Ammonia = 35 ppm w A 6.0 DISCUSSION 6.1 PCR The true-nested PCR method produced much better results than those obtained using the pseudo-nested method. The species specific bands were much swnger than in the pseudo-nested gels. When other bands were present the species specific bands were more intense with the tme-nested method, where as under the pseudo-nested method often ail the bands were of comparable intensity. The poorer quality and higher incidence of failure resulted in abandoning the pseudo-nested method in favor of the tme-nested PCR rnethod. The population estirnates for the spiked controls indicated that cleaning the DNA sample caused a reduction in the amount of DNA. Where both crude and cleaned samples were used the cleaned samples always resulted in a lower population estimate though they were not statistically different at the 95% confidence level. For 72hioparu.s at the highest inoculation rate the population estimate was low, an estimate of log 5.2 k0.7 cells g-'for the cleaned extract at an inoculation rate of log 7.3 *0.1 cells g-'. At the moderate inoculation rate, log 5.2 &O. 1 cells g -', the estimate for the cleaned extract was log 5.0 I0.7 cells g *'. The lowest inoculation rate of T.thioparus used as a standard was log 3 -2*O. 1 cells g -', only the cleaned extract produced PCR amplification products. Using a ten fold dilution senes the population estimate was log 4.0 I0.7 cells g -' while a two fold dilution senes resulted in a population estirnate of log 3.8 I0.3 cells g -'. For Neuropaea only the highest inoculation rate was successflllly run, again the population estirnate was low, an estimate of log 4.2 I0.7 cells g-' for the cleaned extract at an inoculation rate of log 6.6 *O. 1 cells g-'. The detection Iimit for the MPN-PCR methodology used deterrnined based on the average size of environmental sample and the lowest MPN score (only one positive at no dilution) is log 2.0 *0.7 cells g-' dry media The results at the lowest inoculation rate for Tthioparus indicates that accurate population estimates near the detection limit are possible. The upper detection limit for the MPN-PCR methodology based on the average size enviro~unentalsample and the highest MPN score (only one negative at the highest dilution, IO6) is log 8.5 I0.7 cells g-' dry media. The highest population estimate obtained fkom the environmentai samples was log 8.1 *0.7 T.thiopams cells g " dry media. While the resuits for the spiked controls indicate that the populations may have been underestirnated at the higher densities there were differences between the spiked standards and the environmental samples. For the populations which develop within the operating biofilters growth occurs under conditions which preferentialiy select organisms, causing a population shift, thus the nurnber and diversity of other microorganisms will decrease. This is in contrast to the spiking of compost media, where the inoculum is added to the existing population without any corresponding reduction in the number or diversity of the other species. This higher background population could affect population estimates of the higher standards. 6.2 OVERALLPERFORMANCE OF BIOFILTER STUDIES The overall performance of the biofilters are sumrnarized in tables 5 and 6. Al1 removal rates and population estimates were based on a uniform activity throughout the biofilter, samples were taken after the media in the columns had been thoroughly mixed. This prevents any observation of variation in activity with height within the columns. The depth of media was always 20 cm or less. The removal rates achieved are reported in two different manners. The maximum removal achieved during the study represents the peak removal. For DMDS removal the maximum removal ody occurred for a short period of time and in aii but one case (combined treatments during the combined and individual study) was less than the loading rate. The maximum removal of DMDS achieved, 1.7 g-S d " kg -' (dry media), was during the nutrient supplementation study for the biofilter media which had sodium nitrate added as a nitrogen source. This is comparable to the maximum removal rate of DMDS by a compost biofilter achieved by Smet et al. (1996a) of 1.3 g-S d-' kg -' (dry compost). The maximum removal of DMDS achieved by Cho et al.(1 99 1) for a peat biofilter, 0.68 g-S d-' kg-' (dry peat), is comparable to the maximum removals obtained in the studies conducted here without nutrient supplementation. For the studies where biological removal of DMDS was established (excluding the cold study) the range of maximum removals was nom 0.5 1 to 0.95 g-S d -' kg -' (dry media). For ammonia removal, the maximum removal usually persisted for the majority of the study and in al1 cases corresponded to the loading rate. The maximum arnmonia removal achieved during this research corresponded to the highest loading rate, 0.30 g-N d-'kg-' (dry media), this loading was applied to the biofilter treating amrnonia only during the stored media study. Smet er al. (2000) achieved a maximum amrnonia removal rate of 0.83 g-N d-' kg '' (dry compost) while investigating high amrnonia loading of biofilters. The average removal is the average of al1 the removal rates determined during the study. For ammonia the range of average removal rates was 0.079 to 0.24 g N d " kg -' dry media, while for DMDS removal the average range was 0.0039 to 0.94 g S d -' kg -' dry media. What the removal rates do not indicate is the mechanisms involved in the observed removals. 63 PHYSICALCHEMICAL TREATMENT VS BIOLOGICAL TREATMENT Bioflltration relies on physical absorption to transfer the contamuiants fiom the air phase to the liquidlsolid phase, but once there biological transformations are necessary for continued activity of the bed. At any instant it is impossible to detennine if the observed removal is a result of biological activity or is purely physical-chemical. The rapid breakthrough of DMDS, where outlet concentrations equal inlet concentration within minutes of starting the biofilters, compared to arnmonia, where breakthrough took several days, dernonstrates the higher absorptive capacity the biofilters have for ammonia. Physical adsorption is characterized by higher initial absorption followed by reduced absorption as the media becomes saturated. This can be seen in the initial stages of amrnonia removal for al1 of the compost filtea. For the first two or three days there is complete removal of ammonia, at which point amrnonia breakthrough occurs with reducing ammonia removal over a period of a couple of days. Using this initial absorptive penod during the study into the effects of inoculation an absorptive capacity of 1.28 g N kg-' dry compost was determined for compost at 67% moisture, and 1.10 g N kg-'dry compost for compost at 64% moisture. These values compare with the adsorptive capacity of 1.16 g N kg-' dry compost deterrnined by Smet et ai. (2000) using oven dried compost. As well as being able to adsorb to the filter media, arnmonia is soluble in water where it can dissociate to ammonium ions. Moisture content of the filter media has a definite impact on the sorption of arnmonia. If the two absorption capacities stated above (1 -28 g N kg" at 67% moisture and 1.10 g N kg-' at 64% moisture) are calculated based on water content they result in the same value, 0.62 g N 1-' -0. The effect of moisture on the sorption of ammonia is also evident in the response of the filtes to the addition of water to correct the moishire of the media. This is most clearly observed in the biofilters packed with autoclaved media, see Figure 10. Clear increases in ammonia removal are observed following water additions on day 6,20 and 44. On day 6,300 ml of water was added to each of the columns, 0.1 8 g N is the amount of ammonia which the water based sorption capacity would predict would be removed fiom the air for 300 ml of water added to the colurnn. Integrating the ammonia removal for the three days following the addition for the two stenle, non-inoculated columns (the ones ltast Iikely to have any biological removal) it is found that 0.22 and 0.21 g N are absorbed during the three days. One approach to dealing with the two modes of sorption is to treat them independently, calculating the adsorptive capacity of the dry media and that of the water associated with it separately. This is the approach taken by Smet er al. (2000). They calculated an adsorption capacity of the compost using oven dried media, they determined an adsorptive capacity of 1.16 g N kg-' dry compost. To account for the absorption into the water phase they used the Henry's law partitionhg coefficient for ammonia, combining these two values an estimate of 1.28 g N kg-'was set as the minimum absorptive capacity of the moist compost material. While this value compares well with the absorptive capacities determined in this study there are problems with this approach. If the water associated with the media is treated as fiee water able to corne to equilibrium with a gas phase, as descrïbed by Henry's law, then the dissociation of ammonia into ammonium and hydroxyl ions should dso be considered. Due to the neutral to slightly acidic pH range often associated with biofiltration this dissociation of arnrnonia to ammonium ion has a very profound impact on the calculated absorptive capafity of the water phase. Using Henry's coefficient of NH, at 20 OC (H = 5.6 x104) (Smet et al. 2000) and a dissociation constant for amrnonia (K ,= 1.75 x IO-') ( Gerhartz, 1985) with pH of 7 the amount of nitrogen that would be removed fiom the air in the biofilter described by Smet et al. (2000) becomes 11.7 g N kg-'. This is no longer in the range observed in this study, nor in the experimental results of Smet et al. (2000). As the pH becomes more acidic and the equilibriurn shifis even more to the formation of ammonium ions the absorptive capacity based on this mode1 grows. Altematively the adsorption of ammonia by the media can be viewed as a single process where water addition does have an effect. This is the approach taken by Togashi et al. (1986) who considered there to be two distinct means by which annonia cm bind to the media. Peat, and other humic materials, contain strong acidity sites as well as weak acidity sites and other oxygen-containing tùnctional groups. The strong acidity groups can trap ammonia forming ammonium salts in a neutralizing reaction. The