Microbiological oxidation of sulfide and acedate in a denitrifying fluidized bed reactor: Fundamental and engineering aspects

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MICROBIOLOGICAL OXIDATION OF SULFIDE AND ACETATE

IN A DENITRIFYING FLUIDIZED BED REACTOR

f u n d a m e n t a l a n d microbiological a s p e c t s

* U % ' ^

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P e t r u s J o s e p h u s F r e d e r i c u s Gommers

MICROBIOLOGICAL OXIDATION OF SULFIDE AND ACETATE

IN A DENITRIFYING FLUIDIZED BED REACTOR

fundamental and microbiological aspects

PROEFSCHRIFT

t e r verkrijging van de graad van doctor aan de

Technische Universiteit Delft, op gezag van de

Rector Magnificus, P r o f . d r . J.M. Dirken, in het

openbaar te verdedigen ten overstaan van een

commissie aangewezen door het College van

Dekanen op 14 mei 1987 t e 16.00 u u r

door

Petrus Josephus Fredericus Gommers _.

geboren te 's Gravenhage. / '>j

TRdiss

A

Dit proefschrift is goedgekeurd door de promotoren

P r o f . d r . J . G . Kuenen

STELLINGEN BEHORENDE BIJ HET PROEFSCHRIFT VAN P.J.F.GOMMERS

Batch

jn niet geschikt om het vermogen van organismen

bronnen te gebruiken, te bepalen.

trophe microorganismen, die sulfide, zwavel en/of

ren, zijn waarschijnlijk chemolithoheterotrophe

Een na

op de .

dat een

tot migi

Dit betel

situaties

- At

Wa

Het vergelij

geimmobilis.

misleidende

- Bosm:

o*

(9

(1974) Arch.Micr.99,1-14

acteriepopulaties in fluid bed reactoren

fwezigheid van gasstromen.

e invloed van grootte en soortelijk gewicht

van met biolagen begroeide deeltjes leert

uid bed reaktoren niet in alle gevallen leidt

aar substraat arme posities in de reaktor.

e verschillen wel degelijk tot instabiele

al fluidized bed treatment of Water and

D.F. & Atkinson B. (1981) page 26

\capaciteiten

van reaktoren gevuld met

(gelijk specifiek oppervlak noopt tot

^Vat.Res.10, 297-308

6. De hoge celop, ^_^^...—rnföbacillus versutus, gekweekt op mengsels

van acetaat eri-irïïosulfaat, zijn niet uitsluitend te verklaren door het

sparende effect van de assimilatie van acetaat in plaats van C O T .

7. Het feit dat 'fuzzy' wiskundige modellen hun intrede hebben gedaan in

de beschrijving van biologische en biotechnologische processen zegt iets

over de mate van inzicht in deze processen die nu bestaat.

- Dohnai M. (1985) Biotech.Bioeng. 27,1146-1151

8. Een maatregel uitgevaardigd door het Ministerie van Onderwijs en

Wetenschappen om de wetenschappelijke produktiviteit van faculteiten

te toetsen aan het aantal wetenschappelijke publikaties per

onderzoek-manjaar zal leiden tot een stimulering van de economie- en

rechtenfaculteiten t.o.v. de beta- en technische faculteiten.

- Jaarverslagen Nederlandse Universiteiten 1985

9. Hoewel de mengtijd van de klanten van de Bijenkorf te Amsterdam op

zaterdagmiddag in de orde van 100 dagen ligt is dit niet lang genoeg

om stratifikatie van de populatie te veroorzaken.

10. De industriële ervaring dat met een rigoreus doorgevoerde kwaliteits­

bewaking en -controle, kosten gereduceerd kunnen worden pleit voor

een stringente invoering van milieubeschermingsmaatregelen.

11. Leven op Europa (maan van Jupiter) is mogelijk door de

aanwezigheid van gereduceerde zwavelverbindingen, zonlicht en

carbonaten.

- Clarke A.C.(1982) 2010, Granada Publishing, London

- Ruby E.G. et al (1981) AppI.Environm.Micr.42,317-324

12. Tijdens het werken met thiosulfaat dient het onstaan van thio-fanaten

vermeden te worden.

VOOR PA en MA

VOOR JANET

This study was carried out at the Department of Microbiology and

Enzymology of the University of Technology Delft, The Netherlands.

The investigations were supported by the Foundation for Fundamental

Biological Research (BION) which is subsidized by the Netherlands

Organization for the Advancement of Pure Research (ZWO).

VOORWOORD

PROMOVEREN DOE JE ALLEEN, HET WERK MET ANDEREN ! De volgende personen/groepen wil ik in het bijzonder danken: Gijs, voor de vele stimulerende, maar juist ook kritische discussies;

wetenschap bedrijven heb ik van jou geleerd

Karel, voor de technologische inbreng; dimensieanalyse en microbiele fysiologie laten zich niet zomaar verenigen

Willem, zonder jouw hulp, je inventieve aanpak en persoonlijke stijl was het niks geworden

Lesley, some day I will choose between sulfur and sulphur, thanks for putting up with my accent

Bert en Ferry, voor jullie hulp en persoonlijke inbreng in het werk en het bruggehoofd dat jullie vormden tussen Micro en BPT

Seff en Arnold, die vanuit Gist Brocades zorgden dat het werk wat dichter bij het wereldse bleef

Iedereen in de verschillende werkplaatsen, voor de vele fraaie bouwwerken, geduld en behulzaamheid, zelfs voor iets wat ik nooit gebruikt heb Frieda, Anita en Marion, voor het (bijna voorspelbare) paniek-typewerk Hans, voor de energieke intro in de energetica en de rol als praatpaal Bart, voor de extra's, het zooien en de introduktie in het Leidse en

Groningse uitgaansleven

Pa en Ma, voor de basis, het geduld en de nimmer aflatende belangstelling en vertrouwen

en tenslotte Janet, voor de verandering en de o zo noodzakelijke duwtjes in de rug bij de laatste loodjes

MET DANK AAN IEDEREEN DIE MEEGEHOLPEN HEEFT HET BED FLUÏDE TE HOUDEN !!

CONTENTS

CHAPTER PAGE

1 Introduction 3

2 Gas-Phase influence on the mixing 54

in a fluidized bed bio-reactor

3 Thiobacillus strain Q, a chemolitho- 63

heterotrophic sulphur bacterium

4 Biochemical limits to microbial growth 95

yields, an analysis of mixed s u b s t r a t e

utilization

5 Simultaneous sulfide and acetate 123

oxidation in a denitrifying fluidized

bed r e a c t o r . - S t a r t up and reactor

performance-6 Simultaneous sulfide and acetate 155

oxidation in a denitrifying fluidized

bed reactor.-Measurements of activi­

ties and

conversions-7 Summary and concluding remarks 186

Samenvatting en concluderende opmerkingen 195

CHAPTER 1

1.1 INTRODUCTION

The evolution of waste water treatment systems during the last twenty years can be summarized in two trends (table 1):

- an increasing number (and complexity) of reactor designs (eg the introduction of new reactor types in waste water treatment such as fluidized beds and air lift loop reactors)

- an increasing number (and complexity) of specific conversions removing an increasing number of pollutants (eg the addition of the specific removal of sulphur, nitrogen and phosphorous compounds to the traditional BOD/COD REMOVAL)

The first trend has been accompanied and sustained by a considerable research effort from the engineering sciences. The second trend has been stimulated mostly by fundamental research in biochemistry and microbial physiology and ecology. In well defined systems, such as pure culture fermentations, a considerable degree of integration between the engineering and fundamental sciences has taken place (e.g. baker's yeast production, penicillin production). However, in this respect the waste water treatment industry is lagging behind. This is not the result of poor communication between the engineering and fundamental research groups, but is caused by the undefined and complex nature of the microbial populations present in waste water treatment systems. As a result, the engineer has to rely on black box measurements and overall kinetic data, and often does not have sufficiently detailed microbiological knowledge to predict the behaviour of the complex microbial system. Specialized industrial waste waters containing a limited number of compounds might result in less complex situations, and could thus provide the most promising starting point for the integration of engineering and fundamental research.

