Operational Factors affecting AD
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1 Anaerobic Fermentation Environmental Factors Biogas Compact Workshop Postgraduate Programme Renewable Energy (PPRE) Project Planning for Biodigesters in Developing and Industrialized Countries April, 2011 University of Oldenburg, Germany Henri Spanjers Lettinga Associates Foundation Operational Factors affecting AD Composition (biodegradability) Concentration Mixing Deposition in biofilms Adhesion Interception Flocculation Sedimentation Lettinga Associates Foundation 1 1
2 Environmental factors: Contents Temperature + fluctuations Availability of nutrients (N, P, micro-nutrients) ph / Buffer capacity / Alkalinity Volatile Fatty Acids Ammonium Presence of alternative electron acceptors (SO 2-4, NO 3-, etc.) Presence of toxic compounds Risk of formation of inorganic precipitates Risk of formation of scum layers and/or flotation layers Odour problems Lettinga Associates Foundation 2 Temperature Affects metabolic activity of bacteria Affects transfer and solubility of gases Affects settleability of biological solids Reaction decreases with decreasing temperature: 11% slower per o C (Arrhenius) Final degradation extent is proportional to temperature Lettinga Associates Foundation 3 2
3 Temperature I psychrophyllic <20 C II mesophyllic C III thermophyllic >45 C Lettinga Associates Foundation 4 Environmental factors Temperature + fluctuations Availability of nutrients (N, P, micro-nutrients) ph / Buffer capacity / Alkalinity Volatile Fatty Acids Ammonium Presence of alternative electron acceptors (SO 2-4, NO 3-, etc.) Presence of toxic compounds Risk of formation of inorganic precipitates Risk of formation of scum layers and/or flotation layers Odour problems Lettinga Associates Foundation 5 3
4 Nutrients Need to be present in the right quantities Indispensable for good process performance Macro-nutrients are needed in larger quantities Nitrogen Phosphorous Calcium Magnesium Sulphur Micro-nutrients (trace elements) are only needed in very small quantities e.g. certain heavy metals Lettinga Associates Foundation 6 Macro and micro nutrient requirements Requirement of anaerobic sludge for macro and micro nutrients --> amounts in mg/kg dried cell Macronutrients: Micronutrients: N Fe 1800 P Ni 100 K Co 75 S Mo 60 Ca 4000 Zn 60 Mg 3000 Mn 20 Cu 10 (Based on elemental composition of methane bacteria, Scherer 1983) Conversion factors for methane bacteria cell: g VSS*1.4= g COD g TS*0.825= g VSS Lettinga Associates Foundation 7 4
5 N and P requirements The requirement for N and P can be calculated from the cell composition. (i.e % N and approx. 2% P) substrate = mixture of volatile fatty acids growth yield = g/g COD : N : P = 1000 : 5 : 1 C : N : P = 330 : 5 : 1 substrate = non-acidified carbohydrates growth yield = g/g COD : N : P = 350 : 5 : 1 C : N : P = 130 : 5 : 1 Lettinga Associates Foundation 8 Trace metal requirements Fundamental role in microbial growth and metabolism Can be toxic in higher concentrations Cell composition of micro-organism provides idea of needed quantities Present as metal centre in enzymes or as cofactor Different requirements for different processes Often these metals are irreplaceable by other metals Lettinga Associates Foundation 9 5
6 Trace metal - Cobalt Catalytic centre in corrinoid of the enzyme methyltransferase (corrinoid also referred to as cobalamin or vitamin B12) Corrinoids can contain up to 98% of total cobalt present in organism Content of cobalt in methanogens and homoacetogens depends on species, substrate and cultivation condition Co + Lettinga Associates Foundation 10 Trace metal - Nickel In cofactor F430 of the enzyme methyl-com reductase Nickel is also present in many other enzymes, e.g.: urease, hydrogenase, carbon monoxide dehydrogenase Ni + Lettinga Associates Foundation 11 6
