Chapter_6A_Biological_Process_Treatment.pptx

## Anaerobic Biology: A Comprehensive Overview

### Introduction to Anaerobic Biology

Anaerobic biology refers to the study of biological processes that occur in the absence of oxygen. This field encompasses the metabolism of microbes and other organisms that have adapted to thrive in environments devoid of oxygen. The importance of anaerobic biology extends beyond ecological niches; it plays significant roles in global biogeochemical cycles, waste management, bioenergy production, and even food processing.

### The Importance of Oxygen and Anaerobic Environments

Oxygen is a vital element for many organisms, particularly aerobic organisms, which rely on oxygen for energy production. However, numerous environments exist on Earth where oxygen is scarce or completely absent. These anaerobic environments can be found in:

- Waterlogged soils
- Deep ocean sediments
- Anaerobic digesters
- Landfills
- The guts of animals

In these environments, anaerobic microorganisms have evolved specialized metabolic pathways that allow them to utilize alternative electron acceptors such as nitrate, sulfate, carbon dioxide, or organic compounds.

### Types of Anaerobic Organisms

Anaerobic organisms can be classified into several categories based on their energy-generating pathways:

#### 1. **Obligate Anaerobes**
These organisms cannot survive in the presence of oxygen. They rely exclusively on anaerobic respiration or fermentation to generate energy. Examples include:

- **Clostridium spp.**: A genus of bacteria known for producing spores and causing diseases like tetanus and botulism.
- **Bacteroides spp.**: Commonly found in the human gut, involved in the digestion of complex carbohydrates.

#### 2. **Facultative Anaerobes**
These organisms can switch between aerobic and anaerobic respiration depending on the availability of oxygen. Examples include:

- **Escherichia coli**: A common gut bacterium that can ferment sugars in the absence of oxygen or respire aerobically when oxygen is available.
- **Saccharomyces cerevisiae**: Yeast that ferments sugars for ethanol production but can also carry out aerobic respiration.

#### 3. **Microaerophiles**
Microaerophiles require oxygen for their metabolism but at lower concentrations than what is available in the atmosphere. Examples include:

- **Helicobacter pylori**: A bacterium associated with gastric ulcers that thrives in low-oxygen environments.

#### 4. **AEROTOLERANT Anaerobes**
These organisms do not require oxygen for growth but can tolerate its presence. They mainly ferment substances to produce energy. Streptococcus spp. are good examples.

### Anaerobic Metabolism Pathways

Anaerobic metabolism differs significantly from aerobic metabolism primarily in the final electron acceptor used in respiration. The following pathways are common:

#### 1. **Fermentation**

Fermentation is an ATP-generating process that does not involve electron transport chains or external electron acceptors. During fermentation, orga

1. Chapter 6A biological treatment process fundamentals

2. PART 6A1

3. Objectives of biological treatment Transform dissolved and particulate biodegradable constituents in to acceptable end products. Capture and incorporate suspended and non- settleable colloidal solids in to a biological floc or film. Transform or remove nutrients such as nitrogen and phosphorous. Remove specific trace organic constituents. In industrial waste water treatment, the objective of biological treatment may be the reduction of organics, solids and in some cases inorganic solids to acceptable levels. ---------------------------------------------- Some of the contaminants in industrial waste water may be inhibitory. As a result pre treatment may be required.

4. Terminology used for biological wastewater treatment METABOLIC FUNCTION TREATMENT PROCESSES TREATMENT FUNCTION Aerobic ( Oxic ) ProcesseS Suspended growth processes Biological nutrient removal Biological phosphorous removal Anaerobic processes Attached growth processes Carbonaceous BOD removal Nitrification Anoxic processes Combined processes Denitrification Substrate Facultative processes Lagoon processes Combined

5. Microorganisims in wastewater treatment The biomass containing organic matter, nitrogen and phosphorous is slightly denser than water and can be removed by gravity settling. Suspended growth processes: The microorganisims are maintained in suspension by appropriate mixing methods. An example of suspended growth process is the activated sludge procxess . The activated sludge produces flocs that can be removed by gravity. Attached growth processes : The microorganisims are attached to inert packinfg material. The packing material can be rock .gravel, slag, sand ,redwood and plastic. V 1 (Organic material) + V 2 O 2 + NH 3 + V 4 PO 4 3-  V 5 (new cells) + V 6 CO 2 + V 7 H 2 O Where the V i are the stoichiometric coefficients.