weaker acidity groups and other oxygen-containing sites trap ammonia by forming hydrogen bonds with the ammonia, ammonia bound by this means is observed to be released during drymg. While the actual binding sites for amrnonia are on the media the hydrogen bonding which occurs between these sites and ammonia requires sufficient water for the binding to take place. These two diEerent binding mechanisms and the role of water in the reversibfe binding of ammonia may better account for the water mediated absorption observed in the biofilters using sterilized media. Togashi et al. (1986) determined an absorption capacity for peat neutralized to pH 7.25 to be 2.48 g N kg-'dry peat, while sightly higher than those observed during this study the higher humic content and the greater surface area per volume of peat would account for a higher absorption capacity. Similar peaks to those observed in the autoclaved media biofilters can also be seen for the non-autoclaved media following the days of water addition, Figures 7,8 and 9, where they are superimposed on a general increasing trend in removal. This more gradua1 and sustained removal could be attnbuted to biological activity in the bed, though other processes may be contributing to the overail observed removai. The lack of an initial complete removal phase for the DMDS indicates the limited absorption capacity compost has for this compound. The subsequent removal of DMDS when observed is attributed to biological removal. For ammonia there other factors which can contribute to removal. Ammonia cm be chemically scrubbed fiom the air. Under reduced pH conditions the aqueous equilibrium between ammonia (NH,) and ammonium ions (NHC) will shift in favour of ammonium ions. The resulting ammonium ions can combine with the product of nitrification to form ammonium nitrate (NH,NO,) or with the product of DMDS oxidation to form ammonium sulphate ((NH&SO,)- Others have observed that in biofilters treating arnmonia as rnuch as hdf of the ammonia removed is through reaction with nitrate (Smet et al. (2000). While this is a chemical reaction, it is biologically dependent since the necessary reagents are the product of biological activity. Figure 16 shows the speciation of inorganic nitrogen in the biofilter media treating ammonia alone, ammonium ion accumulation is only slightly less than that of nitrate. Figure 15 shows that for combined removal of DMDS and ammonia, ammonium is the only form of nitrogen that accumulates in the filter media. Mile this suggests that chemical scrubbing of the ammonia could account for the removal observed, it does not necessarily preclude biological transformations of nitrogen. 6.4 BIOLOG~CALACTIVITY As stated previously the slow increase in removal with theis indicative ofbiological removal where acclimatizationand population growth are required before maximum removal can occur. This behaviour is observed in the removal profiles for most of the biofilters. in some cases the transition fiom physical removal to biological removal is not distinguishable, such is the case of amrnonia ody treatment shown in Figure 14. A clear drop in ammonia removal followed by a steady increase is seen in the biofilters treating both gases but the ammonia only biofilters show continuous amrnonia removal. Here the acclimation is quick enough that the biological activity is established before the absorptive capacity of the bed is exhausted. Fuaher evidence of biological activity is the accumulation of the products of biologically mediated metabolic pathways, in this case the accumulation of nitrite and nitrate fiom nitrification and sulphate fiom the biological oxidation of DMDS. Analysis of the media for nitrite and nitrate established their production in the biofilters treating amrnonia in isolation. The production of sulphate is implicated in the accumulation of sulphur in the filter media. Since DMDS is not absorbed by the media, sulphur will only accumulate if the DMDS is transformed to a form that will remain in the media, sulphate and elemental sulphur are both possible forms and both require biological action to be formed from DMDS. The most direct evidence of biologicai activity is to monitor microbial populations capable of the expected transformations. This was done by means of the MPN-PCRmethods developed for monitoring T.thiopanrs and N.europaea, the population estimates of these two species are shown in Figures 21 and 23, respectively. The T.thioparus population values demonstrate a clear log growth phase f?om the initial population of log 2.9 cells per g dry media, to a sustained average value of log 7.8 cells per g dry media. This growth was observed both in the biofilters treating DMDS alone and in those treating DMDS and ammonia. In the biofilters treating ammonia alone the population of T.fhioparus dropped below the detection limit of log 2 cells per g dry media (see appendix A). This obvious growth in the presence of substrate, and lack of growth in its absence, is strong evidence that the Zthioparus population is responsible for the observed removal of DMDS. N-europaea dso demonstrated growth in the presence of ammonia while populations dropped below detection in biofilters treating DMDS alone (see appendix A), indicating that it was actively metabolizing available ammonium. The population densities achieved were much lower than those for Tthioparus, starting with an initial value of log 3.2 Neuropaea cells per g dry media and only attainuig a maximum of log 4.8 cells per g dry media. An in situ activity rate can be determined ushg the removal rate demonstrated by the biofilter at the time of sarnpling, the total mass of the media and the population density determined for the sample. The activity rates determined in this way for the T.thioparus populations demonstrate a distinct difference between activity during growth and stationary phases, with growth phase activity approximately three orders of magnitude higher than stationary phase, see Figure 22. The average growth phase activity rate was 4.0 x 1O-" mol DMDS cell" h-' while the stationary phase activity rate averaged 6.9 x 1O-'' mol DMDS cell" h-', on a mass sulphur basis these activities are 2.6 x 10-'O g S cell-' h" and 4.4 x IO-" g S cell-' h" respectively. These values are comparable to the 2.0 x g S ceil-' h-l reported for Tthioparus in peat biofilters (Cho, Hirai and Shoda, 1991) and the 3.26 x 1O-'? g S cell-' h-' reporîed for a biofilter using an encapsulated Thiobacillus strain (Chung and Huang, 1998). Attributhg al1 ofthe observed ammoniaremoval to the activity of N. europaea results in the activity rates illustrated in Figure 24. The average for the arnrnonia only samples fiom day 13 and day 4 1 is 1-30 x 10'10g N cell" h-', assuming a one to one ratio of ammonium to nitrate, i.e. only half of the ammonia removed is actually oxidized, the nitrification rate becomes 6.5 1 x 10'" g N cell-' h-'. This is comparable to the 5.79 x IO'" g N ch-' h-'activity rate for a biofilter packed with beads of Neuropaea encapsulated in alginate calculated from the work by Chung and Huang (1998), although it is a couple of magnitudes higher than most values reported for liquid cultures (see table 3 in introduction). The rate of nitrification is more difficult to access in the case of CO-removalof amm~niaand DMDS. The fact that the N.europaea populations persisted at numbers comparable to those in the ammonia only biofilters, yet diminished below detection in those treating DMDS only, suggests that they were actively nitriQing in the combined treatrnents. Yet the media analysis indicates that no nitrate accumulated in the filter media when combined loading was applied. The absence of nitrate accumulation has often been wrongly correlated with an absence of nitrification. In fact nitrate accumulation is not the nom in nahual systems yet nitrification is ubiquitous. The question of what happened to the nitrate may be partially answered by the results fkom the nutrient supplementation study. Figure 28 shows the response of T.rhiopurus to various nitrogen additions, clearly nitrate has the most rapid and dramatic impact on DMDS removal. While ammonia additions dso increased DMDS removal rates the response was slower than that observed for nitrate, this could be due to the availability of nitrate with arnrnonia being more tightly bound to the filter media. Another part of the solution is evident in the difference in DMDS removal for the combined and DMDS only treatments. Figure 13 shows that the CO-removalof ammonia in the biofilters resulted in substanttiaily higher DMDS rernoval rates. The cumulative effect of this higher removai rate is demonstrated in the amount of sulphur found in the media samptes. At the end of the 99 day run the combined treatments had removed 54% more sulphur, averaging 37.7 g S per column, than the DMDS only treatrnents, which averaged 24.5 g S per colurnn (see appendix B). The T.thiopanrs is obviously responding to nitrogen additions, both in the form of nitrate and ammonia. The question remains, how much of the ammonia removed is undergoing nitrification. 