7 Addition of trace metals The form in which metals will be present depends on: chemical properties of the metal concentrations of anions concentration of complexing agents present ph Examples of wastewaters with usually sufficient amounts of trace metals: domestic sewage agro-industrial wastewater (not all) Lettinga Associates Foundation 12 Environmental factors Temperature + fluctuations Availability of nutrients (N, P, micro-nutrients) ph / Buffer capacity / Alkalinity Volatile Fatty Acids Ammonium Presence of alternative electron acceptors (SO 2-4, NO 3-, etc.) Presence of toxic compounds Risk of formation of inorganic precipitates Risk of formation of scum layers and/or flotation layers Odour problems Lettinga Associates Foundation 13 7
8 Effects of ph on anaerobic digestion Direct effect on enzyme activity (charge / tertiary structure of proteins) Indirect affecting toxicity of various compounds (H 2 S, NH 3, VFA) affecting the availability of nutrients (e.g. by affecting the solubility) affecting the availability of substrates (e.g. at ph<6.1, milk proteins coagulate which makes them less available) affecting the availability of CO 2 (very low pco 2 at ph>8.0.) Lettinga Associates Foundation 14 ph Range methane digestion ph (optimum ranges per process) Hydrolysis Acidogenesis Acetogenesis Methanogenesis of acetate Methanogenesis of hydrogen Lettinga Associates Foundation 15 8
9 Buffer systems Buffering capacity: capacity of solution to resist to ph changes (e.g. added H + will be neutralised by buffer system) N.B.: term for buffering capacity with respect to acids: alkalinity Especially brought about by bicarbonate Buffer system: a mixture of weak acid and its conjugate base. The optimum ph of a buffer depends on the pka/pkb value of the acid/base applied Lettinga Associates Foundation 16 Buffer capacity Lettinga Associates Foundation 17 9
10 Buffer capacity Buffering capacity: ß = -da/dph (A= amount acid added) E.g. for mixture of acetate and acetic acid HAc/Ac-: ß is maximum for ph = 4.75 (50/50% acid.base!) and minimum for 100% HA or Ac -. For ph<3 and >11, the buffering capacities of H + and OH - are most important ß proportional to the total concentration Lettinga Associates Foundation 18 K-values Acid HClO4 HCl HNO3 H3O - H3PO4 CH3COOH H2CO3 - H2PO4 - H2S NH4 + HCO3 - HPO4 2- H2O OH - HA H 2O H O + A Perchloric acid Hydrochloric acid Nitric acid Hydronium ion Phosphoric acid Acetic acid Carbon dioxide Dihydrogen phosphate Hydrogen sulfide Ammonium ion Bicarbonate Monohydrogen phosphate Water Hydroxide -logka =pka ~24 A H 2 Conjugate Base ClO4 - Cl - NO3 - H2O H2PO4 - HPO4 2- CH3COO - HCO3- HS- NH3 CO3 2- PO43- OH - O 2- + O HA + OH Perchlorate ion Chloride ion Nitrate ion Water Dihydrogen phosphate ion Acetate ion Bicarbonate ion Monohydrogen phosphate ion Bisulfide ion Ammonia Carbonate ion Phosphate ion Hydroxide ion Oxide ion -log Kb =pkb Lettinga Associates Foundation 19 10
11 Buffers in AD Useful buffers are active in the neutral ph range (6 8). H 2 2 CO3 / HCO3 / CO3 NaAc / HAc pka = 6.3 pka = 10.3 pka = H 2 PO / HPO NH + / NH 4 3 pka = 7.2 pka = 9.3 ph = pka + log (A - / HA) H 2S / HS pka = 7.1 Lettinga Associates Foundation 20 Buffer index As a function of ph for a solution of the carbonic system (C c mmol/l) in water Buffer index: the concentration of strong acid and/or strong alkali to produce one ph unit change!! Bi = dcb / dph = dc A / dph Lettinga Associates Foundation 21 11
12 Buffer index Buffer indexes as a function of ph for acid/base systems likely to occur in anaerobic treatment plants Lettinga Associates Foundation 22 Bicarbonate alkalinity Bicarbonate is generated in the digestion process itself (metabolism generated alkalinity) CH 3 COO - Na + CH 4 + HCO 3 - If a solution of 5 g NaAc is digested, the produced alkalinity is 5/82 = 60 mmol or 60 meq HCO 3-. Lettinga Associates Foundation 23 12