6. Activated sludge process and aerated lagoons

7. Trickling filters and rotating biological contactors

8. Suspended growth processes Microorganisms responsible for treatment are maintained in liquid suspension by appropriate mixing methods. Developed by Clarke and Gage in Masachusetes and Ardern and Lockett (1914) in England. The microbial suspension is known as Mixed Liquor Volatile Suspended Solids (MLVSS). An important feature of activated sludge is the formation of floc particles, ranging in size from 50 to 200 m which can be removed by gravity.

9. Attached growth processes The microorganisims responsible for the conversion of organic materials to nutrients are attached to an inert packing material. The organic material and nutrients are removed from the waste water flowing past the attached growth also known as biofilm . Packing materials used in attached growth processes include rock, gravel, slag, redwood and a wide range of plastic and other synthetic material. The packing may be submerged or non-submerged. Trickling filter is the most common unsubmerged attached growth process.

10. Microbial metabolism Examples of bacteria metabolism: (a) aerobic; (b) Aerobic autotrophic, (c) anaerobic heterotrophic. & CO 2

11. Heterotrophs – Use organic carbon for the formation of new cell biomass. Autotrophs - Organisms that derive cell carbon from carbondioxide . Autortrophs have low cell yields of cell mass and growth rates. Photrophs – Organisims that are able to use light as an energy source.

12. Chemotrophs – Organisims that derive their energy from chemical reactions. Chemoautotrophs – derive theor energy from the oxidation of reduced inorganic compounds such as ammonia, nitrite ferrous iron and sulfide. Chemohetrotrophs – usually derive their energy from the oxidation of organic compounds.

13. Respiratory metabolism – generation of energy by enzyme mediated electron transport to an external electron acceptor. Fermentative metabolism : The use of an internal electron acceptor for energy generation. Aerobic – Oxygen is used as an electron acceptor. Anoxic – nitrate or nitrite is used as an electron acceptor under anaerobic conditions.

14. Obligate aerobics are organisms that use oxygen for their energy needs. Facultative aerobic – can use nitrite/nitrate as electron acceptors. Obligate anaerobes- exist only in an environment devoid of oxygen. Facultative anaerobes – can shift from fermentative to aerobic respiratory metabolism.

15. Nutrient requirements For municipal wastewaters treatment sufficient nutrients are generally present. For industrial waste waters nutrients may be needed to be added to the biological treatment processes. The lack of sufficient nitrogen and phosphorous is common especially in the treatment of food processing wastewaters high in organic content.

16. Bacterial growth kinetics The Lag Phase – Represents the time required for the organisms to acclimate to their new environment. The Exponential Growth Phase – Bacterial cells are multiplying at their maximum rate there is no limitation due to substrate or nutrients. The biomass curve increases exponentially. The stationary phase – The biomass concentration remains relatively constant over time. The amount of growth is offset by the death of cells. Endogenous phase – The substrate has been depleted so that no growth is occurring. The change in biomass is due to cell death. An exponential decline in biomass concentration is observed.