6.5 COUPLINGOF DhlDS AND AMMONIA REMOVALS If a biofilter being loaded with both DMDS and ammonia established any removal it removed both compounds. Not only did removal of both occur, but they always occurred at relatively the same time and in a sirnilar pattern. Figures 6 through 9 as well as Figures 1I and 12 al1 show the similar pattern of rise in ammonia removal shortly preceding a similar rise in DMDS removal. If these removals are the result of independent processes such a consistent and close coupling would not be expected. As mentioned previously there are severai means of ammonia removal, only one of which is coupled with DMDS removal, the chemicai formation of ammonium sulphate. Using the four to one ratio of ammonia molecules which can be chemicdly removed for every DMDS molecule oxidized, a theoretical ammonia removal rate can be caiculated based on the DMDS removal rate. The following four figures (Figures 33 through 36) are reproductions of Figures 6 through 9 with the addition of this theoretical ammonia removal based on the observed DMDS oxidation. Al1 four show that the formation of ammonium suiphate is suficient to account for the observed ammonia removals. Most significantly the onset of arnrnonia removal predicted fiom the DMDS removd matches that observed, demonstrating the direct link to the onset of DMDS oxidation. The actual ammonia removal is limited to 100%, where the theoretical exceeds this indicates that the sulphate production could chemically remove more ammonia than was loaded to the biofilter. There was only one study where the observed DMDS removal rate was not sufficient to chemically remove al1 of the ammonia loaded to the biofilters, that was the tong term combined removal study. Figures 3 1 and 32 show the inlet and outlet concentrations of DMDS and arnmonia for this study, respectively. Figure 37 compares the actual ammonia removal rates with a theoretical ammonia removal based on chemical reaction with the sulphate, sulphate production is calculated fiom the observed DMDS removed. Unlike the other studies the ammonia removal is only complete between days 42 and 70, it is only during this time that the theoretical removal of ammonia far exceeds the actuai. Even the apparent outlier point on day 152, where the amrnonia removal drastically drops, is well predicted by the theoretical line. The fact that the theoreticaI removal based on chemical reaction can more than account for the actual removal observed indicates îhat no additional removal mechanisms for ammonia, such as nitrification, were significantly contributing to the obsewed arnmonia removal. Further evidence of chemical removal of ammonia for the combined treatments cornes fiom the media anafysis. in al1 but one of the studies where combined treatrnent biofilters were run the media analysis indicates that no nitrate was accumulating in the filter media, while in the biofilters treating ammonia only nitrate accounts for approximately 60% of the inorganic nitrogen accumulating in the media. The only time there was direct evidence of nitrification in biofïiters treating both DMDS and ammonia was in the initial effect of inoculum study. While there was lirnited media analysis during this study the final analysis after 90 days of operation indicates nitrate accumulation, particularly in the inoculated media (see table B 1 in appendix B). As stated earlier the absence of nitrate accumulation does not necessarily mean nitrification is not occ~g,nitrate can be utilized by a wide range of microorganisms. As well as king present in the inorganic foms (ammonia, nitrate and nitrite), large arnount of nitrogen are present in the biofilter media in organic foms. Exchanges between the inorganic and organic pools of nitrogen are constantly occurring, growing cells will incorporate inorganic nitrogen into organic molecules, and upon death these organic molecules will be degraded releasing inorganic nitrogen. In most of the studies conducted evidence of the exchange between these two pools of nitrogen are obscured by the continual loading of ammonia nitrogen. The one study which allows this exchange to be observed is the nutrient supplementation study. In this study there was a one-tirne addition of nitrogen to the system at the beginning. Inorganic nitrogen was added either as nitrate or ammonia. Tracking the inorganic nitrogen in the media during this study (table B3, in appendix B) reveals that where nitrogen supplementation occurred the inorganic nitrogen (nitrate or ammonia) quickly leaves the inorganic pool, presumably incorporated into biomass which accounted for the increased DMDS removal in the supplemented biofilters. By day 15 of the study 85% of the inorganic nitrogen originally present in the media in the supplemented biofilters is missing fiom the inorganic pool. While the non-supplemented biofilters show a continual drop in inorganic nitrogen throughout the study the supplemented biofilters show an increase in inorganic nitrogen at the end of the study as arnmonia begins to accumulate in the media. The difference between the amount of arnrnonia in the supplemented media and the non-supplemented media at the end of the study corresponds to 43% of the added nitrogen in the case of the nitrate addition and 33% when ammonium was the supplement. In the case of ammonium supplementation, ammonium accumulation in the media was not the result of direct deposition into the media but rather the nitrogen was taken up by the biomass, converted to organic nitrogen then after ce11 death was mineralized back to inorganic ammonia. The same cycling fiom inorganic to organic then back to inorganic occurred with the nitrate was added as a supplement. While the accumulation of nitrate in bio filters rece iving ammonia loading c lear 1y indicates that nitrification is taking place, the absence of nitrate accumulation in the combined feed is not proof that nitrification is not occurring. The accumulation of nitrate duing the study into the effects of inoculation demonstrate that nitrification and DMDS removal can occur simultaneously in a compost biofilter. The persistence of a nitriQing population in the biofilters treating both DMDS and ammonia during the combined and individual study is strong evidence that nitrification is also occurring in these biofilters. The long term combined removal study, on the other hand, provides strong evidence that nitrification is not occurring. This study dif5ered fiom the previous two in that DMDS removal was established pnor to the introduction of ammonia. The existence of sulphate in the media prior to the introduction of ammonia rnay have prevented the establishment of nitrification since the amrnonia would be readily removed as ammonium sulphate, the actual substrate for nitrification is ammonia 6.6 AMMONIAINHIBITION OF DMDs REMOVAL The establishment of DMDS removal, when applied as the sole contaminant, during the cold study and the stored media studies (Figures 30 and 26 respective1y)indicates that neither condition inhibited DMDS removal. During those studies the biofilters receiving both DMDS and ammonia failed to establish DMDS removal. The media for these studies was prepared as one batch which was then subdivided between the biofilters. The only condition which varied between columns in each study was the contaminant loading. This clearly demonstrates that the additional loading of ammonia inhibited the removal of DMDS. At the neutral to slightly acidic pHs of the filter media during these studies would cause the ammonia~ammoniumion equilibrïurn in the aqueous phase to favour ammonium ion, thus the ammonium ion may be the actual cause of inhibition. Even once the temperature in the laboratory warmed up during the cold study the biofilters which were receiving both DMDS and ammonia failed to demonstrate any removal. Only ten days af3er the ammonia was turned off did the biofilters begin to demonstrate DMDS removal (Figure 30), providing Merevidence that amrnonia addition inhibiting DMDS removal. Yet once DMDS removai was established the re-introduction of ammonia to the airstream did not inhibit the DMDS removal (beginning of the long tenn combined midy). Smet et al., 2000, showed that dimethyl sulphide (DMS)removal in a biofilter could be completely inhibited with the introduction of ammonia as a second substrate at a loading rate of 140 ppm. The arnmonia addition was for too short a time (six days)for nitrification to become established, so the arnmonia removal observed was attributed to absorption with a conespondhg increase in pH fiom 4.3 to 7.7. Similar to the present study Smet et al. (2000) found that sulphur removal was regained after a period of amrnonia desorption (five days) once ammonia loading was stopped. The current study indicates that amrnonia inhibition of DMDS removal can occur at ammonia concentrations as Iow as 30 ppm when other environmental stresses are present (cold study) and at 54 ppm when no obvious environmentalstresses are present (stored media). Yet no inhibition is exhi bited at amrnonia concentrations up to 50 ppm during the other studies. 6.7 DMDS INHIBITIONOF AMMONIAREMOVAL For the combined treatment during the combined and individual loading experiment, Figure 14, there was a drop in the ammonia removal for the combined treatment which was absent fiom the ammonia only. While ammoniaremoval recovered, and became equal to the complete removal observed in the amrnonia only treatment, it has been shown previously that chemical reaction with sulphate can account for the subsequent ammonia removal. Further evidence of DMDS inhibition of nitrification occurs in the study using the media which had been stored. Ammonia removal was established in isolation but not for the combined treatment. Even after the DMDS is tumed off, the columns which had received combined loading failed to establish arnrnonia rernoval. The ability for chemical removal of amrnonia to account for al1 of the ammonia removal observed in the various combined studies suggests that nitrification does not occur in the presence of DMDS. This complete inhibition of nitrification by DMDS is contradicted by a few pieces of evidence. The accumulation of nitrate in the inoculated columns treating both DMDS and atnmonia during the effect of inoculation study is direct evidence that nitrification can occur in the presence of DMDS. The persistence of the Neuropeae population in the combined treatrnents during the combined and individual study also indicates that nitrification and DMDS oxidation can occur simultaneously. 