Table 1 Wastewater treatment in 1960 and 1980 Compounds Removed Partculates BOD/COD (Colour/Odour) Particulates BOD/COD (Colour/Odour) N-compounds P-compounds S-compounds Heavy metals Trace compounds Microbial Processes used Aerobic Oxidation Anaerobic Digestion Aerobic Oxidation Acidification/Methane formation Nitrification Denitrification Sulfide oxidation Sulfate reduction Phosphor accumulation Trace meat accumulation

Reactors used Activated sludge Trickling Filter Digestion Vessel Lagoons/Oxidation Ponds Activated Sludge Trickling Filter Digestion vessel Lagoons/Oxidation Ponds Upflow fixed bed Expanded/Fluidized bed Sludge Blanket

Rotating Disk Contractor Air Lift Loop Reactor

This thesis concerns the treatment of waste of relatively simple compositions e.g. the effluent of a methanogenic reactor used for treatment of industrial waste. This effluent contains sulfide and lower fatty acids (e.g. acetate). These compounds are removed by denitrification with nitrate in a fluidized bed reactor. Despite its relative complexity this type of reactor was selected because it provides a high conversion capacity (Heynen 1984).

Substantial fundamental microbiological knowledge of both the physiology and ecology of pure and mixed (both defined and undefined) cultures capable of sulfide oxidation (Gottschal and Kuenen, 1980a, 1980b, 1981; Beudeker et al, 1982; Kuenen and Beudeker, 1982) was already available providing a firm basis for this study.

This introduction falls into three sections:

a) The microbial recycling of elements, and its application in waste water treatment, with particular emphasis on the processes of sulfide oxidation and denitrification.

b) Some theoretical and technological aspects of a fluidized bed system. 5) The physiology and ecology of the sulfide oxidizing bacteria, together

with some aspects of bacterial carbon and energy metabolism. This will provide the necessary background for a discussion of biomass production in pure and complex cultures.

1.2 ELEMENT CYCLES AND WASTE WATER TREATMENT

The microorganisms u3ed in waste water treatment systems originate from nature and generally perform the same type of conversions in their habitat. Although these conversions are independent reactions they are part of complete element cycles without which life could not exist. It should be

Figure 1: The strong analogy between the nitrogen and the sulfur cycle (after Kuenen, 1980).

1. assimilatory sulfate redcution 2. assimilatory nitrate reduction 3. desulfurylation by microorganisms 4. amraonification by microorganisms 5. nitrogen fixation to org-NH„ 6. nitrogen fixation to ammonia 7. dissimilatory sulfate reduction 8. denitrification to nitrogen gas 8a. dissimilatory nitrate reduction 9. (lithotrophic) oxidation of sulfide 10. (lithotrophic) oxidation of ammonia

remembered that, although these cycles are usually considered separately, they are closely linked, and cannot proceed in isolation. The system described here utilizes organisms that play an important role in both the nitrogen and sulfur cycles, (fig.1).The conversions in these cycli involve electron transfer reactions. The reduction of sulfate to sulfide (either via assimilatory or dissimilatory routes) coupled with the reoxidation of the sulfide to sulfate has strong parallels with the reduction of nitrate to ammonia, followed by the reoxidation of the ammonia via nitrite to nitrate. Of great importance to the system under study is the interaction between these cycli as the oxidation of sulfide is done with nitrate as the electron acceptor instead of oxygen. Microorganisms which perform the steps illustrated in the cycles have been isolated and studied. In wastewater treatment plants, often only a part of any given element cycle takes place. However, it should be remembered that the reactions in question can also proceed in the opposite direction. Especially when reduced sulfur compounds are to be treated, this phenomenon should be taken into account in the choice or evaluation of desired alternative end products.

1.3 SULFIDE REMOVAL IN WASTE WATEH TREATMENT

Sulfide is a very general pollutant of municipal and industrial wastes, whereas other reduced sulfur compounds such as thiosulfate and thiocyanate usually only occur in isolated, industrial wastes. Thiosulfate is one of the major pollutants of photographic wastewater (± 100 gr/1). Wastewater from coal conversion processes aimed at the production of liquid and gaseous fuels contain both thiocyanate (± 500 mg/1) and hydrogen sulfide (± 1000mg/l). The rapidly increasing capacity for anaerobic methanogenic wastewater treatment presents a new and potentially very large source of hydrogen sulfide-containing wastes. In these anaerobic systems, the sulfur

'/able 2 Toxic effects of H, S (Wheotley, 1979). Concentration PPM * Vol Exposure Period Physiological Effect 100 200 500 1000 0.01 0.02 0.05 0.10 hours 60 min 30 min

irritation of nose and eyes headaches, dizziness nausea, exitcment, insomnia unconsciousness, death

Table 3 Hydrogen sulfide removal processes I. ABSORPTION FROM GASES USING

INORGANIC SUBSTANCES IN SOLUTIONS AND SUSPENSIONS

Potass iiiio Phosphates (3) Sodium Carbonates (3} Iron Compounds (5) Alkali Metal Hydroxides (2) Arsenic Compounds (2) Potassium Carbonate (4) Alkaline Liqui ds not Forming

III. REMOVAL FROM GASES BY 0THBH METHODS

OXIDATION (11)

Homogeneous Catalysis with Amines

N-Methylpyrrolidone as Liquid Phase

Catalytic Oxidation to S0_ Oxidation In Sewage Digestion

Alkaline Liquors from Sulfate Cellulose Pulping Sodium Bisulfite

Sodium Sulfide, Hydroixde and Carbonate

Alkaline Earth Metal Sulfides Cellulose Pulping

Sodium Bisulfite

Sodium Sulfide, Hydroxide and Carbonate

Alkaline Earth Metal Sulfides Sodium Bicarbonate

Ammonium Hydroxide

ABSORPTION FROM GASES WITH ORGANIC COMPOUNDS

ALKANOLAMINES (13) Triethanolaroine

Monoethnnolamiiie + Diethylene IV. Glycol Modified Dipropanolamines Diisopropanolamines Alkanolamines + Morpholinones Addition of Sulfolane 2,2-Dimethyl-l, 3-dioxolane-4-methanol Alkanolamines + Ethers OTHER AMINES (6) ETHERS (4) ESTERS (2) OTHER COMPOUNDS (8)