13 CO 2 / HCO 3- / CO 3 2- as a function of ph Lettinga Associates Foundation 24 CO 2 / HCO 3- / CO 3 2- Balance CO 2 (g) C CO2 (l) = K H. P CO2 CO 2 (l) + H 2 O CO H + H 2 CO 3 HCO 3- + H + Lettinga Associates Foundation 25 13
14 Alkalinity Capacity of the wastewater, or reactor liquid, to take up protons Assessment: amount of acid needed to titrate sample until ph = 4.0 At ph=4.0 all inorganic carbon is present as CO 2 minimum buffer index Original sample ph ~ 7.0 alkalinity practically equivalent with the concentration of bicarbonate bicarbonate alkalinity If other weak acids (VFA!) are present total alkalinity Lettinga Associates Foundation 26 Role biogas CO 2 and reactor alkalinity on reactor ph Lettinga Associates Foundation 27 14
15 Metabolism Generated Alkalinity Sulphate and sulphite 2 4 H2 + SO4 + CO2 HS + HCO3 + 3H 2O 2 3COO + SO4 2HCO + HS CH 3 3COO + 4HSO3 3HCO3 + 4HS + 3H 2O 3 2CH + CO 2 Note: reactor ph = 7-8 Anions have cationic counter-ions Lettinga Associates Foundation 28 Metabolism Generated Alkalinity (digestion of protein) C + 3H7O 2N + 2H 2O CH4 + 2CO2 NH3 + 3H7O 2N + 2H 2O CH4 + CO2 + HCO3 + NH4 C (ph = 7) Assume wastewater contains 2 g/l proteins (~C 3 H 7 O 2 N) MW= 89 g 1 mole C 3 H 7 O 2 N => 1 mole NH 4+ -N 2/89 mole =>2/89 mole NH 4+ -N, or 22 mmole or 22 meq NH 4+ -N This will bind 22 meq HCO - 3 Lettinga Associates Foundation 29 15
16 Metabolisms that do not generate Alkalinity Carbohydrates Sugars Organic acids Aldehydes Ketones Esters Generally: If no cation is released from the organic compound then no alkalinity is generated Lettinga Associates Foundation 30 Environmental factors Temperature + fluctuations Availability of nutrients (N, P, micro-nutrients) ph / Buffer capacity / Alkalinity Volatile Fatty Acids Ammonium Presence of alternative electron acceptors (SO 2-4, NO 3-, etc.) Presence of toxic compounds Risk of formation of inorganic precipitates Risk of formation of scum layers and/or flotation layers Odour problems Lettinga Associates Foundation 31 16
17 Toxicity of volatile fatty acids (VFA) Two inhibitory effects: Toxicity of the VFA Lowering of the ph The undissociated form of VFA is toxic ph dependent Lettinga Associates Foundation 32 Toxicity of volatile fatty acids (VFA) 1.0 Fraction VFAunionized/VFAtot Acetic acid pka = 4.75 Propionic acid pka = ph [HA] [HA] + [A F = (ph pka) = ] Lettinga Associates Foundation 33 17
18 50% Inhibition concentrations of VFA ph acetic acid mg COD l -1 propionic acid mg COD l [VFA] unionized = [VFA] α 0 α 0 = { 10 (ph - pk a) + 1 }; pk a = 4.75 Lettinga Associates Foundation 34 Toxicity of volatile fatty acids (VFA) % IC (g COD/L) Acetic acid 5 Propionic acid ph Lettinga Associates Foundation 35 18
19 Toxicity of volatile fatty acids (VFA) The rate of recovery of a low ph-shock depends on: the ph-value during the shock, the duration of the shock, the concentration of C-2, C-3, C-4, the type of sludge, the extent of adaptation. Recovery may take up to several weeks, even months. In absence of VFA, little effect is observed if ph is lowered down to values of ph: (e.g. with methanol as substrate). Lettinga Associates Foundation 36 Environmental factors Temperature + fluctuations Availability of nutrients (N, P, micro-nutrients) ph / Buffer capacity / Alkalinity Volatile Fatty Acids Ammonium Presence of alternative electron acceptors (SO 2-4, NO 3-, etc.) Presence of toxic compounds Risk of formation of inorganic precipitates Risk of formation of scum layers and/or flotation layers Odour problems Lettinga Associates Foundation 37 19
20 The undissociated molecule (NH 3 ) is the toxic compound: + 4 NH NH3 [NH ] = [NH [NH3 ] F = [NH ] + [NH 3 + H w + ph 10 ] Kb + 10 K 4 ] Ammonium toxicity ph F strongly depends on ph and temperature! Lettinga Associates Foundation 38 Relationship NH 4+ and Hydrolysis during cow manure digestion in CSTR systems (30 C) Cow manure Hydrolysis (%) HRT 3000 (days) Hydrolysis (%) NH4 (mg N/l) 1000 mg NH 4+ -N >4000 mg NH 4+ -N Lettinga Associates Foundation 39 20