6.8 EWDENCEOF HETEROTROPHICNITRIFIERS There is strong evidence that T.thioparus is responsible for the oxidation of DMDS observed in these studies. There is also evidence that this oxidation can be inhibited by ammonia, yet there is Merevidence that DMDS oxidation and nitrification can occur simultaneously. The weakest evidence presented in the results is that the N.europeae is responsible for the observed removal of arnrnonia. While the activity rates determined for the biofilters treating ammonia only are comparable to rates determined for a biofilter packed with encapsulated pure cultures of N. europeae the two environments are very dissimilar, and direct comparison has limitations. Most other estimates predict N-europeae activity rates a couple of orders of magnitude lower than those determined during this study (see table 3). A much lower activity rate would not allow the estimated population of N. europeae to be responsible for the ammonia oxidized in the amrnonia only biofilters. The biofilters treating acombination of ammonia and DMDS had comparable population estimates for N. europeae, in those columns it has been demonstrated that chernical removal is sufficient to account for the observed ammonia removal. For the ammonia only biofilters it is possible that other nitrifiers could be contributhg to the observed ammonia oxidation. Environments where heterotrophic nitrifiers are believed to play a dominant role in the oxidation of arnrnonia are acidic environments and at temperatures above 40°C (Focht and Verstraete, 1981). The material used as biofilter media in this study was compost which had been processed at temperatures above 40°C, it is likely that any nitrifiers which might exist in the media would be heterotrophs. The acidifjing nature of the biological oxidation of DMDS would also favour heterotrophic nitrifiers over autotrophic ones. The only evidence that heterotrophic niûifiers may be contnbuting to the observed removals is indirect and relates to the stored media study. In al1 the other studies using fiesh media there is a likelihood that heterotrophic nitrifiers are present. Allowing the media to air-dry would drastically reduce the indigenous populations. Inoculating with T.thioparus,Neuropaea and N.winogradskyi allowed the establishment of DMDS oxidation and ammonia oxidation in isolation but not when applied in combination. This can be explained by the inhibition of T.thioparus by ammonia and the autotrophic nitrifiers by DMDS, but why does this inhibition not occur in the other studies using media which had not been previously drîed. A partial explanation can be gained by investigating the amrnonia inhibition of T thioparus. The cold study provided the most direct evidence of the inhibition of T.thioparus by ammonia. DMDS removal became established in al1 four biofilters receiving both ammonia and DMDS only derthe ammonia was turned off. Interestingly once DMDS removal was established resurning ammonia loading did not inhibit the DMDS removal. This fact can be explained by considenng the fonns in which ammonia cm exist within the biofilter. In the aqueous phase of the biofilter ammonia can exist as fkee ammonia WH,) or as ammonium ion (NH,'). The compost media is initially neutral to slightly basic, this would cause the ammo~a-ammoniumequilibriurn to favour ammonia. It is fkee ammonia which is considered to be more toxic. The ammonia concentration can be reduced through the action of nitrifiers, by reductions in pH which will shift the equilibriurn towards ionic ammonium, and the availability of reactive compounds such as sulphate. DMDS oxidation is an acid fornllng reaction and the end product is sulphate. It is unlikely that under these conditions nitrification would become established since it is amrnonia not ammonium ions that are the substrate for the nitrifiiers. The fact that resurning ammonia loading did not induce inhibition in the established DMDS oxidation cm be attributed to the presence of sulphates to react with the ammonia and the acidification that occurred during sulphate production. The long term removal of DMDS in these biofilters demonstrates that once established, the chernical removal of amrnonia fiom the system is sufficient to prevent ammonia inhibition. In the studies using fiesh compost there may be sufficient heterotrophic nitrifiers to keep the ammonia levels low enough to prevent inhibition allowing DMDS oxidation to become established. The absence of these heterotrophic nitrifiers in the air-dried media allowed ammonia levels to remain inhibitory. This is purely speculative yet could explain some of the observed results, though one piece of evidence still conflicts. The presence of undetected heterotrophic nitrifiers wouid explain the ammonia oxidation rates in the ammonia only biofilters which seem to be higher than the autotrophic nitrifier populations would be expected to achieve. if this removal was in a large part due to these heterotrophs a lower removal rate would be expected in the ammonia only biofilter packed with media which had been air-dried. But in fact both the average and maximum removal of ammonia are higher for the amrnonia only biofilters during the stored media study than during the combined and individual loading study. The MPN-PCR techniques developed during this study are only suitable for quantimng known, specific species. To identiQ dominant, unknown species from a diverse population of microorganïsms requires a different approach. While selective culturing techniques can idente species present which are able to metaboIize the supplied nutrient they fail to determine if they were active in the natural environment. Analysis of the DNA extracted directly fiom environmental samples is the best determinant of species dominant under the environmental conditions. Denaturing gradient gel electrophoresis (DGGE) of PCR amplified sequences has been explored for monitoring diverse populations in soils. This approach was explored for application to compost based biofilters. A discussion of DGGE, the method used, dong with the results is presented in the following section. Predicted and Obsewed Ammonia Removal Unsterile inoculated A + DMDS +Ammonia + Predicted Ammonia removal Figure 33: Theoreticai amrnonia removal based on DMDS removal. compared to observed ammonia removal for unsterile inoculated biofilter, replicate A. Predicted and Obsewed Ammonia Removal Unstenle Inoculateâ 8 O tO 20 30 40 50 60 70 rime (dam + DMDS - Arnrnonia - Theoretical Ammonia Removal Figure 34: Theoretical amrnonia removal based on DMDS removal compared to observed ammonia removal for unsterile inoculated biofilter, replicate B. Predicted and Obsewed Ammonia Removal Unsterilized A 30 40 The(days) + DMDS + Ammonia +Theoretical Ammonia Removal Figure 35: Theoretical arnmonia removal based on DMDS removal compared to observed ammonia removal, for unsterilized compost biofilter, replicate A. Predicted and Observed Ammonia Removal Unsterilized B 30 40 Tirne (days) + DMDS - Ammonia + Theoretical Ammonia RemovaI Figure 36: Theoretical ammonia removal based on DMDS removal compared to observed ammonia removal for biofilter with unstenlized media, replicate B. Long Term Combined Removal Actual and Theoretical Ammonia Removal O 20 40 60 80 100 120 140 160 180 The(days) Actual Removal (A) Actual Removal (B) -Theoretical Removal Figure 37: Actual ammonia removal rates observed in biofilters treating both ammonia and DMDS as well as theoretical chernical ammonia removal in the form of ammonium sulphate. Theoreticai ammonia removal based on actual DMDS removal. 7.0 PRELIMINARYDGGE INVESTIGATION While the MPN-PCR technique developed during this project works for monitoring known microbial constituents witbin the biofilter environment it does not allow determination of present but unknown species. Amplification of DNA by PCR using universai primers with subsequent separation with denaturing gradient gel electtophoresis (DGGE) has the potential to allow unknown species to be monitored and identified fiom environmental samples. 7.1 INTRODUCTION TO DGGE Denaturing gradient gel electrophoresis (DGGE) is a means of viewing PCR amplified sequences which allows separation based on differences in base pair sequences not ovemll length. The PCR products, al1 of the same length, are nui on a polyacrylamide gel containing a Iinearly increasing gradient of chemical denaturing agents, urea and formamide. An electrical potential is applied across the gel to induce DNA movement. Separation results due to the decreased mobility of partially denatured DNA compared to DNA in the helical form. As the DNA migrates through the gel it is exposed to an ever increasing concentration of denaturing compounds. Denaturing, or melting, occurs in discrete so-called melting domains and is determined by base-pair sequences. DNA having a lower guanine and cytosine (GC) content will denature sooner than higher GC containing fragments. Once denatured the migration of the fragment is virtually halted. These discrete melting domains result in a banding pattern in the gel. DGGE was developed for identimng mutations in genomic DNA, and has ken used to identify single point mutations. The application of DGGE as a means of analyzing bacterial 16s rDNA amplified fiom complex microbial populations by PCR was first described by Muyzer et al. (1993). Incorporation of a GC-rich extension to one of the primea used in the amplification can mod@ the melting behaviour of the hgment, ailowing better resolution. Muyzer et al. (1 993) were able to identifi constituents which represented only 1 % of the total population. It should be noted that each band in a DGGE frorn a complex population does not necessarily represent a single species nor even fragments with the same sequence, they just denature at the same concentration of denaturing agents. To identiQ speci fic species an O 1igonucleo tide probe can be hybridized to the amplified DNA sequences in the DGGE gel, identifying if the species of interest is present. in this manner Muyzer et al. (1993) were able to detect the presence of sulfate-reducing bacteria. As a PCR based technology, DGGE can use the range of selectivity that primer design allows. Kowalchuk et al. (1997) used 16s rDNA pnmers specific to ammonia oxidizers of the P subdivision of the Proteobacteria. The resulting DGGE bands were then isolated and sequenced, in this rnanner they were able to determine that Nitrosopira was much more prevalent than Nitrosornonas in natural samptes. Iwamoto et al. (2000) used a fünctionai gene as the target for their PCR amplification, DGGE analysis of the products allowed them to monitor changes in a specific fùnctional group within a population. The target gene was mmoX which is a marker for methanotrophs that possess the soluble form of methane monooxygenase. Methanotrophs possessing the soluble form of this enzyme have been shown to degrade trichloroethylene (TCE) at a very high rate compared to those with the membrane bound form of the enzyme. Using DGGE analysis of this specific amplified sequence they were able to monitor shifts in this important TCE degrading population during biostimuiation studies. DGGE has been demonstrated to be a relatively quick and reliable rneans of measuring àiversity in complex microbial populations. When coupled with hybridization or sequencing, knowledge about specific species can be gained. This preliminary study is designed to investigate the possibility of using DGGE to monitor changes in the microbial community of biofiltea treating different combinations of contaminants. 