Contact with Liquid Anhydrous

S 02

Catalytic Incineration Oxidation with Iodine in Organic Solvents

Oxidation to SO„ and Recovery of Sulfur

ADSORPTION (2)

Contact with ZnO at Elevated Temperatures

Use of Porous, Impregnated

Charcoal HYDROTREATINQ PROCESS USE OF ELECTROLYSIS H2S RECYCLE IN WHITE LIQUOR

REGENERATION

USB OF ION EXCHANGE RESINS USE OF MOLECULAR SIEVES ADSORPTION AND OXIDATION REMOVAL FROM LIQUIDS

FROM FLUID HYDROCARBONS (4)

Recovery from Other Acidic Gases

Removal by Electrolysis With Molecular Sieves From Benzene

FROM AQUEOUS SOLUTIONS (6) Stripping with Steam Degassing with NH3 Recycle Oxidation to S and ( Use of Nitrogen Gas Using Membranes Use of Distillation

compounds present (usually sulfate) are often reduced to sulfide. The necessity to remove hydrogen sulfide from the effluent originates from its high COD/BOD value and malodorous and poisonous properties (see table 2 ) . A wide spectrum of chemical and physical methods for the removal of hydrogen sulfide from gasses and liquids exists (table 3 ) . These range from gas absorption with aqueous solutions or organic solvents to removal by oxidation or absorption onto ion exchange resins. Most methods were developed in the oil and natural gas industry and they either involve the addition of expensive and environmentally hazardous chemicals, or the use of energy intensive technologies. Biological removal has only been considered by a small number of authors (eg Obayashi, 1985). The products of biological oxidation are elemental sulfur and/or sulfate. No consensus exists as to which product is considered economically most desirable. The advantages and disadvantages of elemental sulfur and sulfate production are shown in table 4. (see also fig.2). Depending on the relative importance of the different criteria a choice can be made. If the production of sulfuric acid is needed, the formation of sulfate may be desirable, but in most cases elemental sulfur would be preferable as an end product, because only then is the sulfur definitively removed from the effluent. However, as long as both microbiological and technological processes for the formation and processing (separation) of elemental sulfur are not available, and the wastewater can be dischargead into the sea or to other water bodies with a naturally high sulfate content, the formation of sulfate is an acceptable alternative. The system used in this study developed for the microbiological conversion to sulfate rather to sulfur as the treatment plant was close to the sea. Possible microbial processes for the microbial oxidation of sulfide to sulfate include (see fig.1) aerobic oxidation, phototrophic oxidation or oxidation with nitrate (denitrif ication). The aerobic process has two disadvantages. Firstly, oxygen most be provided via aeration (introducing a

Table 1 : Advantages and disadvantages of sulfide oxydation products Product SULFATE (S0=) ELEMENTAL SULFUR

(S°)

Advantages NON-TOXIC

MAJOR ION IN SEAWATER SOLUBLE PRODUCT . easy separation from biomass . no build-up in reactor NO EUTROPHIC POTENTIAL EFFLUENT NET REMOVAL OP 'S' POSSIBLE ECONOMIC VALUE NON TOXIC

Disadvantages

EUTROPHIC POTENTIAL - EFFLUENT NO NET REMOVAL OF 'S' NO ECONOMIC VALUE IN SOLUBLE PRODUCT . difficult separation from biomass . build - up in reactor EUTROPHIC POTENTIAL SLUDGE WASTE

110

Billions of lb

100.

90_

Consumption

80.

Production

70_

7

J L

J I I L

1980

1985

1990

7

1995

Figure 2: Global consumption of sulfur outpaces production (data for non communist countries only).

Redrawn from Chem. & Eng. News (1985) nov. 4, anonymous, sulfur market tightening world wide.

gas phase with extra transport restrictions). Secondly, during the process two equivalents of acid are produced per equivalent of sulfide oxidized H_S + 2 0 , + S07 + 2H ) . The phototrophic oxidation, though feasible on the laboratory and pilot plant scales, presents scale-up problems with respect to the efficiency of illumination. Another drawback of the phototrophic oxidation is the high biomass production per molecule of sulfide oxidized, Also if elemental sulfur is the chosen end product, there is the disadvantage that in many of the practical systems tested so far, the sulfur is often almost exclusively intracellular (Kobayashi et al, 1983). Anaerobic sulfide oxidation with denitrification produces less acid than the aerobic oxidation and can proceed completely to sulfate (Kuenen, 1975), but the supply of nitrate may be expensive. The system proposed in a recent patent (Mulder, 1982, E.P.A. 0051.888) uses the nitrogen already present in the waste water to this purpose (fig.3, step 3 ) . The nitrogen compounds (almost exclusively ammonium) are biologically converted to nitrate in an aerobic reactor (nitrification, step 4 ) . The nitrate-containing water is mixed with the sulfide-containing effluent from the methanogenic reactor. It is clear from fig.3 that this process has the advantage of the simultaneous removal of nitrate (denitrified to nitrogen gas) and conversion of sulfide to sulfate. As this process is based on denitrification some aspects of this conversion in waste water treatment systems will be discussed.

1.4 DENITRIFICATION

Denitrification is one of the three biological processes involved in nitrate conversion. The other two processes, assimilatory and dissimilatory nitrate reduction, yield ammonium as the end product, while denitrification converts nitrate to nitrogen gas via such intermediates as nitrite, nitrous oxide and nitric oxide (Robertson and Kuenen, 1984). The biochemical and physiological

CO,/CH,/H,S

2 A 2

Gas

J J

CH

4

/C0

2

/H

2

S

Gas

t i n

Wastewater

Kj-N.S0|7C0D

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1

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Spent Air

Cleaned

Wastewater

T

00

Air

Fatty Acids

S

2

7Nr£

Methane Formation

2

Recycle NO"

s

2

;ml

Nitrification Nitrification

3 4

I

Reactions in Column. 3

S

=

Organic

Compounds

N0

3

-

sa-co

2

*

N

2

/

Figure 3: Integral wastewater treatment plant as described in E.P.A. 0051.888.

properties of denitrification have been studied extensively and reviewed recently by Knowles (1981), Payne (1981) and Delwiche (1984). Generally, the physiology of denitrification is very similar to aerobic physiology, (eg with respect to the function of the respiratory chain) and denitrifying species are also able to use oxygen. The yields obtained with denitrifying bacteria are generally lower than those found when the same species is grown aerobically. The amount by which the nitrate-based yield is lower than that of the oxygen-based culture varies with the structure of the electron transport chain involved, but denitrifying yields 60S! of those obtained aerobically are not uncommon. In many of the known denitrifying organisms the presence of oxygen inhibits denitrification, but this is not always the case (Robertson and Kuenen, 1984). The use of of denitrification in waste water treatment was reviewed by Winkler (1984). It is usual to remove oxygen from the effluent in order to provide optimal denitrifying conditions. This review, however, deals almost exclusively with heterotrophic (organic) denitrification . and not with denitrification supported by reduced sulfur compounds.

Instead of compiling a comprehensive review of denitrification only two subjects will be discussed here. These are the use of fixed biomass reactors for waste water denitrification and the different electron donors which can be used. In both cases, the emphasis will be placed on the use of reduced sulfur compounds.