17. Batch process biomass growth phases with changes in substrate and biomass versus time

18. Measuring biomass growth Estimate of biomass concentration can be obtained from BOD, COD, VSS, DNA, ATP, Protein content. Example of cell yield: 3C 6 H 12 O 6 + 8O 2 +2NH 3  2C 5 H 7 NO 2 + 8CO 2 + 14H 2 O 3(180) 8(32) 2(113 )

19. By similar calculation, the COD of glucose from the reaction: C 6 H 12 O 6 + 6O 2  6CO 2 + 6H 2 O COD =  (O 2 )/ (C 6 H 12 O 6 ) = 6(32 g/mole) / 3(180 g/mole) = 1.07 g O 2 / g glucose Also Y = 0.39 g cells/ g COD used . The observed yield is less than the theoretical yield due to energy requirements apart from cell synthesis. C 5 H 7 NO 2 + 5O 2  5CO 2 + NH 3 + 2H 2 O COD of cell tissue =  (O 2 ) / (C 5 H 7 NO 2 ) = 5(32)/(113) = 1.42 g O 2 /cells. COD utilised = COD cells + COD of oxidised substrate. COD of oxidised substrate = oxygen consumed Oxygen consumed = COD utilized – COD cells = (1.07 g O 2 /g glucose)(3mole * 180 g glucose/mole) – (1.42 g O 2 / g cells)(2moles*113g cells /mole) = 577.8 g O 2 - 320.9 g O 2 = 256.9 g O 2  

20. Oxygen consumed/Glucose used as COD = (256.9 g O 2 )/(3 moles)(1.07 g COD/g glucose)(180 g glucose/mole) = 0.44 g O 2 / g COD used. However, this is apparent from the original equation: 3C 6 H 12 O 6 + 8O 2 +2NH 3  2C 5 H 7 NO 2 + 8CO 2 + 14H 2 O 3(180) 8(32) 3(113) Because: Oxygen used/ Glucose as COD = 8(32) g O 2 /mole /3(180) g/mole(1.07 g COD / g glucose) = 0.44 g O 2 / g COD used.

21. PART 6A2

22. Microbial growth kinetics Concentration of organic matter is defined mostly by bCOD or COD ( b stands for biodegradable) and UBOD . They comprise of soluble, colloidal and and particulate components. Biomass solids are measured through TSS and VSS. The mixture of recycled solids and influent waste water is measured by MLVSS and MLSS . The solids are composed of biomass , non-biodegradable volatile suspended solids ( nbVSS ) and inert inroganic suspended solids ( iTSS ).

23. Rate of utilization of soluble substrate Rate of Substrate Utilization, Where r su = rate of substrate concentration change due to utilization, g/m 3 .day k = maximum specific substrate utilization rate, g substrate / g microorg.day X = biomass (microorganism) concentration , g/m 3 S = Growth-limiting substrate concentration in solution g/m 3 K = Half velocity constant, substrate concentration at one-half the maximum specific substrate utilization rate, g/m 3

24. Maximum specific growth rate ,  m Maximum specific growth rate ,  m = kY k = Maximum specific substrate utilisation rate, g/g/day  m = Maximum specific bacterial growth rate, g/ g.day Y = True yield coefficient = gm biomass produced/g substrate utilized(consumed).

25. Other rate expressions r su = -k r su = - kS r su = - kX r su = - kx (S/K s )

26. Rate of biomass growth with soluble substrate r g = - Yr su - k d X Where  = specific biomass growth rate, g VSS/ g VSS.day

27. Typical kinetic coefficients for the activated sludge process Coefficient Unit Value Typical Range k Gbs COD /g VSS.day 2-10 5 K s mg/L BOD 25 -100 60 mg/L bsCOD 10 -60 40 Y mg VSS / mg BOD 0.4 - 0.8 0.6 mg VSS / mg bsBOD 0.3 - 0.6 0.4 k d g VSS / g VSS. day 0.06 - 0.15 0.10

28. Rate of oxygen uptake r o = - r su - 1.42 r g Where: r o = Oxygen uptake rate , g O 2 /m 3 .day r su = rate of substrate utilization, g bs COD/m 3 .day 1.42 = the COD of cell tissue, g, gbsCOD / gVSS r g = rate of biomass growth, g VSS/m 3 .day

29. Effect of temperature k T = k 20  (T-20) Where: k T = reaction rate coefficient at temperature T, o c k 20 = Reaction-rate coefficient at 20 c.  = temperature activity coefficient. T = temperature, c.