7.2 DGGE METHODOLOGY The environmental and genomic DNA samples were the same ones used for the MPN-PCR analysis. A single PCR reaction was conducted using the universal prirners 530fU and 985rClamp (the reverse primer contains a 40 base pair GC rich clamp on the 5' end). The PCR reaction mixture and thermocycling procedures were the same as during the MPN-PCR analysis. The PCR products were purified using the QIAquick PCR purification kit fiom Qiagen. The purified PCR products were run on a 8% acrylamide gel. The chemical denaturants used were formamide and urea, the gel had a linear denaturing gradient fiom 45% to 65%. Gels were run at 60°C with an 80 mV potentiai for 16 hours. 7.3 DGGE RESULTS Figure 38 shows the results of the DGGE analysis of the PCR products from the amplification of the T.thioparus and N-europueacultures as well as initial and final filter media samples fiom the combined and individual biofilter experiment. 7.4 DISCUSSIONOF DGGE RESULTS The DGGE analysis of the PCR products did work, distinct bands are visible and Figure 38: DGGE gel with denaturing gradient ranging fiom 45% to 65%. Samples are 1) Tthioparus genomic DNA, 2)N-europaea genomic DNA, lanes 3 through 9 are media samples nom the biofilters, 3) May 17 initial media for al1 biotilters, lanes 4 through 9 are samples from the various biofilters taken on July 3 1,4) and 5) are combined treatments A and B respectively, 6) and 7) are ammonia only treatrnents A and B respectively, and 8) and 9) are DMDS only treatments A and B respectivley. distinct differences are observable between the different treatrnents. Lane 1 shows that three bands result fiom the amplification of the T.thioparus genomic DNA, while unexpected this is not un-heard of. Bands corresponding to these are the only bands visible in lanes 8 and 9, the PCR products for the biofilters treating DMDS only. The N. europaea (lane 2) produces a diaise band which appears faintly in lanes 6 and 7, products fiom the biofilters treating ammonia only. Perhaps the most significant result is the distinct band in lane 5 which does not correspond to either of the control species and is not present in any of the other lanes (though another gel of these products showed this band in lane 4 as well). Lane 3, the initial filter media, shows the presence of bands which are not visible in any other lanes, these could represent species which dominate in the compost but are not selected by the addition of eitber DMDS or amrnonia. While the DGGE results themselves are far fiom conclusive they do indicate that this approach could be applied to the biofilter environment to track changes in populations over time. Al1 of the lanes show a surprisingly limited nurnber of bands, this could be partially due to the primers used, the variability of the 16s rDNA sequence amplified, as well as the effect of the GC-rich clamp, or it could reflect a lack of diversity present in the samples. While low diversity would be expected in al1 of the biofilters which had been treating contaminants for an extended penod of time (lanes 4 through 9), the initial media could also have limited diversity since it was compost which was obtained when it was still exhibiting elevated temperatures. DGGE could prove to be a good starting point for observation of microbial communities in the biofilter environment. DGGE bands could be sequenced to identifi dominant species under different loading conditions. This approach has the advantage of not requiring any a priori knowledge of the species present, nor is it limited to studies where inoculums or pure cultures are used. The research conducted during this study has indicated several areas of research which are worth pursuing. The impact of the presence of co-contaminants on the treatment of the individual contaminants by the biofilter illustrates the need for Merstudy on treatrnent of mixed gases. Of particular interest is impact of the presence of amrnonia on the removd of other contaminants. The MPN-PCR method for quantifying microbial populations in filter media samples is a valuable analytical tool which could be used in a wide range of applications. Further development ofthis method, with application to soi! and sediment samples, is recommended. As well as expanding the range of samples, the range of species tested for should also be expanded, of particular interest would be expanding the range of nitrifiers which could be quantified. Finaily the DGGE analysis conducted in this study, though only explorative, demonstrates a non-selective means of monitoring microbial populations within the biofilter media. This approach should applied to future biofilter studies and be coupled with identification techniques to provide a better understanding of the underlying microbial populations and their response to different contaminant loading during biofiltration studies. A quantitative PCR method was successfully developed which allowed accurate determinations of T.thiopanrs and N.europaea from samples of bio filter media. The fully nested MPN-PCR method had a detection lirnit of log 2.0 h0.7 cells per dry gram of media. The major obstacle to successful PCR amplification of DNA extracted fkom environmental biofilter media was the presence of high arnounts of humic acids. This was overcome through the development of a DNA extraction method to minimize the extraction of the hurnic acids, cleaning of the crude extract with Sephadex spin colurnns, and the addition of BSA and formamide in the PCR reaction mixture. Collectivety these measures allowed PCR amplification using the DNA extract without any dilution. This quantitative PCR method has broad applications for monitoring populations in soi1 and sediment samples as well as compost and other high organic media. The population determinations conducted with the MPN-PCR method allowed in situ population determinations. T.rhioparus populations were observed to grow fiom an initial inoculation of log 2.9 * 0.7 cells g -' (dry media) to a maximum of log 8.1 k 0.7 cells g -' (dry media), sirnilar growth was observed in biofilters treating DMDS either in isolation or with amrnonia added as a CO-contaminant.N europaea populations remained more modest, only ranging from the initial inoculum size of log 3.2 * 0.7 cells g -' (dry media) to a maximum of log 4.8 * 0.7 cells g '' (dry media). Again the populations were comparable in biofilters treating amrnonia in isolation and those treating both DMDS and ammonia. Both T.fhioparur and N. europaea populations dropped below the detection limit in the media from biofilters which were not treating their respective substrates, DMDS and ammonia. In situ activity rates for T.thioparus clearly demonstrate a drop in activity fiom 2.6 x 1O-'' g S cell-' h-' during growth phase to 4.4 x 10'" g S cell-' h'l during stationary phase. The biofiltration studies demonstrate that T.thiopanrs is inhi bited by ammonia at concentrations as low as 50 ppm. The inhibition is attributed to fiee ammonia concentrations and can be overcome if conditions favour ammonium ion formation. Chernicai reaction of ammonia with the sulphate produced through DMDS oxidation by T.thioparus was shown to be sufficient to remove the ammonia loaded to the biofilters, preventing inhibition of the DMDS oxidation. The autotrophic nitrification initiated by N. europaea demonstrated inhibition by DMDS concentrations of 5 1 ppm. The combined removais of DMDS and ammonia observed were attributed to either chernical removal of the ammonia through the formation of ammonium sulphate, or due to the presence of nitrifiers which were not inhibited by DMDS. Heterotrophic nitrifiers were proposed as a likely possibility. 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Plant and Soi1 170 (1 995): 1 15-22. raûk Al: T.mkpsnn padstian atifnates for biofitîer 8amples and standards uslna tub nested I Dayr Sampk Extraction Sample Hmn from Date Cdumn Weîwî. %Moistutu Dry wt. vdume volume Diluhion MPN Conlidona start (O) (9) (ul) (ul) mries Score lador 3.65€+03 1.07EtOB 5.9lEtM 3,28Et08 3.11Et03 3.91€+07 B. 15Et08 1.13EtO8 73 30-Jul 6 1 3.9 60.6 1.54 500 1 1O Mection ttm~for sampier I Jsing averans dry weiaht. 1.61 500 1 10 Standards Name Inoculation i desnad 7.3 r uude 5.3 r deaned 5.3 I crudo 3.3 1 dsaned 3.3 1 deaned 3.3 I crude Nol inoculaleci l deaned Not lnoculaled letection limit for standard8 :rude 2leaned JD = no( determined Ppf WWWW OQWO 7 '.? * "? Y) Q C> C) - Dayr Smpk Extraction Sampk ltom Date Cdumn Wetwt. %Molrtum Dry wt. vdume vdume Diluiion rtart (QI @) (ul) aeries I (ui) 4 51 1.96 5000 1 10 3.7 51.4 1.80 5000 1 10 3.7 51.4 1.80 5000 1 1O 3.7 50.7 1.82 5000 1 10 3.2 50.7 1.58 5000 1 10 3.7 52.6 1.75 5000 1 10 3.5 55.4 1.56 5000 1 10 - Standards Name Inoculation lo~(celdgl 6 uude 7.3 6 dsaned 7.3 '8bk M: N,eurupaea popuiatio tstlmates for blofilter sampies usina one spscir~cand one univenal primer ln Vis sscond PCR. Nitmsomonas europaea , . [,yqg.;; Oayr Same Exlracîion Same Confident. liml ConMeno limlt from Date Cdumn Wet wt. %Molslure Dry M. vdume vdume Dilution Initial MPN :onfidencc & xmsr uppr Iower upwr start (0) (0) (ui) (ul) seriea Dilution Score factor :'lm)1 (cdldg) (cdldp) ;Ta. . W.. {kp) ,1*= i ' -.L Pi. + * = i' . 4 51 1.96 5000 1 10 100 9 1 4 67 w+ûS 4.97EtO4 1 OBEtW :' 1'$#', 4.7 6.0 3.7 51.4 1.80 5000 1 10 100 23 4.67 %.@W. 1.37Et05 2.9OEtM , :Stg'q 5.1 6.6 3.7 51.4 1.80 5000 1 10 100 3.5 4.67 @,73EW'2.08EtD) 4.54Et05 i 4.3 5.7 3 7 50.7 1.82 5000 t 10 1O0 1466 4.67 d1Meh7.:8.60Et08 1.88Et08 . ,?'!.' 6.9 8.3 3 2 50.7 1.58 5000 1 10 100 14 4.67 4.&tOS 9.50EtW 2.07Et06 ' .$,pi- , 5 O 6.3 3.7 52.6 1.75 5000 1 10 sl. ii: 3 5 55.4 1.56 5000 1 10 1O0 42 4.67 1,wtûO 2.88Et05 6.28€+06 5.5 6.8 i $1 1.75 5000 1 10 100 3 5 4 67 9.98EW 2.14E+04 4.66Et05 ; 6:O . 4.3 5.7 Controls Dilutions O 10' 102103 C 800 bp - ZOO bp - Dilution MPN Score Figure Al: PCR results for sarnple May 17, 1999 al1 columns, (A) fust PCR results, (B) second PCR with T.thioparus specific primers, (C) second PCR with Ns.europaea. Controls are 1) T.thiopams, 2)Ns. europaea, 3) negative contro!, 4) Controls 1 2 3 CO O 10 l 10 10 Dilution Dilution 11O011O111O211031104110511061 I~onfr01~ MPNScoreo3 03 3 1 1 0 0 Il23 Dilution ~10°~101~102~10311041105 11061 /~ontrols MPNScore.3 1 O 0 O O 0 O O 11234 Figure A2: PCR results for sample May 3 1, 1999 colurnn 2. (A) first PCR results, (B) second PCR with T.thioparus specific primers, (C) second PCR with Ns. europaea. Controls are 1) T.thioparus. 