.DENITRIFICATION IN WASTE WATER TREATMENT SYSTEMS USING FIXED BIOMASS.

The use of fixed biomass reactors for anaerobic processes such as methane production and denitrification has received much attention as the main disadvantage usually encountered with this type of reactor (diffusion

limitation of gaseous substrates e.g. oxygen) is not relevant. This process has evolved from laboratory via pilot plant to full scale plants and can achieve very high conversion rates compared with other systems (table 5 ) . This is mainly due to the high biomass concentrations attained in the reactor (5-40 gr./L). The specific conversion rates (per unit of biomass) are comparable in most systems (table 5 ) . Table 6 gives an extensive, but not complete, overview of fixed biomass systems used for denitrification reported in the literature. A wide range of nitrate concentrations (1-7550 mg N0„ -N/l) has been used, mostly depending on the intended application. Two groups can be recognized, the first concerned with reducing the highly concentrated nitrate levels in specific industrial waste waters to a manageable amount and second a group concerned with bringing down the nitrate concentration to a level low enough to avoid adverse effects on the environment (<10 mg/1). Although no strict linear relationship between the influent nitrate concentration and the achieved loading rate exists, the increase of the removal rates with influent concentration is unmistakable (figure 4 ) .

The 'heterotrophic' reactors show distinctly higher removal rates than the 'inorganic' reactors (figure 5 ) , while for "mixed type" influents (both organic and inorganic, EPA 00051.888 and chapter 5 of this study) intermediate values are found. The most likely cause of these differences is the variation in the biomass concentration in these systems because of the different biomass yields.

ELECTRON DONOHS FOR DENITRIFICATION

The type of electron donor used by denitrifying organisms can vary widely among the organic compounds (Rhee and Fuhs, 1978; Francis and Callahan, 1975; Skrinde and Bhagat, 1982) but also includes reduced sulfur compounds

Table 5 : Comparison of Reactor types in wastewater treatment systems REACTOR TYPE CONTACT PROCES FIXED/PACKED BED U.A.S.B. FLUIDIZED EXPANDED BED BIOMASS CONCENTR. 8/1 0.5 - 2 5 - 1 5 5 - 2 0 10 - 40

REPORTED REMOVAL RATES METHANE FORMATION kg COD/m.d. 3 - 5 3 - 10 5 - 2 0 20 - 50 DENITRIFICATION kg N/m .d 1 - 3 1 - 5 3 - ? 5 - 6 5

Tabel 6 : Reported reactor loading rates for denitrifying fixed biomass reactors. Electron 1 donor aspar-tate methanol ethanol B.O.D. methanol methanol settled sewage ethanol methanol methanol methanol methanol methanol molasses nitrate cone.in feed in mg N/1 30 200 250 500 1000 1500 2500 7-15 20-130 30-1*0 250 500 1-20 1500 20-30 32 1.2 21.5 18.7 55.0 11*50 kg N/n? . day reactor loading 1-2 3.5 13-65

8

19 26 50 50 1.8-1».8 32 3.1 5.2 15 32 3.8-T.U 60 3.6 1».7 6.2 5-5 7.2 13.5 10.6 38.lt Temp °C 5-20 1 22-30 10-15 20 8-15 27-31 10-20 25-38 10-20 .22 20 19 25 20 38 Reference Nilsson 1980 Vissoughi 1982 Hancher 1978 Cooper "1982 Gasser 1975 Cooper 1980 Walker 1981 Gauntlett 1981 Bosman 1981 Hawkins 1981 Jeris 197U Jeris 1975 Bosman 1978 Remarks Pure culture Ps.den.in alginate fixed bed fluid bed fluid bed eff1.10-20 mgN/1 fluid bed fluid bed fluid bed fluid bed fluid bed fluid bed high nitrate effluent expanded bed fluid bed fluid bed fluid bed Electron donor methanol sulfur Thiosul-fate sulfide Thiosul-fate Thiosul-fate sulfur sulfide sulfur Hydrogen sulfide +acetate sulfide +acetate nitrate cone.in feed in mg N/1 20-1» 10 30 25 25 1000 l»00-1l»00 50 25 30-lt0 60 50 20 kg N/mJ . day reactor loading 0.05-0.55 0.12 0.025 0.08 0.075 1.6-5.6 0.017 0.06 1.5-3.0 0.13 2.5 5.0 Temp °C 25-33 30 30 30 30 28 20 1 1 35 35 Reference Polprasert 1986 Batchelor 1978 Bisogni 1977 Steinmul-ler1980 Claus 1985 Driscoll 1978 Gaid 1980 Kurt 1981» Patent EPA 0051. 888 thi s study Remarks filter mixed vessel packed bed packed bed packed bed packed bed packed bed fluia bed fluid bed fluid bed

Figure 4: The acchieved loading rates for fixed biomass denitrification

reactors is higher for nitrate influent concentrations larger

than 100 mg N/l.

<D

Reactor Loading

KgN(m

3

day)

_1

< 1

1-10

10-30

> 30

Nitrate Influent Concentration

N/ i \ - 1

<100mg

N

(l )

>100mg

N/ . x-1

N

(l )

26%

70%

4 %

0 %

ft

10%

25%

3 0 %

35%

Applied

Reactor

Loadingrate <|

Kg NCL_N(m

3

.dayr

10 H

10

o

5-Heterotrophic

O This Study o

* ♦Patent

x Autotrophic

x

x

10 20 30 5 50

60~

Electron donor

o Heterotrophic

X Sulphur compound

oMixotrophic

(organic* sulfide)

70 80 90 100

Influent N0£(mgN.l )

Figure 5: Denitrification loading rates in fluidized/expanded or fixed

(Bisongi and Driscoll, 1977; Timmer ten Hoor, 1976; Wood fc Kelly, 1983; Justin & Kelly, 1978; Robertson & Kuenen, 1983) and hydrogen (Kurt et al. 1984). Some representative compounds are shown in tabble 7 together with their electron donating capacity. This diversity is illustrated also by the large number of bacteria that are recognized as denitrifiers (Bergey, 1974; Payne, 1981). Organisms using heterotrophic or autotrophic (inorganic) electron donors possess different assimilatory pathways for the formation of biomass. Reduced organic compounds are partially used as a source of carbon for the formation of biomass. During autotropic growth on the inorganic electron donors, inorganic carbon (C0„) serves as the carbon source. The assimilation usually proceeds via the Calvin cycle (Kuenen and Beudeker, 1982). The system studied in this thesis is neither solely autotrophic nor heterotrophic as both acetate and sulfide are present in almost equimolar concentrations. When these substrates are present in limiting concentrations, mixotrophic growth can take place (Kuenen & Beudeker, 1982).Only a limited number of organisms are known to be able to simultaneously use organic and inorganic compound as electron donors for denitrification.These include Thiosphaera pantotrpha (Robertson and Kuenen, 1983) which can use reduced sulfur compounds or hydrogen together with an organic compound. Aerobic mixotraphy is more widespread (Kuenen & Beudeker, 1982). Indeed, T. versutus and P. denitrifacions are both able to grow mixotrophically if oxygen is provided, but cannot use reduced sulfur compounds as the electron donor for denitrification. A good insight into the physiological behaviour of these types of organisms is an important part of the understanding of the behaviour of a complex wastewater treatment system. In discussing some general aspects of the processes for the removal of sulfur and nitrogen compounds from wastewaters, three areas of special interest for the system described in this thesis can be distinquished: - the application of fluidized bed reactors (par. 1.5).