30. Modelling suspended growth treatment processes Rate of accumulation of microorganisms within the system = Rate of flow of microorganisms in to the system - Rate of flow of microorganisms out of the system boundary + Net growth of microorganisms within the boundary

31. Accumulation = Inflow - Outflow + net growth

32. Assuming steady state conditions ( dX / dt = 0) and using the relation r g = - Yr su - k d X The inverse of the term on the left is called the average solids retention time (SRT)

33. - r su /X is known as the specific substrate utilization rate U Where  = V/Q is the hydraulic retention time

34. Effluent dissolved substrate concentration

35. biological nitrification A two step process in which ammonia (NH 4 -N) is oxidised to nitrite (NO 2 -N) and nitrite is oxidised to nitrate (NO 3 - ). Ammonia exerts oxygen demand and is toxi to fish. Nitrogen should be removed to control eutrophication . If waste water has to be reused, ammonia should be removed (example ground water).

36. Process description For suspended growth process nitrification can be designed along with BOD removal. A two stage system can be designed in the presence of toxic and inhibitory substances. Designed systems have much longer hydraulic and solid retention times. . In attached systems most of the BOD must be removed before nitrifying organisims can be established. Therefore, a separate attached growth system is designed after BOD removal.

37. Biological nitrification (A) single sludge and (b) two sludge

38. Stoichiometry of biological nitrification 2NH 4 + + 3O 2  2NO 2 - + 4H + + 2H 2 O 2NO 2 - + O 2  2NO 3 - Total Oxidation reaction : NH 4 + + 2O 2  NO 3 - + 2H + + H 2 O Alkalinity Requirement: NH 4 + + 2HCO 3 - + 2O 2  NO 3 - + 2CO 2 +3H 2 O Biomass Synthesis reaction: NH 4 + + 1.863O 2 + 0.098CO 2  0.019C 5 H 7 NO 2 + 0.98NO 3 - + 0.0941H 2 O + 1.98H +

39. Growth kintics for nitrification Nitrification is also affected by the level of dissolved oxygen present. Hence the specific growth rate includes the dissolved oxygen term: Where:  n = Specific growth rate of nitrfying bacteria, g new cells/g cells.day  nm = maximum specific growth rate, g new cells/g cells.day N = Nitrogen concentration, g/m 3 K n = The half velocity constant, substrate concentration at half the maximum substrate utilization rate, g/m 3 k dn = Endogenous decay coefficient g VSS/ g VSS.day . DO = dissolved oxygen concentration, g/m 3 K = Half saturation coefficient for DO , g/m 3

40. Environmental factors Nitrification declines below pH 6.8 Optimal nitrification occurs at pH values at pH between 7.5 and 8.0. Alkalinity may be added in the form of lime, soda ash, sodium bicarbonate or magnesium hydroxide. Toxic compounds such as solvent organic chemicals, amines, proteins, tannins, phenolic compounds, alchols , cyanates , ethers carbamates , benezene can inhibit the nitrification process. Metals are known to cause significant to complete inhibition of nitrification. Complete inhibition occurs at Nickel concentration of0.25 mg/L, chromium of 0.25 mg/L and copper of 0.1 mg/L.

41. Biological denitrification The biological reduction of nitrate to nitric oxide, nitrous oxide, nitrogen gas is termed denitrification . Biological denitrification is used where there are concerns of eutrophication and ground water contamination. Denitrification with nitrate recycling in to anoxic tank is called substrate denitrification . When the anoxic tank follows the nitrification tank it is called post anoxic denitrification .

42. (a) Substrate driven denitrification (b) Endogenous driven

43. PART 6A3

44. Micrbiology A fluidized bed reactor under anaerobic condition oxidize NH 4 + by NO 2 - to N 2 gas and a small amount of nitrate. ( Anamox process). Most of the hetertrophic bacteria responsible are facultative aerobic organisims with the ability to use oxygen as well as nitrite And nitrate. The autotrophic nitrifying bacteria such as Nitrosomonas europea can use nitrite to oxidize ammonia with the production of nitrogen gas. Ammonia oxidation with the reduction of nitrite under anaerobic condition has been shown at temperatures above 20 c in the Annamox process.

