2. Literature Review
2.3 Importance of nutrients in anaerobic digestion
2.3.2 Growth and functioning of Microorganisms
2.3.2.1 Macronutrients
Nutrients are necessary for the actual growth and metabolism of the microorganisms (Chen et al., 2008; Fermoso et al., 2009; Burgess et al., 1999). An absence of these will severely limit the substrate utilization rate (Speece, 1996). Six macronutrients are required by biological cells for metabolic processes such as synthesis of proteins, lipids, carbohydrates and nucleic acids (Burgess et al., 1999). They are carbon, oxygen, hydrogen, nitrogen, sulphur and phosphorous. Besides carbon, the macronutrients required in the highest concentration for growth are nitrogen and phosphorous (McCarty, 1964; Burgess et al., 1999). These macronutrients may be absent in industrial waste streams. Addition in the appropriate quantities must be done as a lack of these decreases the microbial population. This results in an increase in the hydraulic retention time of the influent as more time is required for the breaking down of the complex material (Burgess et al., 1999).
There exist a number of different ratios of COD of the wastewater stream to macronutrients required by microorganisms. COD:N:P ratios of 100:10:1 (Beardsley and Coffey, 1985 as cited in Burgess et al., 1999), 250:7:1 (Franta et al., 1994) and 100:20:1 (Metcalf and Eddy, 1991 as cited in Burgess et al., 1999) have been presented in literature. It has also been suggested that nitrogen requirements may be determined from the cell growth and the fraction of nitrogen in the cells.
McCarty (1964) describes using the average chemical formulation of biological cells of C5H9O3N.
Based on that, the nitrogen requirement is about eleven percent of the cell volatile solids weight.
Phosphorous requirements have been found to be one-fifth of the nitrogen requirement. This translates into two percent of the cell volatile solids weight.
Although differences in the amounts of nitrogen and phosphorous to be added to an anaerobic system do occur, the roles of these macronutrients in biological treatment processes are well known (Wood and Tchobanoglous, 1975). Carbon requirements for microorganisms are also unambiguous.
Certain microbes are unable to metabolise complex, synthetic compounds as their sole carbon source and consequently require a readily degradable form of carbon for efficient functioning (Singleton, 1994). The addition of these forms of carbon (examples include glucose, glutamate and organic acids) assist in maintaining the effectiveness of the microorganisms (Leahy and Colwell, 1990). Sole addition of these carbon sources to wastewater systems that lacked nutrients increased the degradation of other pollutants (Gonzalez and Hu, 1991; Hendriksen et al., 1992).
20 2.3.2.2 Micronutrients
The function of micronutrients in biological treatment processes are not as well defined as the roles of the macronutrients (Wood and Tchobanoglous, 1975). This is due to the complex nature of the chemical and biochemical interactions during anaerobic digestion, as well as the difficulties involved in measuring trace quantities (Burgess et al., 1999). Unlike macronutrients, a theoretical amount of micronutrients required by the microorganisms has not been established (Burgess et al., 1999). The one certainty is that the supplement needs to be comprehensive to cater for all the microorganisms present in the sludge.