Table 7 t Electron donors for denltrifIcatlon . 10 electrons + 6 H O + 2 NO* ->■ N + 12 OH TYPE ALCOHOL CARBOHYDRATE ORGANIC ACID N. ALKANE BENZENE-DERIVATE INDUSTRIAL WASTE INORGANIC COMPOUNDS EXAMPLE METHANOL GLUCOSE ACETATE PROPANE BENZOATE MOLASSE SULFIDE THIOSULFATE SULFUR HYDROGEN

mmole electrons donated PER mMOLE 6 24 8 20 30

-8 8 6 2 PER GRAM 0.19 0.13 0.13 0.45 0.25 0.07 0.24 0.05 0.19 1

GRAMS ELECTRON DONOR NEEDED TO REMOVE lg NO " - N 1.9 2.7 2.7 0.8 1.5 5.1 1.5 7.1

1.9

0.4

- the behaviour of different physiological groups of sulfur oxidizing bacteria (par. 1.6).

- the effect of mixed electron donors on biomass yields of microorganism (par. 1.7).

The relationships between the different chapters are demonstrated in the following schematic presentation, and are based on two basic questions concerning waste water treatment reactors:

- how much sludge (biomass) growth occurs

- what conversion capacity can the reactor achieve under various conditions.

<a

'SLUDGE GROWTH -

^

a a

a

Biomass yields dual substrates I 5 I Biolayer formation I 5 I Sludge production

<2>

WASTEWATER ANAEROBIC TREATMENT

9

H2S

<2>

DENITRIFICATION in fluid bed reactor electron donors: sulfide + acetate FUNDAMENTAL KINETICS

°»

m

EMPIRICAL

Mixing in fluid bed reactors Type of organism with r " ^ l acetate & sulfide 1 P Reactor performance I 5 I | 6 | Biomass activities

1.5 FLUIDIZED BED REACTORS IN WASTE WATER TREATMENT

The advantages and disadvantages of different reactor types for waste water treatment applications have been reviewed frequently during the last ten years (eg Henze & Harremoes, 1983; Rittmann, 1982; Switzenbaum, 1983; Olthof & Oleszkiewicz, 1982). For this reason, only fluidized bed reactor willbe reviewed here.

ADVANTAGES ASSOCIATED WITH FLUIDIZED BED REACTORS

-high biomass concentration, and therefore high volumetric loading rates

-high liquid velocities can be used

-no blockage problems need to be expected if suspended solids are present in the wastewater to be treated

DISADVANTAGES RELATED TO FLUIDIZED BEDS

-liquid recycling is frequently necessary (with extra pump capacity) -suspended solids will not remain in the reactor and are not

likely to be removed

-process control is relatively complex

The reactor used in this study differs in one main respect from fluidized bed reactors already described in the literature. The biolayer is 2-3 mm instead of 50-200 pm thick. Due to these thick biolayers, three aspects of fluidized bed reactor performance must be discussed in more detail. These are the fluidization behaviour of the particle, the mixing of the liquid and solid phases, and the diffusion of substrates into the biolayer.

Before addressing these subjects, a comparison of the original pilot plant reactor (operated at Gist Brocades, Delft) with the laboratory-scale model is presented. Technical data and a few important dimensionless parameters are shown in table 8. The use of dimensionless parameters to facilitate the direct comparison of reactors of different scales is commom and, the good agreement between the parameters for the two systems confirms that the laboratory model is representative of the larger system. One dimensionless parameter should be added to the traditional ones. This is the dimensionless concentration gradient in the reactor. This is necessary to create comparable selection conditions for the different physiological types of bacteria believed to play a role in the reactor. As is explained in paragraph 1.6, the ratio of organic to sulfur compounds experienced by the organisms plays an important role during the selection/competition between these organisms. These ratios may be different at different heights in the reactor,due to different consumption rates of the organic and of the sulfur compounds. It was therefore reasoned that if the concentration gradients (dimensionless) were comparable in the pilot plant and the laboratory reactor, the selected gradients of organisms in the two reactors would be comparable. The essential assumptions and basic chemical technological knowledge for the calculation of this were:

- the fluidized bed reactor was considered to be a plug flow reactor (r = Vj * A * dc)

- the conversions in the reactor were described as a function of the biomass surface (and not of the volume).

(r = f(cx).a.A.dx and a = 6(l-«.)/dp)

- the microbial population would express the same kinetic relationship in both the pilot plant and the laboratory reactor

Ta'ule 8 : TECHNICAL DATA AND DIMENSIONLESS PARAMETERS OF THE LABORATORY AND THE PILOT PLANT FLUIDIZED BED REACTORS USED

LENGTH (m) DIAMETER (m)

3 VOLUME (m )

DIAMETER SAND PARTICLES mm DIAMETER BIOMASS COVERED mm

PARTICLES WASTE FLOW 1/h SUPERFICIAL LIQUID cm/s VELOCITY REYNOLDS NUMBER Re = n

!

GALILEO NUMBER r* - _ ■ ua •• ~ ni GEOMETRY NUMBER H/D PILOT PLANT 12 0.2 0.4

1

4

•W000 •<- 1 40 640.10 60 LABORATORY

3

0.04 3.6.10"3 0.5 3.5 ->-40 ■^0.6 21 400.10 75

d(c /c ) 6 * (1- t.) * (c ) H X O 1 X

«

d(x/H) cQ dp * vx symbols u s e d : c X c _o X H Cl F ( c d P v1 A ( m o l e . 1 ) ( m o l e . 1 ) (m) (m)

(-)

x) ( k g . s- 1. (m) ( m . s ) i 2\ (m )

concentration at height x in the reactor concentration entering the reactor height in the reactor

total height of the reactor liquid volume fraction -2

) kinetic function substrate consumption particle size

superficial liquid velocity area of reactor section

As no expression for the kinetics is known, the parameter cannot be calculated. However, the variable part of it (H/dp*v.) can be compared for the two reactors (the kinetics, initial concentration of the substrate and the liquid fraction in both systems are assumed equal). For the pilot plant

3

this variable part equals 300*10 s/m (seconds per meter), while for the 3

laboratory model this parameter is only slightly smaller 150*10 s/m. Based on this information, it was assumed that the population selected in the laboratory reactor would be comparable to that in the pilot plant reactor. FLUIDIZATION Biomass remains in a fluidized bed reactor despite high fluid flows because it is attached to carrier particles present in the reactor. Depending on the ratio of biomass to carrier volume, the density of the particle will differ, and therefore also the minimal superficial liquid

velocity to fluidize the particles will be different. If the biomass layer thickness exceeds the carrier particle size by a factor of two, the density of the particle as a whole can be assumed to be equal to that of the biomass even with heavy carrier particles such as sand (calculated densities shown in fig.6). With these thicker biolayers, it is important to note that the minimal fluidization velocity will increase with increasing biolayer thickness because of the increasing particle size) instead of decreasing, as is the case with thin biolayers (because of the decrease of the overall density).