21 Ammonium toxicity Fraction free ammonia on total ammonium is in dependency of ph and temperature Dealing with ammonium toxicity in practice: lower the ph lower the temperature dilution of the waste water pre-treatment (stripping!) Lettinga Associates Foundation 40 Adaptation to ammonium toxicity Lettinga Associates Foundation 41 21
22 Adaptation to ammonium toxicity 50% inhibition concentrations of NH 4+ -N for methanogenic activity NH 4+ -N ph T Substrate Sludge Adaptation* Ref.** (mg l -1 ) ( C) > C2:C3 granular none (1) C2 granular 300 (2) H 2 M. formicicum none (3) C2 digested domestic 700 (4) C2:C3:C4 digested pig manure 815 (5) C2:C3:C4 digested pig manure 2420 (5) C2 digested pig manure 3000 (6) * The concentration of NH 4+ -N to which the sludge was previously adapted to. ** References: (1) Dolfing & Bloeming 1985; (2) Koster & Lettinga 1983; (3) Hobson & Shaw 1976; (4) Kugelman & McCarty 1965; (5) Van Velsen 1979; (6) Kroeker Lettinga Associates Foundation 42 Environmental factors Temperature + fluctuations Availability of nutrients (N, P, micro-nutrients) ph / Buffer capacity / Alkalinity Volatile Fatty Acids Ammonium Presence of alternative electron acceptors (SO 2-4, NO 3-, etc.) Presence of toxic compounds Risk of formation of inorganic precipitates Risk of formation of scum layers and/or flotation layers Odour problems Lettinga Associates Foundation 43 22
23 The biological sulphur cycle Lettinga Associates Foundation 44 Biochemical properties of C and S Similarities: Appearance in various states of oxidation, C: (-4) (+ 4) CH 4 CO 2 S: (-2) (+ 6) H 2 S H 2 SO 4 Can both act as electron acceptor / donor in anaerobic systems Reduced end-product is gaseous Possible formation of precipitates: MeS, CaCO 3 Bacteria using either S or C as electron acceptor may compete for the same substrate Lettinga Associates Foundation 45 23
24 Sulphate, sulphite and nitrate as electron acceptors 1. Oxidation with oxygen 2. Oxidation with sulphate 3. Oxidation with sulphite 4. Oxidation with nitrate COD + O 4e + O 2 2 COD + SO H O + CO 2O e + S S COD + SO 6e + S S COD + 2NO3 5e + N 5+ N H S + CO 2 2 H S + CO 2 N + CO Lettinga Associates Foundation 46 Sulphate, sulphite and nitrate as electron acceptors Conclusions: 1 mol SO 4 2- ~ 2 mol O 2 1g SO 4 2- => 0.67 g COD 1 g SO 3 2- => 0.6 g COD 1 g NO 3- -N => 20/7 g COD = 2.86 g COD (1 g NO - 3 => 0.65 g COD) Lettinga Associates Foundation 47 24
25 Anaerobic Conversion of Organic Matter with Sulphate Organic Polymers proteins carbohydrates lipids Mono- and oligomers amino acids, sugars, fatty acids Volatile Fatty Acids Lactate Ethanol H 2 / CO 2 Acetate CH 4 / CO 2 H 2 S / CO 2 Sulphate reduction Lettinga Associates Foundation 48 Problems associated with sulphate reduction Negative effect on energy balance of anaerobic reactor Lower methane yield per unit organic waste Biogas treatment required (H 2 S removal) Maintenance costs Increase Malodour Corrosion to engines, boilers, reactor parts, pipes,.. Effluent polishing required S 2- increases effluent COD (1 mol S 2- = 2 mol O 2 ) Bulking of activated sludge (successive post-treatment) Lettinga Associates Foundation 49 25
26 Odour H 2 S: colourless, flammable, toxic, heavier than air. In low concentrations: "rotten eggs. Low-level exposures usually produce local eye and mucous membrane irritation At 300 ppm the olfactory nerve loses sensitivity. At first the "rotten egg" odour is detected but on the second or third breath, the odour is no longer noticed. At 600 ppm, breathing is inhibited, as the lungs fill with the gas. Exposures of ppm or greater usually result in death The Netherlands: MAC-value = 10 ppm (15 mg/m 3 ) Lettinga Associates Foundation 50 Corrosion under water Model of electrochemical and microbial activities in corrosion of carbon steel (after Nielsen, 1993 and Hamilton, 2002) Lettinga Associates Foundation 51 26