45. Stoichiometry of biological denitrification Reduction using nitrate as an electron acceptor: NO 3 -  NO 2 -  N 2 O  N 2 The electron donor can be 1) the bsCOD in the influent wastewate 2) the bsCOD produced during the endogenous decay and 3) an exogenous source such as methanol and acetate. Waste water Based C 10 H 19 O 3 N + 10NO 3 -  5N 2 +10CO 2 +3H 2 O +NH 3 + 10OH - Methanol Based 5CH 3 OH + 6NO 3 -  3N 2 +5CO 2 +7H 2 O +6OH - Acetate Based 5CH 3 COOH + 8NO 3 -  4N 2 +10CO 2 +6H 2 O + 8OH -

46. Oxygen equivalent of using nitrate and nitrite as electron acceptor For Oxygen 0.25O 2 + H + + e -  0.5H 2 O For nitrate: 0.20NO 3 - + 1.2H + + e -  0.1N 2 + 0.6H 2 O For nitrite 0.33NO 2 - + 1.33H + + e -  0.67H 2 O + 0.17N 2 Oxygen equivalent: (0.25*32 g O 2 )/mole divided by (0.24*14 g N/mole) = 2.86 g O 2 / g NO 3 - -N

47. bsCOD r = bsCOD syn + bsCOD Where bsCOD r = bsCOD utilized, g bsCOD /day bsCOD syn = bsCOD incorporated in to cell synthesis, g bs COD/day bsCOD = bsCOD oxidized, g bsCOD /day. The bsCOD syn is calculated from the net biomass yield bsCOD syn = 1.42Y n bsCOD r Y n = net biomass yeld , g VSS / g bsCOD r bsCOD r = bsCOD o + 1.42Y n bsCOD r bsCOD = (1 – 1.42Y n ) bsCOD r From the equivalent of Oxygen with nitrate nitrogen bsCOD = 2.86NO x

48. 2.86NO 3 - = (1 - 1.42 Y n ) bsCOD r Or

49. Growth kinetics for denitrification The substrate utilization rate r su for the anoxic/aerobic process is modified by a term to show a lower utilization rate in the anoxic zone as follows: Where  = fraction of denitrfying bacteria in the biomass g VSS/ g VSS The  fraction is not necessary in the post anoxic process where the biomass consists mainly of the denitrfying bacteria. The nitrate concentration controls the substrate utilization only at low nitrate concentrations, near 0.1 mg/L

50. The general formula taking in to account nitrate, denitrfying bacteria and dissolved oxygen is: Where K o ’ = DO inhibition coefficient for nitrate reduction, mg/L K s-NO3 = half velocity coefficient for nitrate limited reaction, mg/L The pH is generally elevated due to denitrification . A decrease in denitrification has been reported when the pH was reduced from 7 to 6 in batch unacclimated tests.

51. Biological phosphorous removal Phosphorous is used generally to control eutrophication . |Chemical treatment using alum or iron salts are generally used to control phosphorous. The advantages of biological phosphorus removal are reduced chemical costs and less sludge production as compared to chemical precipitation. Phosphorus accumulating organisms (PAOs) are encouraged to grow and consume phosphorous in a reactor configuration that provides competetive advantage for PAOs over other bacteria. An anaerobic reactor is placed ahead of the activated sludge tank and is provided with a recirculated sludge.

52. Removal mechanism Numerous bacteria are capable of storing excess amounts of phosphorus as polyphosphates in their cells. Under anaerobic conditions PAOs will assimilate ferementation products ( eg . Volatile fatty acids) in to storage products within the cells with the cocncomitant release of phosphorous from stored polphosphates . Under aerobic conditions, energy is produced by the oxidation of storage products and polyphosphate storage within the cell increases.