MIXING Studies of mixing of both liquid and solid phases in fluidized bed reactors have concentrated on reactors containing either small (diameter

-3

<500 pm) or high density (>2000 kg.m ) particles. As the degree of mixing can substantially influence reactor performance, and the degree of solids mixing determines the conditions experienced by the organisms on the individual particles, a number of measurements have been made (chapter 2) to elucidate the mixing behaviour of the system studied.

DIFFUSION As interest in fixed film reactors has increasted, more and more work on the transport of substrates and bacterial products within biolayers has been done. It is generally assumed that for biolayers thicker than 50 pm , diffusion can limit substrate conversion rates. Diffusion coefficients in biolayers have been measured for several molecules. For nitrate and nitrite,values in biolayers are generally between 50 and 100 % of that in

water (Kissel, 1985). Because the biolayers in the system described in this thesis are very thick, diffusion limitations will certainly play an important role, but because of the large number of compounds entering and leaving the layer,and the lack of reliable micro-electrodes for nitrate and the other compounds involved, it will be very difficult to quantitatively

Figure 6: Relationship between the ratio of biolayer thickness to sand particle diameter and the resulting density of the particle (assuming sand 2600 kg.m"3 and biomass -1080 kg.m- ) .

Particle density

Kg(m3)-

1

1400

1300

IO

(O

1200-^

1100

Biomass __

Densi t y

1080 Kg(m

3

)

J

1000

—t—

4

T

- 1 —

10

- 1 —

12

14

_^ Biolayer Thickness/Sandparticle

Diameter

estimate the effect of diffusion on the overall performance of the system studied. In this situation we have chosen not to investigate the effect of diffusion on the microbial activity in the reactor. However, as described in chapter 6, an experimental set-up was designed to measure the conversion rates of complete particles taken from the reactor. Assuming the effects of diffusion to be comparable, this approach allowed comparison of these experiments with the behaviour of the reactor as a whole.

1.6 PHYSIOLOGY AND ECOLOGY OF SULFIDE OXIDIZING BACTERIA

Reduced inorganic sulfur compounds produced in the environment are subject to biological oxidation in the absence and presence of light under aerobic and anaerobic conditions. Although phototrophic bacteria can play an important role in sulfur compound oxidation in nature, they will not be discussed here as the system studied does not allow phototrophic growth.The non-phototropic (or colourles) bacteria involved in the oxidation of reduced inorganic sulfur compounds include organisms with widely different types of physiology and morphology ranging from specialist obligate chemolithotrophs, via facultative chemolithotrophs which may grow mixotrophically, to specialist heterotrophs, some of which may benefit from the oxidation of reduced sulfur compounds. These terms are defined in table 9.

The known obligate chemolithotrophs belong to the genera Thiobacillus and Thiomicrospira. These organisms are able to generate energy only from the oxidation of reduced inorganic sulfur compounds such as sulfide, thiosulfate and elemental sulfur and not from the oxidation of organic compounds. Organic carbon is synthesized by these bacteria from C0„ via the Calvin cycle, but exogenous organic carbon compounds may contribute maximally to 20-30 % of the total cell carbon.

Table 9 : Important characteristics of the different physiological types of colourless sulfur oxidiring bacteria.

OBLIGATE CHEMOLITHOAUTOTROPH FACULTATIVE CHEMOLITHOAUTOTROPH CHEMOLITHOHETEROTROPH HETEROTROPH ENERGY SOURCE reduced sulfur compounds

+

+

+

-organic compounds

-+

+

+

CARBON SOURCE

co

2

+

+

-organic compounds

-+

+

+

Facultative chemolithotrophs are not only able to grow autotrophically with reduced inorganic sulfur compounds as their energy source, but are also capable of heterotrophic growth. Such bacteria belong to the genera Thiobacillus. Sulfolobus. Thermothix. Thiosphaera. Beggiatoa and Paracoccus■ Several Thiobacillus species are able to utilize mixtures of inorganic and organic compounds simultaneously (mixotrophic growth). Depending on the ratio of inorganic and organic substrates, C0„ may serve as an additional carbon source.

Heterotrophic bacteria able to oxidize reduced inorganic sulfur compounds may be divided into two groups, those that obtain energy from the oxidation process and thosis that at first sight do not measurably benefit from it. The former group includes members of the genera Thiobacillus and Pseudomonas. For organisms that do not obtain energy from oxidation of sulfide, it may still be advantageous to oxidize sulfide because of its toxicity. In some of the Beggiatoa and Thiospira species and in Thiobacterium which lack catalase, the oxidation of sulfide may protect the cells from reactive,harmful, oxygen species such as hydrogen peroxide (Nelson ad. Castenholz, 1981; Dubinins and Grabovich, 1983). Beggiatoa oxidizes sulfide to intracellular sulfur which can be further oxidized to sulfate, or can serve as an electron acceptor for the oxidation of organic compounds under anaerobic conditions.

The physiological characteristics of the different types of bacteria and their interactions with each other, with the abiotic environment and with eukaryotes have recently been reviewed by Kuenen 8. Beudeker (1982), Kelly & Kuenen (1984) and Kuenen, Robertson and Van Gemerden (1985). On the basis of these capabilities, and also on the basis of extensive competition experiments (Gottschal & Kuenen, 1980a, 1980b, 1981) a model has been proposed for the ecological niches of the several types (fig.7). As can be seen, the facultative chemolithotrophs are favoured by conditions with

roughly equal turnover rates of organic and sulfur compounds. This was confirmed with simple mixed culture and enrichment experiments in aerobic chemostats (Gottschal & Kuenen, 1980a, 1980b, 1981). The applicability of this model to anaerobic (denitrifying) situations including homogenous suspensions in chemostats and the heterogenous conditions existing in a fluidized bed reactor remained to be tested. From the pilot plant which was receiving aproximately equimolar amounts of sulfide and acetate, no obligate chemolithotrophs could be isolated, while facultative chemolithotrophs were present as a major group of the population (Robertson & Kuenen, 1983b). The fact that pure culture results could be extrapolated to allow predictions to be made about the system under study illustrates and underlines the possible application of fundamental physiological principles in the study of waste treatment systems.

One of the organisms isolated, Thiosphaera pantotropha (Robertson & Kuenen, 1983a), showed some interesting parallels to the behaviour of the pilot plant. In the pilot plant reactor, although it used nitrate as the main electron acceptor, oxygen was often present. One of the surprising characteristics of both the population as a whole and Thiosphaera pantotropha in pure culture was the ability to denitrify under aerobic conditions (Robertson & Kuenen, 1983a, 1984a+b). It has been hypothesized that the capability to denitrify under aerobic conditions would offer a selective advantage over denitrifiers that do not possess this trait if oxygen was either limiting or fluctuating over short period. Future competition experiments with simple mixed cultures of an 'aerobic' denitrifier and a 'non-aerobic' denitrifier will be necessary to prove this. Compared to the knowledge of obligate and facultative chemolithotrophic bacteria, the knowledge of the heterotrophic sulfur oxidizing organisms is rather scarce. Heterotrophic oxidation of reduced inorganic sulfur compounds appears to be predominant in certain environments, for instance in soils

Molar ratio of inorganic sulphur compounds to organic substrates

I T

o o 1

Obligate chemolithotrophs

Mixotrophs

Chemolithoheterotrophs

Heterotrophs able

to oxidize

Sulphur compounds

I

Figure 7: Model predicting the occurrence of sulphur-oxidizing bacteria as a function of the relative turnover rate of reduced inorganic sulphur compounds and organic substrates during energy-limiting growth conditions in fresh water environments (Kuenen & Beudeker, 1984).