27 Corrosion above the water level sulfuric acid attack Concrete sewer pipe Iron influent distribution box of a UASB treating domestic sewage Corrosion due to microbiological and chemical oxidation of H 2 S to H 2 SO 4 H 2 S reduction of oxidized S-compounds to H 2 S by SRB uncorroded concrete Lettinga Associates Foundation 52 Sulphate Rich Wastewaters Pulp and paper industry Food industry Tanneries Photographical industries Leachates Wash water flue gases Sulphur containing xenobiotics Lettinga Associates Foundation 53 27
28 Sulphur in Manure Livestock Type Solid Manure (lbs/ton) Sulphur Content Liq. Manure (lbs/1000 Imp. gal) total available total available Dairy Beef all types 1.7 Feedlot Swine all types Feeder Poultry Lettinga Associates Foundation 54 Inhibition phenomena Sulphide Only undissociated H 2 S is toxic Diffuses freely through cell membrane Denatures proteins and enzymes (sulphide cross-linking) Effect internal cell ph Inhibition of methane producing bacteria Sulphite Sulphate: Non toxic!! Acute toxicity to methanogens Enrichment Culture: Lag Phase of > 2 h by 25 mg/l Unadapted Sludge : IC50 = mg/l Prolonged exposure: adaptation because 'of SRB populations adapting to higher concentrations Full scale: up to 800 mg/l satisfactorily Lettinga Associates Foundation 55 28
29 ph dependent toxicity H2Sgas H2S aq H 2 S liq = K H * ph 2 S H 2 S HS + H Fraction of total dissolved sulphide present as undissociated H 2 S: f H2S, (30 C). + f H2S = H 2 S/(H 2 S + HS - ) = 1/(10 ph-pka + 1) Lettinga Associates Foundation 56 Inhibition by sulphide Sulphide inhibition at various ph, T, and different sludge types: H 2 S and total-sulphide (TS) concentrations causing a 50% inhibition of acetotrophic methanogenesis. Sludge type ph T H 2 S TS Ref ( C) (mg/l) (mg/l) Suspended Granular Oleskiewicz et al. 1989, 2. Visser et al. 1993e, 3. Koster et al Lettinga Associates Foundation 57 29
30 Competition between SRB and MPB: Hydrogen Clear kinetic and thermodynamic advantage of SRB over MB Lettinga Associates Foundation 58 Methods of dealing with sulphate reduction In anaerobic digesters Preventative Remove sulphate from waste Biochemical inhibitors Corrective No operational problems when H 2 S concentration < 150 mg/1 and COD/SO 4 2- > > Always successful!! However, COD/SO 4 2- < > Methanogenic process can fail Possible action: Reduction of reactor sulphide concentration by: Separation of sulphide production and methanogen production Sulphide removal in methanogenic reactor: e.g. sulphide stripping Lettinga Associates Foundation 59 30
31 Toxicity relieve: sulphide stripping by produced biogas H 2 S g H 2 S l HS - + H + CH 4 COD + SO 4 2- H 2 S-stripping by biogas at high COD/SO 4 -ratio s Lettinga Associates Foundation 60 Combined effect of stripping and ph A A B C A) COD:SO 4 ratio = 10 B) COD:SO 4 ratio = 5 C) COD:SO 4 ratio = 2 a= ph 6.5 c= ph 7.5 b= ph 7 d= ph 8 Lettinga Associates Foundation 61 31
32 Preventive methods: biochemical inhibitors Molybdate Leads to ATP depletion ATP + SO 2 4 ATP + MoO 4 APS 2 APMo Disadvantages Molybdate Competitive Inhibition > High Concentrations Required Non-specific Inhibition of MPB Antibiotics: Gentamicin Transition metals: Cu/Fe, Zn/Fe, Mn/Fe Lettinga Associates Foundation 62 Methods for H 2 S concentration reduction Methods to reduce the H 2 S concentration in methanogenic reactors 1. Stripping of H 2 S from the solution 2. Dilution of wastewater (also with effluent) 3. Increase of the ph of the reactor Lettinga Associates Foundation 63 32
33 H 2 S removal from biogas GTZ procedure for biogas plants: air injection in a floating gas storage air purified biogas biogas S 0 Digester Lettinga Associates Foundation 64 Sulfur deposition at effluent gutter Aviko, Steenderen Lettinga Associates Foundation 65 33