2)Ns.europaea, 3) negative control and 4) Ns. europuea. Control 1 2 3 /10° 101 10t103Diluti~m Dilutions )10° 1101 1102 )Io3 1 104 1 los1 1061 IControls No MPN Score was recorded il234 Fiyn A3: PCR results for sampie May 3 1, 1999 colurnn 5. (A) First PCR results, (8) second PCR with T.thioporus specific primers, (C)second PCR with Ns. europaea specific primers. Controls are 1) T.thioparus,2) Ns-europaea,3) negative control, 4) T.thioparus and 5) Nkeuropaea. Note: No population estimate for T.thioparus was made since a band corresponding to T.thioparus is present in control2. Control 1 2 3 ! 10° 10 10 10 Dilutions Figure A4: PCR results for sample May 3 1, 1999 column 8. (A) Fust PCR results, (B) second PCR with T.thiopurus specific primers, (C)second PCR with Ns-europaea specific primers. Controls are 1) T.thiopams, 2) Ns.europoea, 3) negative control, 4) T.thiopams and 5) Ns.europaea. Control 1 2 3 / 100 io IO :Dilutions tions 110° 110~1 10% 1 103 1 1041 los 1 106 1 1 Controls ScoreeOe3 3. 3. 3 03BO. 123 Figure AS: PCR results for sample June 29, 1999 column 1. (A) First PCR results, (B) second PCR with T.thioparus specific primers, (C) second PCR with Ns-europaea specific primers. Conîrols are 1) T.thioparus, 2) Ns.europaea, 3) negative control, and 4) Nxeuropaea. Control 1 2 3 Il00 10 Dilutions Figure A6: PCR results for sample June 29, 1999 column 6. (A)First PCR results, (B) second PCR with T. fhioparus specific prirners, (C)second PCR with Ns-europaea specific prirners. Controls are 1) T.thioparus, 2) Ns.europoea, 3) negative control, and 4) Ns.europaea. Control 1 2 3 /10° 101102103 Dilutions Dilutions 1 10° 1 10 ( 102 1 j Controls MPN Score@ O O O 1 1 2 3 Figure A7: PCR results for sample June 29, 1999 column 7. (A) First PCR results, (B) second PCR with T.thioparus specific primen, (C) second PCR with Ns.europaea specific primers. Controls are 1 ) T.thioparus, 2) Ns. europaea, 3) negative control, and 4) ï2hioparus. Note: oniy three dilutions were run with Ns.europaea primers since a small population was expected. 1000 bp + Control 1 2 3 /10° 10' IO2 IO3 Dilutions T.thioparus PCR failed Figure A8: PCR results for sarnple June 29, 1999 colurnn 8. (A)First PCR results, (ES) second PCR with T.thiopums specific pnmers, (C) second PCR with Ns-europaea specific primers. Controls are 1) T.thioparus, 2) Ns-europaea, 3) negative control, and 4) Ns.europaea. Note: the second PCR using ï3hioparu.s specific pnmers failed, for the second PCR using Ns.europaea primers only three dilutions were run since a small population was expected. Control 1 2 3 1 io O io 1 la :Dilutions Dilutions 110" 1 101 1 O 1 IO3 1 IO4/ 1051 1061 Conhols MPNScore* O O 1 O O O O 12 3 5 Figure A9: PCR results for sample July 30, 1999 column 1. (A) First PCR results, (B) second PCR with T.thioparus specific primers, (C) second PCR with Ns.europaea specific prirners. Controls are 1) T.thiopams, 2) Ns.europaea, 3) negative control, 4) T.thioparus and 5) Ns. europaea. Control 1 2 3 lioO10 Dilutions Dilutions IIOo [ 101 1 1OZ 1 103 1 10'1 1051 1061 kontrols MPNScoreeO 03 3. 3 3 2.0 1123 Dilutions 110° 10' 1 IO2 1 IO3 1 10'1 1051 1061 ( Contr~ls MPNScoree O 1 1 0 0 0 O 12 34 Figure A10: PCR results for sample Jdy 30, 1999 column 2. (A) First PCR results, (B) second PCR with T.thioparus specific primers, (C)second PCR with Nieuropaea specific primers. Controls are 1) T.thioparus, 2) Ns.europaea, 3) negative control, and 4) Ns.europaea. CO~WOIS 1 2 3 \owl MPN Score O O O Controls @ilutions Dilutions 1 10°l10 '1 10 10311041105 11061 11234 MPN Score 3 3 O O O O O Controls Figure Al1: PCR results for July 30, 1999 colurnn 5. (A) First PCR results, (B) second PCR with T.thioparus specific pnmers, (C) second PCR with fieuropaea. Controls are I ) T.thioparus, 2)Ns.europaea, 3) negative control, and 4) Ns.europaea. No te: on1 y three dilutions were run wi th T.thioparus primers since srnall popdation was expected. Control 1 2 3 1100 10 Dilutions Controis 12 3 11 10° 1 10' 1 10' 1 10' 1 Dilutions Io O O O O MPN Score Figure A12: PCR results for sample July 30, 1999 colurnn 6. (A) First PCR results, (B) second PCR with T.thiopanrs specific primers, (C) second PCR with Ns-europaea specific primers. Controls are 1) T.thioparus, 2) Ns-europaea,3) negative control, and 3) Ns.europaea. Note: only 4 dilutions were run with T.thi0paru.s primers since a small population was expected. Controls 213 214567 Dilution 1 10° 1 101 1 103 1 1 lo4 [ los 1 Score 3 3 O Omoo Figure A13: Second PCR results for standard 2 using Nxeuropaea specific primers NmO l9Of and Nm 448r . (A) Results using crude extract, (B) and (C) results for extract which had been cleaned using sephadex spin columns and concentrated 10 fold. Controls are 1) negative control, 2) Ns.europaea, 3) negative control (run for second PCR only) 4) T.thiopams genomic DNA, 5) Ns. europaea genomic DNA, 6)T.thioparus which had been previously amplified with universal primers and 7) Ns.europaea which had been previously amplified with universal primers. Dilution 100 1 10' 1 10: 1 103 ( 104 1 IOs ( IO6( 123 4Controk Score m2m3240m1O 01 - -- 7----- , .r;. Dilution 1 100 1 IO1 1 10' 1 103 1 IO4 1 los 1061 123 4Controls Score 03l 3 l 3 O .O 00 0.' Figure A14: Second PCR results for standard 6 using T~hitioparusspecific primers Ttp 223f d Ttp 833r. (A) Results using crude DNA extract, (B) results for DNA extract which has been cleaned using sephadex spin column and concentrated 10 fold. Controls are 1) T.thioparur (nested), 2) negative control (nested), 3) T.thiopauus (second PCR only), and 4) negative control (second PCR only). Controls 1 123456 Dilution 1 10° 1 10' 1 10' 1 1 lo3 1 lo4 1 Score O 3 2 .Oe1 Dilution 1 10° 1 101 1 10' 1 IO3 1 104 1 los 1 1061 12 3 4Controls Score a2a3~2a200aOaOo Figure AIS: Second PCR results for standard 7 using T.thioparus specific pnmers Ttp 223f and Ttp 833r. (A) and (B) results using crude DNA extract, (C) results for DNA extract which has been cleaned using sephadex spin column and concentrated 10 fold. Controls are 1) T.fhioporus (nested), 2) negative control (nested), 3) T.thioparus (second PCR only), 4) negative control (second PCR ody), 5) T.thioparus (nested),and 6) negative control (nested) . Sample 8 Cnide Dilution Score /OeOmO Sarnple 1 8 Cleaned 1 Controls ~ilutionj2O 12' 12' 1 z3 1 24 1 2512 1 1234 Score 10rn3.3. leO*leO I Figure A16: Second PCR results for standard 8 using T.hioparus specific prirners Ttp 223f and Ttp 833r. Gel (A) shows results for both the crude DNA extract and the DNA extract cleaned using sephadex spin column, both at 10 fold serial dilutions. Gel (B) shows the results for the cleaned DNA sample run at 2 fold dilutions. Controls are 1) T.thioparus (nested), 2) negative control (nested), 3) T.thiopanrs (second PCR only), and 4) negative control (second PCR only). Sarnple 1A I, 1B 12 3 5 Controls Dilution 1 10° 1 101 1 10: i 1O0 ( 10' 1 1021 IO4 1 Score wOoOl O /Oo 000~0~1 Figure A17: Second PCR results for standards 1 A, uninoculated cmde extract, and 1 B, uninoculated cleaned and concentrated extract. Gel A shows results using T.thioparus specific primers Ttp î23f and Ttp 833r. Gel B shows results using Ns-europaea specific primers NmO l9Of and Nm 448r. Controls are 1)T. thiopanrs (nested), 2)Ns.europaea (nested), 3) negative control, 4) T.thioparus (second PCR only) and 5) Ns. europaea (second PCR on1 y). Figure A18: PCR results for samples May 17, al1 columns, and May 3 1, column 1. Gel A shows results from the fist PCR using universal primers EU 49f and EU 1070r. Gels B and C show result fiom the second PCR using EU 49f and 22hiopurus specific primer Ttp 833r. Gels D and E show results fiom second PCR using EU 49f and Ns-europaea specific primer Nm 448r. (See following tables for identification of lanes and MPN scoring.) Table AS: First PCR results for samp1es May 17, al1 columns and May 3 1, column 1. Gel 1 A 1 May 17, al1 May 31, col. 1 Controls 1 Dilution 10' 10' 102 10' 10' 102 T.p- M.e. Nb-w- Negative 1 2 3 567 9 10 11 12 d Table Ad: Second PCR results using T.thioparus primer for samples May 17, al1 columns. and Mav 3 1. coli May 1 7, al1 May 3 1, column 1 1 Dilution 1 Band al1 negative 1 Score $econdPCR results using Akeuropaea primer sarnple May columns. I May 17, al1 columns IDilution 1 Band 1 score Table AS: S Gel -- - Sample May 3 1, column 1 Dilution Lane Band Score sampie COL 2 I COI. 5 Ii Dilutions .IO: io3i04 105 ko: io~ro~12 3 Controls Figure A19: PCR results for sample May 3 1, 1 999 column 2. (A) First PCR results. (B) Second PCR with universal primer EU 49f and ï3hioparu.s specific primer Ttp 833r. (C) Second PCR with universal primer EU 49f and Nieuropaea specific primer Nm 448r. Controls are 1) T.thioparus, 2) Ns-europaea,3) negative control (nested) and 4) negative controI (second PCR only). Controls 1234 Dilution1 10' 1 IO3 1 IO4] Score O 3 3 Figure A20: PCR results for sample May 3 1,1999, colurnn 6. Gels A and B show second PCR products using the universal primer Eu 49f and the T.thioparus specific primer Ttp 833r. Gel C shows the second PCR results using the universal primer Eu 49f and the Nxeuropaea specific primer Nm 448r. Controls are 1) T.thioparus, 2) Ns.europaea, 3) Nb. winogradskyi, 4) negative control, 5) T.thioparus and 6)negative control. Controls 7891234 123456 Dilution1 1021103 1 1041 1 los 1 1061 Score O 3 3 11* Figure A21: PCR results for sample May 3 1, 1999, column 7. Gels A and B show second PCR products using universal primer EU 49f and the T.thioparus specific primer Ttp 833r. Gel C shows the second PCR products using the universal primer EU 49f and the Ns.europaea primer Nm 448r. Controls are 1) T.thioparus, 2) Ns.europaeu, 3) Nb.winogradskyi, 4) negative control, 5)T. thioparus, 6) negative control(5 and 6 were run in second PCR only), 7)8) and 9) were environmental samples at 10 2, 10 ' and 10 dilutions respectively which had T. thioparur genomic DNA added to them. Dilutions iô2iô3i04 11 2 3 4 C ontrols Dilutions 110' 1 10' 1 10 1 10' 1 10 '1 10' 1 10'1 Controls Saorc O O O O O -0. O* 1234 Figure A22: PCR results for sample June 29, 1999 column 2. (A) First PCR results. (B) Second PCR with universal primer EU 49f and T.thioparus specific primer Ttp 833r. (C)Second PCR with universai primer EU 49f and Ns.europaea specific primer Nm 448r. Controis are 1) ï2hioparu.r, 2) N~europaea,3) Nb.winogradskyi and 4) negative