53. Processes occurring in the anaerobic zone and anoxic zones Anarobic Acetate is produced by fermentation of bsCOD which as defined earlier, is dissolved degradable organic material that can be assimilated easily by the biomass. Using energy available from stored polyphosphates, the PAOs assimilate acetate and [produce intracellular polyhydroxybutrate (PHB) storage products. Some glycogen contained in the cell is also used. Concurent with acetate uptake is the release of orthophosphate

54. Anoxic Stored PHB is metabolized, providing energy from oxidation and carbon for new cell growth. Some glycogen is produced from PHB metabolism. The energy released from PHB oxidation is used to form polyphosphate bonds in cell storage so that soluble orthophosphate (O-PO 4 3- ) is removed from solution and incorporated in to polyphosphates within the bacterial cells. Cell growth also occurs due to PHB utlilization and the new biomass with high polyphosphate storage accounts for phosphorous removal. As a portion of the biomass is wasted, stored phosphorus is removed from the bio-treatment reactor for ultimate disposal with the waste sludge.

55. Microbiology of phosphorous removaL Phosphorous is impoprtant in cellular energy transfer mechanism via adenosine triphosphate (ATP) and polyphosphates.. ATP is converted to ADP with phosphorous release. Phosphorous is stored in the cells as polyphospahte in volutin granules. High concentration of ortho -phosphate in the anaerobic zone is an indication that phosphorous release has occurred in this zone.

56. The PAOs prefere low molecular weight fermentation substrate which is available in the anaerobic zone. The polyphospahtes because of their polyphosphate storage ability can assimialte COD in the anaerobic zone which other bacteria can not. Extended contatct time in the anaeroboc zone results in the release of O-PO 4 3- which is known as secondary release.

57. Biological Phosphorous removal. (a) typical reactor configuration. (b)Electron microscope image of PHB storage. (c) Polyphosphate storage granules.

58. Fate of soluble bod and phosphorus in nutrient removal reactor

59. Anaerobic fermentation and oxidation Have low biomass yields and energy released can be used. Most digestion is mesophilic ( 30 to 35 c). Thermophilic digestion (50-60 ) can be used which also achieves high pathogen kill. Anaerobic treatment is suited to high strength wastes often as pre treatment to subsequent aerobic units.

60. Process description Three basic steps are involved a)hydrolysis b) fermentation ( acidogenesis ) c) methanogenesis . In hydrolysis particulate material is converted to solouble that can be hydrolysed further to simple monomers. In fermentation, amino acids, sugars and some fatty acids are degraded further . The oproducts of fermentation are acetate, hydrogen, CO 2 , and propionate and butrate . In methanogenesis is carried out by a group of organisms called the methanogens . They split acetate in to methane and carbon dioxide. The second group use hydrogen as electron donor and carbon dioxide as electron acceptor to produce methane.

61. stoichiometry of anaerobic fermentation and oxidation 4H 2 +CO 2  CH 4 + 2H 2 O 4HCOO - + 4H +  CH 4 + 3CO 2 + 2H 2 O 4CO +2H 2 O  CH 4 +3CO 2 4CH 3 OH  3CH 4 +CO 2 +2H 2 O 4(CH 3 ) 3 N + H 2 O  9CH 4 +3CO 2 +6H 2 O +4NH 3 CH 3 COOH CH 4 +CO 2 The stoichiometry relates COD with methane production. From the above equation, the COD per mole of methane is 2(32 g O 2 per mole) = 64 g O 2 /mole. The volume of methane per mole at standard conditions (0 c and 1atm) is 22.414 liters. So the CH 4 equivalnt of COD converted under anaerobic conditions is 22.414 /64 = 0.35 L CH 4 / g COD.

62. Anaerobic process schematic of hydrolysis, fermentation and methanogenesis

63. Carbon and hydrogen flow in anaerobic digestion process

64. Thank you