34 Environmental factors Temperature + fluctuations Availability of nutrients (N, P, micro-nutrients) ph / Buffer capacity / Alkalinity Volatile Fatty Acids Ammonium Presence of alternative electron acceptors (SO 2-4, NO 3-, etc.) Presence of toxic compounds Risk of formation of inorganic precipitates Risk of formation of scum layers and/or flotation layers Odour problems Lettinga Associates Foundation Metabolic Mechanisms of Toxicity (Cell Level) Reversible Lower activity Examples: Substrate exclusion due to precipitation or adsorption phenomena (LCFA, fat) Nutrient limitation due to precipitation or coagulation (heavy metals, phosphate) Toxicity due to accumulation of intermediate compounds, e.g. H 2, VFA (substrate or product inhibition) Lettinga Associates Foundation 67 34
35 2. Physiological Mechanisms of Toxicity (Cell Level) Damage to (sub)cellular components Complete but reversible inhibition of the metabolism Slow recovery of the cell activity Examples: Denaturation of enzymes (e.g. formaldehyde, low ph) LCFA effect on membranes 3. Bactericidal Damage to the entire cell, leading to cell death (e.g. chloroform) Irreversible Recovery only by growth Lettinga Associates Foundation 68 Toxicity in anaerobic treatment Two groups of inhibitory agents: 1. Compounds which - in low concentrations - are usually present in biological systems and which are e.g. related to substrate conversion and growth substrates (intermediate) products nutrients trace elements, etc. 2. Compounds which are usually absent in biological systems. Often resulting in disturbance of the cell metabolism at very low concentrations, e.g. chlorophorm, cyanide Lettinga Associates Foundation 69 35
36 CH 4 production with/without toxicant Methane production biomass in standard medium (control) biomass + toxicant Time Lettinga Associates Foundation 70 Degree of toxicity I: Dependent on the concentration Concentration dependent effects exist for some compounds. Three ranges can be distinguished: Stimulatory Non-toxic Toxic Stimulatory and non-toxic ranges occur relatively widely with natural compounds, generally present in biosystems (Na +, K +, Ca 2+, Mg 2+, NH 4+ ) Absent with strong (man-made) toxicants immediate toxicity Lettinga Associates Foundation 71 36
37 Degree of toxicity I: Dependent on the concentration A S stimulation non-toxic concentration inhibition EF3009 Lettinga Associates Foundation 72 Degree of toxicity II: Immediate toxicity Some toxic compounds inevitably kill, independent of the concentration. e.g.: Formaldehyde Sulphite Cyanide Chlorinated carbohydrates Features: toxic at low concentrations hardly any adaptation of the biomass sometimes conversion (CN, SO 3 2-, formaldehyde) Lettinga Associates Foundation 73 37
38 Degree of toxicity III: effective concentration Degree of toxicity often determined by physico-chemical appearance of toxicant: Solubility of the compounds: Heavy metals are highly toxic in free metal form (Me 2+ ), but nontoxic when precipitated as metal sulphide! Soluble LCFA are more toxic than precipitated. Electric charge of the compound: Non-ionized (small) molecules can freely diffuse across the membrane: toxicity of weak acids and weak base is ph dependent Lettinga Associates Foundation 74 Important parameters/phenomena 1. Concentration: effective <=> total degree of dissociation formation of slowly soluble precipitates 2. Antagonism (suppression of toxicant) and synergism (enhancement) Toxic cation Antagonist Synergist Na K K Na Ca Mg NH 4 NH 4 Ca Mg Anaerobic sludge: IC50 = 6-40 g Na+/l Ca Mg Na K Na K NH 4 Mg NH 4 Ca Most data: IC50 = 8-10 Na+ g/l NH 4 Na K Ca Mg Lettinga Associates Foundation 75 38