control. Dilutions 10: 10) 104 / 1 2 3 4 Conîrols ppp Dilutions 110' 1 IO3 1 10'1 10' 1 1061107 1 10'1 /conVols Score 3 1 O O O 00. Oa ; 12 34 Figure A23: PCR results for sarnple June 29, 1999 column 5. (A) Fint PCR results. (B) Second PCR with universal primer EU 49f and ï2hioparus specific primer Ttp 833r. (C) Second PCR with universal primer EU 49f and Ns.europaea specific primer Nm 448r. Controls are 1 ) T thioparus, 2) n% europaea, 3) Nb. winogradskyi and 4) negative control. Figure A24: Both gel A and B show second PCR results using universal primer EU 49f and Tthiopms specific primer Ttp 833r. The samples are standard 6A and 6B at various dilutions, (see following tables for identification of lanes and MPN scoring). Table A9: MPN sconng for T.thioparus of standard 6A run using pnmers Eu 49f and Ttp Sample Standard 6A Dilution Lane in gel Band Score Table A10: MPN sconng for T.thiopams ofstandard 6B nui using primers Eu 49f and Ttp 833r. Dilution Lane in gel 1score Table BI: Media analysis for inoculurnn effects expriment. Time NH4-N N03-N N02-N Total Org. Mat. pH Column Treatment (days) mglkg rnglkg mglkg Sulphur % % 7 serile inoculated 4 non-sterile inoculated 4 non-sterile inoculated 1 non-sterile 2 non-sterile 3 non-sterile inoculated 4 non-sterile inoculated Table 62: Media analysis for combined and individual loading experiment, Combine rreatment NH4-N N03-N N02-N lnorganic Total N mglkg mgkg mghg -N mglkg1 % DMDS and Ammonia (A) DMDS and Ammonia (B) Ammonia only (A) Ammonia only (B) DMDS only (A) DMDS only (B) Table 83: Media analysis for nutrient supplementation experirnent. Netwt, MC Dry wt. rime NH4-N NO3-N N02-N Combined Total N Nitrogen Sulphur Sulphur pH :olumn Treatment I %wet wt. g Iays mghg mglkg mglkg -N rng/kg % g Nlcol. % g S lcol. 1 Control (a) 2 Control (b) nitrate (a) 4 Sodium nitrate (b) sulphate (a) 6 Ammonium sulphate (b) sulphate plus inhibitor (a) 8 Ammonium sulphate plus inhibitor (b) rable 84: Media analysis for cornbined and individual loading expriment using stored media - Combined Column Treatment Wet wt. MC Dry wt. Time NHCN N03-N N02-N Inorganic-N Total N Total S 1 pH g %wetwt. g Da~s mglkg m@g mglkg Wlkg % - (NH4.Nü3.NO2) (exduding NO)\ 1 DMDS and Ammonia (A) 1148 60 459.2 O 135.9 31.78 13.08 181 1.32 14 971.5 26 2.47 1000 1.72 34 1226 8.75 4.5 1235 1.86 2 DMDS and Ammonia (8) 1205 62 457.9 O 135.9 31.78 13.08 181 1.32' 14 1166 25.61 2.93 1195 1.12 34 1226 8.75 4.5 1235 1.86 5 Ammonia Only (A) 1204 61 469.56 O 135.9 31.78 13.08 181 1.32 14 2156 3634 4.5 5790 1.75 34 842 1631 4.5 2473 1.83 Ammonia Only (8) 1297 63 479.89 O 135.9 31.78 13.08 18t 1.32 14 1689 3017 cl,5 4706 1.O8 34 52.25 17.2 4.5 69 1.57 DMDS Only (8) 1277 62 485.26 O 135.9 31.78 13,08 181 1,32 14 78.72 12 4.5 9 1 1.22 I able 153: nneala anaiysis for ng term c mbined rt noval study. Combined rreatrnent Dry wt. Time NH4-N N03-N N02-N Inorganic-h Total N Total S 9 Day s mglkg mglkg mglkg mglkg Y0 % DMDS and ammonia (A) 725 705 686 667 628 609 589 DMDS and ammonia (6) 722 679 657 613 591 569 DMDS only (A) 724 703 682 662 DMDS only (B) 724 682 662 Table C 1: List of primer sequences used and sources Primer Narne 1 Sequence 5' to 3' Melting 1 Source 1 1 * Temp Universal Primers TAC ACA TGC AAG TCG AAC GG Designed during this study. TT(AT) ACA CAT GCA ACT CGA ACG G Designed during this study. GGA CTT AAC CCA ACA TCT CAC GA Designed during this study. ACG GCT ACC TTû TTA CGA CTT Weisburg et al., 1991. Thiobacillus thioporus 16s-rDNA speci fic primers Ttp 194f 1 TCG AGC GGC CGA CGT CTG AT 61 OC 1 Designeci during this study. Ttp 223f 1 CCA CCC GCC CAC CTC TCA T 59°C 1 Modificaiton of Ttp 194f. Ttp 240r 1 ATC AGA CGT CGG CCG CTC GA 6 1OC 1 Reverse version of Ttp 194f Ttp 833r 1 GCT TCG TTA CTA AGG GAT TTC A 57°C 1 Designed during this study. Nitrosonzonas europaca 16s-rDNA specific primers NmO 190f 1 CAA CAC CTT CCC CTA AAG Pommerening-Roser et al. t 996 ~m 459r 1 TCA CAA GGA TTC ATT GCA ACT TT Designed during this study. Nm 448r 1 TTC ATT GCA ACT CTT TCT TTC CC Redesign of Nm 459r NmOr 1 CAT CTT TCG ATG CGT TAT Pommerening-Roser et al, 1996 f - forward primer, r- reverse primer, Bold - primers used in population estimates, * Melting temp based on AT and OC content. able C 1 cont.: List of primer sequences used and sources. 1 I I Narne Sequence 5' to 3' Melting Source Temp. Nitrobacfer winogradskyi 16s-rDNA specific primers Designed for this study. [ CGT AGA GTT TGG CCG TGT CTC ( 61°C Redesign of Nb 3 12r. FGPS 1269r 1 m Tm- GAG ATT TGC TAG 1 41°C Degrange et al. 1995 Nitrosospiru 16s-rDNA specific primer NspOr 1 GGT ATT AAC CGT GAC CGT 1 49OC Pommerening-Roser et al. 1996 Nitrosomonas europaea ammonia monooxygenase sequence speci fic primers NmAmo 9f 1 CCC GTT ATT CCA ATC TGA CCG Hastings 1997. (Referred to as AMOF 1) NrnAmo 103f 1 GCA GAA GTT GCG CïT GGG GTA C Hastings 1997. (Referred to as AMOF2) ------NmAmo 1821r 1 CAG AAT GGC AAG TAC CCA GGT G Hastings 1997.(Referred to as AMORI) NmAmo l844r 1 CCA CCC CAT ACC AGC GCC A Hastings 1997.(Referred to as AMOM) Universal 16s-rDNA primers used for DDGE 530f U 1 CTG CCA GC(AC) GCC GCG G 1530~ ILane1991. 985r Clamp ggc gcc cgg cga ccg gcc cgc ggs 8% gca 47OC Designed for this study using reverse of l cgg ggg cCC GTC AAT TC(AC) TTT 1 universal primer 9261fiom Lane, 199 1. 1 (AG)AG TTT 1 1 f - fonvard, r- reverse, lower cass - gc rich clamp for DGGE, Bold - primers used for final resulis. Squencing results for 7'. thioporus using foiward primer EU49f. Wcd Mby 16 l1:31100l i.\lb.lwq -No. 1 5' II II II 41 II 61 71 II 91 lCNCNTTNNOA OCNCTTTCTT CTNGTCOAOA GTOOCAAACO OTNAATTANN NCTTCONACC NTCCCNATTA TTOOOOHNTA NCOCCAOCNN AAAOCTOTOC GNONAANNCT CGNGAAAOAA OANCAOCTCT CACCGTTTGC CANTTAA'TNN NOAAOCNTGO NAGGONTAAT AACCCCNHAT NOCOQTCONN TTTCOACACO 5' 11 II II 41 JI 61 71 81 91 101 TANTACCOCA T ACOCCCTOA OOOGOAAA(IN UGGOOATCQC AAOACCTCAC GTTATTCOAO COOCCOACOT CTOATTAûCT AOTTOOTOOO OTAATOOCCT ATNATOGCOT ATOCGOGACT CCCCCTTTCN CCCCCTAQCG TTCTQGAOTO CAATAAOCTC OCCGOCTQCA OACTAATCOA TCAACCACCC CATTACCOaA J' S. II II 3I 41 JI 61 II II 91 lolACCAAOOCGA CGATCACITAG CQOCITCTOAO AGOATOAtl!C GCCACACT(lCi AACTGAOACA COOTCCAOAC TCCTACOOOA OOCAOCAOTO 000AATTTTO TQOTTCCGCT OCTAOTCATC GCCCAGACTC TCCTAIITAGO COOT(ITQACC TTOACTCTGT OCCAOOTCTG AGGATQCCCT CCOTCOTCAC CCCTTAAAAC 5' II II 31 41 JI 61 71 II 91 WIIOACAATOGOO GCAACCCTOA TCCAGCCATT CCOCGTOAOT OAAaAAOGCC TTCOOOTTOT AAAOCTCTTT CAGCTOOAAC OAAACOOTAC OCTCTAAcAT CTOTTACCCC CGTTOQOACT AOGTCGGTAA GGCOCACTCA CTTCTfCCGa AAOCCCAACA TTTCOAOAAA OTCOACCTTO CTTTOCCATO COAOAfTOTA Y II II 3I 41 JI 61 71 81 91 AOCOTOCTAA TQACOGTACC OOCAOAAGAA OCACCOGCTA ACTACOTOCC ANCAGCCOCG GTAATACOTA OGOTOCNAOC GTf AAfCOOA ATTACTOOOC TCGCACOATT ACTGCCATGO CCGTCTTCTT CGTOGCCOAT TQATOCACGG TNGTCOOCOC CATTATOCAT CCCACûNTCO CAATTAOCCT TAATOACCCO Y II II Il 41 JI 61 71 II 91 )gIOTAAAQCOTO CNCANOCOQA TTOTTAANCA AANATGTOAA ATCCCCOOCT TAACCTOOOA ATOOCATTTT TOAACTUCAN TNTAAAATNC NTTANAOOOO CATTTCGCAC GNGTNCGCCT AACAATTNOT TTNTACACTT TAGGGQCCOA ATTQOACCCT TACCGTAAAA ACTTOACOTN ANATTTTANO NAATHTCCCC 9 II 21 II 4 I 51 61 71 II #I a,QOTOQAATTC CACNTNTNCN NNNAAATTCC TTNAAATTTN OANOAACACC NATNONNAAA NONANCCCCC TNOONNTNAN ACTTNCCCTT NATNTTNCNA CCACCTTAAG GTONANANGN NNNTTTAAOO AANTTTAAAN CTNCTTOTOO NTANCNNTTT NCNTNG0GOO ANCCNNANTN TOAANOOOAA NTANAANONT P II 2 I 31 41 JI 61 71 II 91 AANCNNNONT THNNANANNN NNNTTTNNNN TNCCCCTNOT NNTNCCCCCC CCCTTNAAAN ANTNTTNNAT NTNTTTTTTT NNOOOAANTN NAAATNCCTT mLTTNGNNNCNA ANNNTNTNNN NNNAAANNNN ANOOUGANCA NNANOOGOCIG OGGAANTTTN TNANAANNTA NAHAAAAAAA NHCCCTTHAN HTTTANOGAA 9 II 11 II 4 1 JI 61 II Il 91 WITNNNNNNNNA ANCN ANNNNNNNNT THON Seqwncing rcsults for T.thioparus using reverse primer Eu 1OïOr. 9 II 31 II 41 Jl LI 11 81 SI 10NNNAAANNN AATNTTTAAT TTACACOAOC TOACOACAOC NTGCAOCACC TOTOTTCCOO TTCTCTTTCO AOCACTCCCO CATCTCTûCA OOATTCCOOA CNNNTTTNNN TTANAAATT A AATOTOCTCO ACTGCTOTCQ NACOTCOTOO ACACAAOOCC AAOAOAAAOC TCOTOAOOOC OTAOAOACOT CCTAAOOCCT 9 Il II II 41 $1 LI 71 II 91 CA1OTCAAT 0 NTAOOTAAOO TTTTTCOCOT TOCATCOAAT NONTCCACAT CATACACCQC f TOTOCOOOT CCCCOf CAAT TCCTftOAOT TTTAAtCTTO QTACAOTTAC NATCCATTCC AAAAAOCOCA ACOTAGCTTA NCNAOOTOTA OTATGTOOCO AACACOCCCA OOOOCAOTTA AOOAAACTCA AAATTAOAAC O II a I 11 41 51 61 II II 91 10ICOACCOTACT CCCCAOOCOO TCAACTTTCA COCOTTAQCT TCQTTACTAA OOOATTTTCA CTCCCCCAAC AACCAAQTTO ACATCQTTTT AOOOC(1TûOA OCTOOCATOA OOOOTCCOCC AOTTOAAAliT GCtICAATCOA AOCAATOATT CCCTAAAAQT OAOOOOOTTO TTQQTTCAAC TOTAOCAAAA TCCCOCACCT Y Il 2 I II 41 51 61 11 II 91 jdlCTACCAOOQT ATCTAATCCT OTTTOCTACC CACOCTTTCO TACATOAGCO TCAOTOOCAT CCCAOOOOOC TOCCTTCOCC ATTQNOTTCC NNCACAtTTT OATOGTCCCA TAOATTAOOA CAAACOATOG GTGCOAAAOC ATOTACTCOC AGTCACCOTA 000TCCCCCO ACOOAAOCOO TAACNCAAQO NNOTOTAAAA O II Il 31 41 51 61 71 II 91 ,,,,ACNCATTTNA CNOTTACANC OTGOAANNNC NCCCCCCCNT NTOACOCACT NNAAACTOOC NNNTTNAAAA ANOCCNTTCC NANONNAAOC CCCONOOOAT TONOTAAANT ONCAATOTNO CACCTTNNNO NOOQGQGONA NACTGCGTOA NNTTTOACCO NNNAANTTTT TNCOONAAOO NTNCNNTTCO OOOCNCCCTA 5' II II II 41 SI 61 11 81 91 slTTNAAAANNN TNNTAAAAAN NNONNTOONC NNNNCNNANN NCCCNATANN NNNNNAANNC AANTTTTNNN ANNATTTTTN NNCNNACCNO NNNtJONNTNN NOOONTATNN NNNNNTTNNû Squencing results for N.europea using fornerd primer Eu49f. S II 11 31 41 JI 61 71 II 91 AANTNNNNNN NNNAOOCTTC THNNTCTCCC COAOAOTOOA OACACOOTTA ATTANTCCNT NOAACCTNHT CNTNAATNOO ONANTANCCC TTCAAAAOAA 'TTNAI.