(Wainwright, 1984) or in the Black Sea (Trudinger, 1967; Tuttle & Jannasch, 1972). In these environments in which substantial oxidation rates of sulfur compounds occur no chemolithotrophic populations were found, which also suggests an important role for the heterotrophic oxidation of sulfur compounds. Due to lack of selective enrichment methods and repeated difficulties with the cultivation of potential heterotrophic sulfur oxidizers, these organisms have been paid relatively little attention. A short overview of this group of bacteria is therefore presented in the following paragraph.

HETEROTROPHIC, REDUCED SULFUR COMPOUND OXIDIZING MICROORGANISMS The number of heterotrophic organisms capable of the oxidation of sulfide, thiosulfate or elemental sulfur is quite large and includes both prokaryotes and eukaryotes (table 10). However, the number of heterotrophic organisms known to generate metabolically useful energy from the oxidation of reduced sulfur compounds is very small. The only well described species in this group Thiobacillus perometabolis (London & Rittenburg, 1969), has recently been cultured under autotrophic conditions (Katayama-Fujiura et al, 1982; Harrison, 1983). Experiments in our laboratory confirmed this and Ribulose bisphosphate carboxylase (RuBP-case) measurements confirmed that carbon could be obtained from C0„ (P.Gommers, unpublished results). Tuttle (1980) and Tuttle and Jannasch (1977) studied large numbers of heterotrophic bacteria able to isolate thiosulfate which had been isolated from the sulfide-oxygen interface of the Black Sea. Work with one strain (Tuttle, 1980) clearly demonstrated that the organism could use thiosulfate as an additional energy source for organic carbon assimilation. However, the fact that the oxidation of thiosulfate preceded only to tetrathionate, a compound unlikely to occur in nature, suggested that this organism was not representative of the heterotrophic sulfur compound oxidizing bacteria. A

Table 10 : Heterotrophic microorganism* able to oxidize thiosulfate,sulfur or sulfide. ORGANISM Thiotrix nivea Thiobacillus rubellus Pseudomonas aeruginosa Streptomyces species Streptomyces species Streptomyces species Undetermined Unditerrained 9 acid-forming 74 base-forming Marine Fseudomonads Isolate nr.B Isolate C-3 and A 50 Pseudomonas fluorescens Thiobacillus trautweinii Arthrobacter aurescens Arthrobacter simplex Arthrobacter species Bacillus licheniformis Bacillus brevis Bacillus species Flavobacterium species Micrococcus species Debaryomyces species Saccharomyces species Brevibacterium species Achromobacter species Mycobacterium species Pseudomonas species Pseudomonas aromatica Pseudomonas pycyanea Pseudomonas fluorescens Pseudomonas putida Pseudomonas species Isolate 3A !:Pseudomonas aeruginosa Pseudomonas fluorescens Achromobacter stutzeri Escherichia coli Hyphoraicrobium S Hyphomicrobium EG Thiobacillus Q Unidentified Soil Amoeba Soil Fungi Asteriomyces crucicatus Sporotrichium thermophile Fusarium solani Alteraria tenuis Aureobasidium pullalans Cephalosporium sp. Penicillium decumbens Sporotrichum thermophile Debaromyces sp. Rhodutorula sp. Saccharomyces sp. Alteraria tenuis (H2S) Sphaerotilus natans REFERENCE Larkin 6. Shlnaberger , 1983 Mlzoguchi et . al., 1976 Schook & Berk, 1978 Wainwright et. al. , 1984 Yagi, 1971

Uierenga, 1966 Guittonneau, 1935 Ruby et.al., 1981

Tuttle & Jannasch, 1972 & 1973 Starkey,1935

Trudinger ,1967 Gleen & Quastel, 1953 Trautwein, 1921 Vitolins & Swaby, 1968 Vitolins & Swaby, 1968 Vitolins & Swaby, 1968 Vitolins & Swaby, 1968 Vitolins 6. Swaby, 1968 Vitolins & Swaby, 1968 Vitolins & Swaby, 1968 Vitolins 6. Swaby, 1968 Vitolins & Swaby, 1968 Vitolins & Swaby, 1968 Vitolins & Swaby, 1968 Vitolins & Swaby, 1968 Vitolins & Swaby, 1968 Vitolins & Swaby, 1968 Sijderius, 1946 Sijderius, 1946 Sijderius, 1946 Sijderius, 1946 Sijderius, 1946 Sijderius, 1946 Starkey, 1934 Starkey, 1934 Starkey, 1934 Starkey, 1934 de Bont et. al., 198 van Suylen & Keunen, 1986 Gottschal & Keunen, 1980 Gommers & Keunen, 1986 Arkesteyn, 1980 Lloyd et.al. , 1981 Wainwright, 1984 Wainwright, 1984 Wainwright, 1984 Wainwright, 1984 Wainwright, 1984 Wainwright, 1984 Wainwright, 1984 Wainwright, 1984 Wainwright, 1984 Wainwright, 1984 Wainwright, 1984 Wainwright, 1984 SRerHBh5V.et.al., 1957 Skermans et.al., 1957

recent isolate from a chémostat enrichment (Gottschal & Kuenen, 1980) was capable of the oxidation of thiosulfate completely to sulfate. The organism could not be grown autotrophically and was therefore selected for further study. The main objective of this work was to increase the knowledge of the physiology of this type of organism so as to be able to compare it with both the obligate and the facultative chemolithotophs. Possible energy generation from the oxidation of reduced sulfur compounds, the lack of autotrophic potential under a variety of conditions and the oxidation capacities for a number of sulfur compounds under different environmental conditions are especially important.

As is described above, the ratio of sulfur to organic compound turnover rates determines the type of organisms selected in fresh water environments (see Kuenen, Robertson and van Geroerden, 1985). An important selection parameter in competetition experiments is the biomass production yield. A good understanding of the influence of mixotrophic conditions on biomass yields is necessary to predict and influence biomass production in the actual waste treatment reactor.

1.7 MICROBIAL GROWTH YIELDS

Growth yields of microorganisms and the processes that influence them have been a subject of continuous interest since the first study published on this subject by Monod (1942). Several reviews on this topic have been published (Stouthamer, 1979; Roels, 1980; v.Dijken and Harder, 1975; Westerhoff, et. al. 1982). The influence of many external factors (e.g. pH, temperature, growth rate, salt concentration, additives and nitrogen source) has been established for individual cases and three major groups of effects can be distinquisted.

In general, it can be concluded that any type of stress experienced during growth results in less than maximal growth yields. This is most likely caused by increased activity (often referred to as maintenance) of the organisms to maintain optimal conditions inside the cell wall.