39 Important parameters/phenomena 3. Adaptation habituation of the cell metabolism in-growth of organisms with the ability of degrading/ detoxifying the inhibitory agent in-growth of organisms with a high tolerance to the inhibitory agent 4. Sludge retention time systems with high sludge retention times are less sensitive to toxic compounds (due to mass-transfer limited substrate conversion) sufficient activity may be preserved under inhibited conditions Lettinga Associates Foundation 76 Categories of toxic compounds Inorganic compounds NH 3 H 2 S SO 3 2- salts heavy metals O 2 Organic compounds low weight VFA high weight VFA apolar phenolic compounds resins tannins xenobiotics chlorinated hydrocarbons formaldehyde cyanide antibiotics Lettinga Associates Foundation 77 39
40 Classification of toxic compounds (1) 1. Compounds that can dissociate (ph dependent toxicity) e.g. VFA, H 2 S, NH 3 2. A-polar compounds e.g. Long chain fatty acids (LCFA), apolar penolic compounds, such as wood resins, and lignin derived compounds 3. Polar compounds e.g. tannins, phenolic amino acids, caramel compounds 4. Ionic compounds e.g. alkali metal ions, earth-alkali metal ions, anions like chloride, SO 3 2-, SO 4 2-, CN -, heavy metals 5. Xenobiotics e.g. chlorinated organic compounds,aldehydes, aromatic compounds, detergents, antibiotics 6. Precipitating compounds and compounds that adsorb Lettinga Associates Foundation 78 Classification of toxic compounds (2) 5. Xenobiotics e.g. chlorinated organic compounds,aldehydes, aromatic compounds, detergents, antibiotics 6. Precipitating compounds and compounds which adsorb Lettinga Associates Foundation 79 40
41 ph dependent toxicity The toxicity is determined by the non-dissociated molecule. Both the total concentration and the ph determine the free non-dissociated (toxic) concentration of these potentially inhibitory compounds. e.g.: NH 3 H 2 S VFA NH4 NH3 + H + H 2 S HS + H + CH3COOH CH3COO + H + Lettinga Associates Foundation 80 Other potentially toxic compounds Chlorinated carbohydrates highly toxic, especially CCl 4, CHCl 3, and CH 2 Cl 2 (already inhibitory at 1 mg/l) stripping (biogas production) reduces toxicity in case of shock loadings: recovery time is affected by the concentration acidification is relatively insensitive to chlorinated hydrocarbon toxicity higher chance of ph drop in the reactor. Cyanide highly toxic at very low concentrations (acidogens are far less sensible) adaptation can occur CN- can be degraded microbiologically adapted sludge rapidly loses its adapted character Lettinga Associates Foundation 81 41
42 Other potentially toxic compounds Formaldehyde causes denaturation of proteins biodegradable at low concentrations Sulphite highly inhibitory at concentrations > 100 mg/l adaptation thanks to growth in of SRB Oxygen highly toxic for obligate anaerobes O 2 toxicity is seldom a problem in practice, thanks to the presence of facultative aerobes Aromatic compounds Apolar compounds more toxic: cell membranes Hydrogen bonds (polyphenols such as tannins): denaturation Lettinga Associates Foundation 82 Causes: Toxicity of aromatic compounds Polarity Apolar compounds are more toxic as a consequence of their destructive effect on cell membranes. Hydrogen bonds Poly-phenols (e.g. tannins) are toxic as a consequence of the hydrogen bonds they form with cell proteins, which often results in denaturation of these proteins. Molecular size Compounds with a molecular size higher than 3000 g/mole will not be toxic since these molecules are too large to pass the cell envelope. Lettinga Associates Foundation 83 42
43 Important factors Adaptation Antagonism and synergism Dissociation of compounds Polarity of compounds Size of the compounds Occurrence of precipitation and/or adsorption Occurrence of (bio)reactions Occurrence of physical processes, i.e. stripping Lettinga Associates Foundation 84 43
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