(NNNNN NNNTCCOAAO ANNNAOAOGO OCTCTCACCT CTOTOCCAAT TAATNAOONA NCTTOOANNA ONANTTANCC CNTNATNOOO AAOTTTTCTT 5' II 2 I 31 41 JI bI II 81 9L IOITOTOCTANTA CCOCHTHTCT CTOAGOAGAA AAOCOOOGOA TCOCAAOACC TTOCOCTAAA OOAOCOOCCC OATOTCTOAT TAOCTAOTTO OTOOflOTAAA ACACOATNAT OOCONANAOA QACTCCTCTT TTCCiCCCCCT AOCGTTCTOO AACGCOATTT CCTCOCCOOO CTACAOACTA ATCOATCAAC CACCCCATTT 5' II 1I 31 JI JI 61 Il 81 91 IglGliCTTACCAA GGCAACGATC AOTAGTTOOT CTOAOAOOAC OOCCAACCAC ACfOOOACTO AOACACOOCC CAOACTCCTA COOOAOOCAO CAOTOOoOAA CCOAATOOTT CCOTTOCTAG TCATCAACCA GACTCTCCTQ CCGOTTOOTO TOACCCTGAC TCTOTOCCOO OTCTOAaOAT OCCCTCCaTC QTCACCCCTT Y II II II II JI 61 II 81 91 AATAATTOTO ATT T ATOACG OTACCGACAO AAAAAOCACC OOCTNACTAC OTOCCANCAO CCOCOOTAAT ACOTAOOOTO COAOCOTTAA TCOOAATTAC T TATTAACAC f AAATACTOC CATGOCTOTC TTTTTCGTOO CCOANTOATO CACGOTNOTC GGCOCCATTA TOCATCCCAC QCTCQCAATT AOCCT T AAfO 9' II 1I 31 41 51 61 11 81 91 JdlTGOOCGTAAA OOOTOCOCAN OCNOTCTTGC AAOTCAAATG TGAAAACCCC CGOCTTAACC TOOOAATTOC OTTTOAAACT CAAOOCTAOA OTOCACANAN ACCCGCATTT cccmxaru CQNCAOAACQ TTCAGTTTAC ACTTTTQOOO QCCOAATTOO ACCCTTAACO CAAACTTYOA OTTCCOATCT CACQTOTNTN O 1I 11 31 41 51 61 71 II 91 OCiOAGTOOAA TTCCTOTNTA CAANNAAATG CNT NANATNT NOAAAACCCC CATGNNAAOO NNCTCCCTOO Of NNCCTNCC CTCTNCCCAA ACNTOOOAOC CCCTCACCI'T AAGGACANAT OTTNNTTTAC ONANTNTANA NCTTTTOOOG OTACNNTTCC NNOAOaQACC CANNOOANOO OAOANOOOTT TONACCCTCO Sequcncing nsults for N.eu*opaea using reverse primer Eu1070r. 9 II 1I fl 41 II 41 II II 91 lDl TTTCOCOTTO CAT COAATTA ATCCACATAA TCCACCOCTT GTOCOOOTCC CCQTCAATTC CTTTOAOTTT TAATCTTOCO ACCOTACTCC CCAOOCOOTC AAAaCGCAAC OTAOCTTAAT TAQOTGTATT AGGTGGCOAA CACOCCCAOO OOCAOTTAAO OAAACTCAAA ATTAOAACOC TCiOCATOAOO OûTCCOCCAO Y II 1I 3 1 41 SI 61 71 Il 91 MITCCCCACOCT TTCOTOCATO AOCGTCAOTO TCAACCCAQO OAOCTOCCTT CQCCATCOOT OTTCTTCCAC ATCTCTACOC ATTTCACTOC TACACATOOA AGOOOTOCGA AAQCACGTAC TCGCAGTCAC AQTTOUGTCC CfCüACOGAA GCOGrACiCCA CAAOAAOGTO TAOAQATOCO TAAAOTOACO ATOTOTACCT Y II 11 11 41 II bI II Il 91 10IATTCCACTCC CCTCTOCTOC ACTCTAGCCT TOTAOTTTCA AACGCAATTC CCAGOTTAAO CCCGOOOCTT TCACATCTOA CTTOCAAOAC COCCTaCOCA T AAGGTOAGO OGAGACGACO T GAOATCQOA AC AT CAAAOT .TT OCOTT AAG GGTCCAATTC OQGCCCCOAA AGTOTAOACT OAACOTTCTO OCOOACOCOT Y II 11 II 41 II 61 II 81 91 )glCCCTTTACQC CCAGTAATTC COATTAACOC TCOCACCCTA COTATTACCG COOCTOCTOO CACOTAOTTA OCCGGTGCTT TTTCTGTCOO TACCaTCATA GOGAAATOCO GGTCATTAAO GCTAATTOCO AOCOTOOQAT GCATAATOOC GCCGACOACC OTGCATCAAT COOCCACOAA AAAOACAGCC AtOOCAOTAT Y II 21 3I 41 31 61 II 81 91 601AATCACAATT ATTCATTOCA ACTCTTTCTT TCCOACTAAA AOAGCTTTAC AACCCOAAOO CCTTCTTCAC TCACOCOOCA TOOCTOOATC AAOCTTTCOC TTAOTOTTAA TAAOTAACGT TOAGAAAOAA AOGCTOAT TT TCTCGAAATQ TTOOOCTTCC OOAAOAAOTO AOTOCOCCOT ACCOACCTAO TTCOAAAOCO 5' II 2 I 31 41 $1 61 71 II 91 CCATTOTCCA AAATTNCCCA CfOCTGCCTN CCaTAGCiAGf CTOGOCCONG TCTCANTCCC AOTOTOQCJTN OOCCOTCCTN TCAAACCAAC TACTOATCQO mlGGTAACAOGT TTTAANOOGT GACOACOOAN OGCATCCTCA OACCCOOCNC AGAOTNAQOG TCACACCCAN CCOOCAOOAN AOTTTOOTTO ATOACTAOCC 9 II II II JI SI 61 71 81 91 M,TQOCTTOOTA AOCCTTTACC CCACCAACTA GCTAAtTAAA AATNOONCOO TCCTTTACQC AAOOOCTTOC OAHCCCCTOC TTTTTTCTTA AANAA ACCaAACCAT TCGQAAATQG OOTClOTTtiAT CtIATTAATTT TTANCCNCiCC AGOAAATQCQ TTCCCGAACQ CfNOOOGACû AAAAAAOAAT TTNTT Squcncing results for N. winogrodrkjd using forward primer Eud SI 61 71 II 91 lCNTTOANAAA ACATTNNCTC TNTNNOCNNA QATTOCCGAC COUANNATTA TTTTTTNONA CCNNCCCCCN NOOCOaOOON TAACTACTCC AAANAOTONC ONAACTNTTT TOTAANNGAO ANANNCONNT CTAACOOCTO QCCTNNTAAT AAAAAANCNT OONNOOOOON NCCOCCCCCN ATTOATOAOO TTTNTCACNO 5' II II 31 41 SI 61 11 #I 91 ,OITNNTACCCiNA TACACCCTAC GOCOONAANG GQOGOATCOC AAOACCTCTN ACTATTOOAO COOCCOATNT CNOATTANCT AOTTOQTQQQ OTAAAOOCTC ANNATOOCNT ATOTOOOATO CCOCCNTTNC CCCCCTAOCO TTCTQOAOAN TOATAACCTC OCCOQCTANA ONCTAATNGA TCAACCACCC CATTTCCOAQ 9 II II II 4 $1 61 II 81 91 ACCAAOOCAA CNATÇCNTAO CTNGTTTGAN AGOACCACCA CTCCACACTO GQACTUANAC ACOONCCATA CTCCTACOOO AOOCAOCACT TOûOQAATff 'O1 TOGTTCCGTT ONT AGONAT C OANCAAACT N TCCTOOTGOT OAGCITOTOAC CCT OACT NT0 TOCCNUOT AT GAOOATûCCC TCCQTCOTOA ACCCCTTAAA O II II 31 41 SI 61 II 81 *I MITONACAATOO GOOAAACCCT OATNCAOCCA TNCCNCOTOT ATQATOAAOO CCTNNNQOTT GTAAANNACT TTNOOCAOAC AAOAAAAOOT TCCTCCTAAT ACNTOfTACC CCCTTTOOOA CTANOTCOOT ANOONOCACA TACTACTTCC OOANNNCCAA CATTTNNTOA AANCCOTCTO TTCTTTTCCA AOOAOOATTA 5' 1 I II 31 41 $1 61 11 II 91 ACNAGOTACT QNTNACNONN TTTTCANAAT AAOCACCGGC T AACT ACOTO CCAOCAOCCQ COOT AATACC TATNOOTOCA AOCOTT AATC NNAATTACfO TONTCCATOA CNANTONCNN AAAAOTNTTA TTCOTOGCCG ATTOATGCAC OGTCGTCOOC OCCATTATOO ATANCCACOT TCOCAATTAO NNTTAATOAC 9 II II I1 41 JI 61 71 II 91 UllaOCOTAAANC OTOfOfNOOC ONTTCOOAAA OAAANATOTO AAATCCCANO OCTCAACCTT OOAACTOCAT TTTTAACTOC COACNTTNAÛ TATOTCANAO CCOCATTTNO CACACANCCQ CNAAOCCTTT CTTTNTACAC TTTAOQOTNC COAOTTOaAA CCTTOACOTA AAAATTaACO OCTONAANTC ATACAOfNTC 9 II Il 31 41 II 61 71 II 91 60100000TANAA TTCCNCCTTT TNNCCNNNNA AAATGCCTTT NATNTNTOOA ANNAATNCCC OATOOCNAAA OOCANNCCCC CNTNOQATTA NTNCNTNACC CCCCCATNTT AAGONOOAAA ANNOONNNNT TTTACGGAAA HTANANACCT TNNTTANOaO CTACCONTTT CCOTNNOOOO CiNANCCTAAT NANONANTOO Y II 2 I II 41 51 61 11 II 91 tOICCTNAAAACH CNAAAAANCN TNOOOONNC GOANTTTTGN ONTTTTTNGN ANCCCCNNO Sequcncing resulis for N.winograds&yi using reverse primer Eu 1O7Or. Md May 16 11:35 2001 r.Wnb.wg Pyr No.I 9 II J I 3 I 41 SI 61 II II 91 lNNAANNNNTN NNNOTONCAC OAOCTOACOA CAOCCATOCA OCACCTQTQT TCCOONTNTC TNOCNANCAC OOACCAAATC TCTTACOQNT TfNCAOACAt NNTTNNNNAN NNNCACNGTG CTCGACTOCT GTCGGTACGT COTGOACACA AOOCCNANAO ANCONTNOTO CCTOQTTTAO AOAATQCCNA AANQTCTOTA Y II 2 I II 41 51 68 71 II 91 10IOfCAAOOOTA OGTAAOOTTT TTCQCOTTGC ATCOAATTAA TCCACATCAT CCACCOCTTO TOCOOOTCCC CGTCAATTCC TTTOAOTTTT AATCTTOCûA CAGTTCCCAT CCATTCCAAA AAOCQCAACG TAOCTTAATT AGQTGTAGTA OGTOGCOAAC ACOCCCAaOO OCAOTTAAOO AAACTCAAAA TTAOAACOCT O II II 31 II $1 61 7I fil II 10ICCQTACTCCC CAQOCOONNA ACTTNACGCO TTAOCTQCOC T'ACTAAQOCC TAACGBCCCC AACAQCTNOT TOACATCOTT TANOOCOTOO ACTACCAoQO QOCATQAGOO QTCCQCCNNT TOAANTOCGC AATCOACOCO ATBATTCCOO ATTOCCWOO TTGTCOANCA ACTOTAOCAA ATNCCOCACC TQATOOTCCC 5' II II 31 41 SI 61 71 II 91 10ITATCTAATCC TGTTTOCTCC CCACOCTTTC OTGNCTQAOC OTCAOTATTA TNCCAOOOOO CNOCCTTCOC CATCOOTOTT CCTNCACATA TCTACOCAtT ATAOATTAOO ACAAACOAGO OOTOCOAAAO CACNOACTCO CAOTCATAAT ANGGTCCCCC ONCGOAAOCO OTAOCCACAA OOANOTOTAT AOATOCOTAA Y II JI 31 41 51 4I 71 II II 10ltCACTOCTAC ACOTOOAATT CTACCCCCCT CTOACATACT CTAONTCOOC AOTNNAAAAT OCAOTTCCAA NOTTQAOCCC TOOOATTTCA CATCTTTCTT AOTOACOATO TOCACCTTAA GATOOOGOOA GACTOTATGA GATCNAOCCO TCANNTTTTA COTCAAOOTT NCAACTCOOO ACCCTAAAaT OTAOAAAOAA Y II 1l II 41 JI 61 71 II II UllTNNQAACCGC TACACACNCT TTACOCCCAA NAATTNCNAT NAACOCTTOO CCCCTACOTA TTACCONOoN TONTCiUNACN NANTTAOCCO OOûCTTATTN ANNCTTOQCG ATGTOTGNOA AATOCGOGTT NTTAANONTA NTTGCOAACC OOOOATGCAT AATOOCNCCN ACNACCNTON NTNAATCOOC CCCOAATAAN 9 II 21 II 41 51 6 1 71 II 91 601TNNNNNNACC CONNNNNANN NNNNNNNATN ANNAANONNN NNNNNNNNTN NNNOOCNNAA ANMNNCTNNN NNNNCCNNNN OOONNNTNNN NNNNNNNCNN ANNNNNNTOO OCNNNNNTNN NNNNNNNTAN TNNTTNCNHN NNNNNNNNAN NNNCCONNTT TNNNNOANNN NNNNQQNNNN CCCNNNANNN NNNNNNNONN Y II 2 I 3 I II JI 6I 71 Dl 91 N,NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNANAANNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNC NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNTNfTNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNO Table C 1 : ATCC Culture Medium 290 S6 for Thiobacillus thioparus. Cheniical Formda 1 Mass Agar (if required) Distilled water 1 .O litre CaCl, and FeCl, autoclaved seperately then added to rest. Table C2: ATCC Culture Media 480 for Nitrobacter winogradskyi, solutions can be made up and stored at room temperature for severa Components added to make final media (for individual components see below) I Solution A 1 Solution B Solution D Solution E Solution F Distilled water 1.O litre I Solution A Reagent Amount CaCI, 2.0 g Distilled water 100.0 ml Solution B 1 Reagent 1 Arnount Distilled water 100.0 ml Solution C Reagent Amount Chelated Iron 0.1 g Distilled water 100.0 ml Solution D 1 - Reagent Amount - N%Mo0,.2H20 0.1 g MnC 12.4H20 0.2 g I CoC12.6H20 0.002 g I ZnSO4.7H,O 0.1 g I CuS04SH20 0.02 II Distilled water 1 .O Iitre 1 Solution E I I 1 Reagent Amount 1 Distilled water 1 100.0 ml 1 Solution F i Reagent 1 hount &WO4 1.74 g Distilled water 100 ml Table C3: Nih.osornonas europaea media. There are three components to this media which are prepared and autoclaved separately then combined. Stock solutions can be made up ahead of theand stored. The recipe makes 1.5 litres of media. Reagent MgSO, (1 .O M stock solution) CaC12 (1 .O M stock solution) FeSO, (1 .O M stock solution) CuSO, (50 mM stock solution) Distilled water 1.2 litres Part II I I 1 Reagent Amount Distilled water 1 0.3 litres 1 Adjust pH to 8.0 using ION NaOH before autoclaving. Part III Make up 500 ml of 5% (wh) NqCO, anhydrous and autoclave Final media Combine Parts I and II with 12 ml of part III and inoculate. Colorimetric Nitrite Analysis - Griess-Ilosvay method Reagents 1) Diazotizing reagent 0.5 g sulphanilarnide in 100 ml 2.4 M HCl (stored at 4°C). 2) Coupling reagent 0.3 g of [N-(1-naphthy1)-ethylenediarnine] hydrochlonde in 100 ml of 0.12 M HC1. (stored in arnber bottie at 4°C). Procedure A) To I ml of sample add 20 pl diazotizing reagent, mix and wait 5 minutes. B) Add 20 p1 couplhg reagent, allow colour to develop (recommended 20 minutes). C) Measure absotbance at 540 m. Protocol for Sephadex Spin Columns 1) Materials required, 5 cc syringes, sterile 15 ml polypropylene tubes, and glass wool cut into 2-3 cm lengths. 2) Take off caps of 15 ml tubes, remove bottom pIug of 5 cc syringes and place syringes in 15 ml tubes. 3) Use the containment hood to prevent glass wool fiom getting in your eyes and lungs, pull plunger fiom syringe and add glass wool, replace plunger, you want a 2 mm thickness of glas wool in the bottom of the syringe. 4) To a 250 rdbottle add 6 g Sephadex and 120 ml TEN buffer (pH 8), swirl. 5) Remove, carefùlly, plungers fiom the syringes. 6) Add 5 ml of Sephadex to each syringe, cover each syringe top in foil. 7) Centrifùge the spin columns for 1 min at 900 rpm. 8) Discard the TEN bufTer in the bottom of the 15 ml tubes. 9) Add 1 ml of TEN buffer to the Sephadex column and repeat (step 7). 10) Add another 1 ml of TEN buffer to the Sephadex and repeat (step 7). 11) Discard the TEN buffer in the bottom of the 15 ml tubes. 12) Place spin columns in an autoclavable rack, place foil over al1 tubes and autoclave for 20 min on liquid cycle (mut be autoclaved the sarne day they are made). 13) Store in a plastic bag at 10 OC for 1-2 months. To Use. 1) Place syringe with Sephadex in new 15 ml stenle tube. 2) Carefully add DNA sarnple to top of Sephadex column, single drops in centre of column. 3) Spin at 900 rpm for 1 min. 4) Discard syringe, retain cleaned DNA in bottom of 15 ml tube. Preparation of Genomic DNA from Bacterial Cultures Starting with 1.5 ml of culture. 1) Pelletize cells by centrifüging, discard supernatant, resuspend pellet in 567 pl of TE baer (50 mM Tris, 50 mM EDTA, adjusted to pH 8.0). 2) Add 30 pl of 10% SDS (sodium dodecyl sulfate) and 3 pl of 20 mghl proteinase K, mix and incubate 1 hr at 3 7 OC. 3) Add 100 pl of 5 M NaCl and mix thoroughly, add 80 pl CTAB/NaCl solution, mix, incubate 10 min at 65 OC. 4) Add equal volume chlorofodisoarnyl alcohol, rnix and centrifuge 4 to 5 min. 5) Transfer the supematant to a fiesh tube. 6) Add equal volume phenoVchloroform/isoamyl alcohol, mix and centrifuge 5 min. 7) Transfer the supematant to a fiesh tube. 8) Precipitate DNA using 0.6 vol. isopropanol, centrifuge to form DNA pellet, discard supernatant. 9) Resuspend DNA in sterile ultra-pure water.