The type of electron acceptor used influences the growth yield (Stouthamer, 1980). Generally yields increase with increasing redox potential of the electron acceptor.

Linton and Stephenson, (1978) discussed the effects of type of electron donor on the basis of data from a large variety of studies and showed a clear increase of the growth yield with increasing energy content of the electron donor. These types of effects on biomass yields have often been used to optimize (=minimize) biomass production in wastewater treatment systems.

The denitrification reactor described in this study involves growth attached to a carrier (fixed biomass) while studies on microbial growth yields have almost exclusively dealt with growth in suspended cultures such as shake flasks and chemostats. Characteristic differences between culture methods that might effect microbial growth yields are shown in table 11. The differences in methods appear to be quite drastic but, as is also shown in table 1 ) , the averages of published growth yields for denitrifying populations grown with the different culture methods do not show large differences. However, the large variability in the individual yields for each culture method demonstrates that factors (such as electron donor type) do exert a larger influence on the growth yield. As mentioned above, the influence of the mixotrophic growth conditions caused by the presence of two electron donors acetate and sulfide is not. only of academic interest but also has important implications for the practical wastewater treatment systems. Excessive biomass production is considered highly undesirable and in fact, many systems are optimized for low biomass production. Therefore

understanding and control of biomass production during mixotrophic growth conditions should provide practical quidelines for the optimal operation of the actual wastewater treatment plant.

GROWTH WITH MIXED ELECTRON DONOR SUBSTRATES Mixed electron donor substrate regimes (as here, where sulfide and acetate are involved) can be divided into three categories (table 12). The acetate sulfide combination belongs in the third category as acetate can function as the carbon source (assimilation) and as the electron donor (dissimilation) while sulfide can function only as an electron donor. In other words, acetate is heterotrophic substrate and sulfide an autotrophic substrate- The simultaneous use of heterotrophic and autotrophic substrates has been defined mixotrophic growth (Rittenburg, 1969; Kuenen and Beudeker, 1982). The effect of the autotrophic substrate on the utilization of the heterotrophic substrate can generally be described as redistribution among assimilation and dissimilation. As more autotrophic substrate is added, the amount of energy available to the organism increases, so that the organism can use a larger fraction of the heterotrophic substrate for assimilatory purposes. Important aspects of this type of growth are:

1. The maximum amount of the heterotrophic substrate which can be converted to biomass.

2. The amount of energy from the autotrophic substrate that replaces energy from the dissimilation of the heterotrophic substrate.

Although several elaborate studies on the effect of mixed substrates in aerobic chemostats exist, a theoretical evaluation of these two aspects is still lacking.

Table 11 : Characteristics of culture methods which Blight influence the growth-yield therein , arid published average growth growth-yield measured for devi­

trification. CULTURE METHOD GROWTH RATE GROWTH MODE SUBSTRATE C O S C ( i n t h e r e a c t o r ) BIOMASS CONC. BIOMASS COMPOSITION DIFFUSION L I M I ­ TATION SOLUBLE SU3STATES MEASURED GROWTH YIELD FOR D I N I T -RIFYING SYSTEMS k g VS / k g H 03 -* VOLATILE SOLIDS \ dry weight BATCH .MAXIMUM .NOT CONSTANT .SUSPENDED .FUNCTION OF TIME •FUNCTION OF TIME .FUNCTION OP TIME ■HOT LIKELY 0 . 9 ( 0 - 3 - 2 . 1 ) H n A CHEHOSTAT .CONSTANT .CONTROLLED D .SUSPENDED .CONSTANT .LOU -CONSTANT .CONSTANT .NOT LIKELY 1 . 2 ( 0 . 2 - 2 . 5 ) ETEROTROPHIC UTOTROPHIC EL 0.9 ( 0 . 4 - 1 . 6 )

ACTIVATED SLUDGE JpLUlDUED BEO

.CONSTANT .CONTROLLED WAS OUT .ATTACHED ( F L O C S ) .CONSTANT .LOW .CONSTANT .CONSTANT . d i f f e r e n c e » i n s i d e f l o e .POSSIBLE 1.0 ( 0 . 6 - 1 . 4 ) ELECTRON DONO ECTRON DONORS .FUNCTION OF PLACE IN REACTOR H .'COHTKULLEB' WASH OUT .ATTACHED (BIOLAtER) ■ FUNCTION OF PUCE IN REACTOR .FUNCTION OF PLACE IH REACTOR ■difference* betvee p a r t i c l e s depends on s o l i d n i x i n g ■difference i n s i d e p a r t i c l e s .DEPENDS ON BIOLAYERTHICKNESS 1 . 2 ( 0 . 5 - 2 . 6 1 ) RS 0 . 9 ( 0 . 7 4 - 1 . 2 )

TBb!e 12; MIXED SUBSTRATE CASES DISTINGUISHED BY THE POTENTIAL OF EITHER OF THE SUBTRATES (A «nd B) TO BE USED FOR BOTH ASSIHILATORY AND DISSIHILATORY PURPOSES OR FOR DISSIMILATORY

PURPOSE ONLY. 1 2 3 ASSIMILATION AND DISSIMILATION A B A DISSIMILATION ONLY A B B EXAMPLE GLUCOSE + ACETATE FORMATE + HYDROGEN SULFIDE ACETATE + HYDROGEN SULFIDE

1.8 ORGANIZATION OF THIS THESIS

The aim of the studies presented in this thesis was to increase both the engineering and the microbial understanding of a denitrifying, fluidized bed reactor in which sulfide and acetate were simultaneously oxidized. This introduction has presented the necessary background information on the subjects discussed in more detail in chapters 2 to 6. The subjects in these chapters can be read separately or in sequence. As well as being superficially described here, the interrelationship of the variou s chapters will be discussed in Chapter 7.

Chapter 2 deals with mixing phenomena in two and three phase fluidized bed reactors containing particles with a density only slightly higher than water, such as that described in chapters 5 and 6. Knowledge of both the liquid and solids mixing should increase the understanding of the overall kinetics of substrate removal and also the possible mechanisms of selection of microorganisms in the microbial communities of a biofilm.

Chapters 3 and 4 deal with two microbiological aspects of the system described in chapters 5 and 6. In the third chapter the physiological properties of a recently isolated, chemolithoheterotrophic species are presented. This study complements the existing understanding of obligate and facultatively chemolithotrophic metabolism, so that basic information on three major physiological types involved in sulfide oxidation is now available. In the fourth chapter, the effects of mixotrophic growth conditions such as those in the system described in chapters 5 and 6 on microbial growth yields are discussed. Based on a limited number of elaborate studies on this subject with pure cultures, a theoretical study was made of the influence of an auxiliary energy source (i.e. sulfide) on the assimilation and energy generation from a carbon and energy substrate (i.e. acetate) and is described here.

As already mentioned, chapters 5 and 6 describe the work done on the fluidized bed system built in the laboratory. Chapter 5 deals with overall

reactor performance (mass balances, redox balances), while chapter f> concentrates on the conversion capacities of the bioniass enriched for in the reactor. In these chapters, findings of the chapters 2, 3 and 4 are used to describe phenomena observed in the reactor.

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