Metals play a vital role in the biological processes of living organisms (Fermoso et al., 2009). More than twenty five of the elements in the periodic table have an essential biological role (Franzle and Market, 2002). The trace elements required include manganese, zinc, cobalt, molybdenum, nickel, copper, vanadium, boron, iron, iodine, selenium, chromium and tungsten (Fermoso et al., 2009;
Burgess et al., 1999; Speece, 1996). The table below provides a description of some of the general roles of the trace elements in microbial systems (Burgess et al., 1999), highlighting the essential nature of micro-metals:
21
Table 3: Role of micronutrients in microbial systems
Micrometal Requiring microorganisms
Function References (cited in Burgess et al., 1999) Iron (Fe2+ and
Fe3+)
Aerobic bacteria, Aspergillus niger
Growth factor Lilly and Barnett (1951); Mahler and Cordes (1966)
Iron (Fe3+) Possibly all organisms Electron transport in cytochromes Rasmussen and Nielsen (1996); Knauss and Porter (1954) Synthesis of catalase, peroxidise, aconitase Wood and Tchobanoglous (1975)
Iron reducing bacteria Ion reduction for floc formation Nielsen (1996)
Zinc Bacteria Metallic enzyme activator Mahler and Cordes (1966)
Activity of carbonic anhydrase and carboxypeptidase A
Wood and Tchobanoglous (1975); Cardinaletti et al. (1990) Stimulates cell growth Speece et al. (1983); Shuttleworth and Unz (1988)
Cobalt Bacteria Metallic enzyme activator Wood and Tchobanoglous (1975); Mahler and Cordes (1966)
Structural constituent of cofactor vitamin B12
Jansen et al. (2007) Magnesium Heterotrophic bacteria Enzyme activator Srinath and Pillai (1966)
Manganese Bacteria Enzyme activator Wood and Tchobanoglous (1975)
Copper Bacteria Enzyme activator Gökçay and Yetis (1996)
Chelates other substances and reduces their toxicity
Vandevivere et al. (1997)
Nickel Cyanobacteria and
Chlorella
Stimulates enzymes in methane production Gökçay and Yetis (1996) Methanogenic
anaerobes and activated sludge cultures
Maintenance of biomass Gökçay and Yetis (1996)
Calcium Aerobic bacteria Cell transport systems, osmotic balance and aids flocculation
Nielsen (1996); Shuttleworth and Unz (1988) Thiothrix and Zoogloea Improves flocculation by increasing growth
rates
Geradi (1986)
22 The majority of the metals form part of the active site of enzymes that catalyze anaerobic reactions and transformations (Fermoso et al., 2009). The table below provides a description of the functions of some trace elements in these enzymes (Fermoso et al., 2009):
Table 4: Roles of trace metals in enzymes involved in anaerobic reactions and transformations (Fermoso et al., 2009).
Element Functions Element Functions
Cu - Superoxide dismutase
- Hydrogenase (Facultative anaerobes) - Nitrite reductase
- Acetyl-CoA synthase
Ni - CO-dehydrogenase - Acetyl-CoA synthase
- Methyl-CoM reductase (F430) - Urease
- Stabilize DNA, RNA - Hydrogenase Co - B12-enzymes
- CO-dehydrogenase - Methyltransferase
Se - Hydrogenase
- Formate dehydrogenase - Glycin reductase Fe - Hydrogenase
- CO-dehydrogenase - Methane monooxygenase - NO-reductase
- Superoxide dismutase - Nitrite and nitrate reductase - Nitrogenase
W - Formate dehydrogenase - Formylmethanofuran- dehydrogenase
- Aldehyde-oxydoreductase - Antagonist of Mo
Mn - Stabilize methyltransferase in methane producing bacteria - Redox reactions
Zn - Hydrogenase
- Formate dehydrogenase - Superoxide dismutase Mo - Formate dehydrogenase
- Nitrate reductase - Nitrogenase
V - Nitrogenase - Chloroperoxydase - Bromineperoxydase
23 A high concentration of metals in solution does not guarantee that these metals will be absorbed by microorganisms or form part of the active site of enzymes. Factors such as bioavailability of the metals in the aquatic environment and biouptake by the cells play an important role.
Nutrient deficiency is signaled by a high volatile fatty acid (VFA) concentration. High VFA in the effluent is also the indication for toxicity. Once it has been determined that the nutrients are sufficient and bioavailable, the problem of toxicity should be investigated (Speece, 1996).
2.3.2.3 Vitamins
A vitamin is an organic compound that is required by an organism as a vital nutrient in limited quantities. The role of vitamins and their effects have not been as widely studied as that of macro and micronutrients. Vitamins are not as crucial to the growth and functioning of the microbes as the addition of other nutrients, where addition would enhance a process but elimination would not be detrimental. Many systems function well without the addition of vitamins. This is due to the ability of the microorganisms to synthesize some of the vitamins (Lemmer et al., 1994). However, in the case of Co, the addition of vitamin B12 may be a substitute for the addition of a metal salt. Fermoso et al., 2010 investigated the effect of supplementing a cobalt deprived system with vitamin B12
pulse additions and compared this to the addition of cobalt as CoCl2. Both maintained full methanol degradation in the reactors but a higher specific methanogenic activity was observed in the vitamin B12 supplied reactor.
Vitamins that are required and have a defined role in the system include vitamins K, B1, B2, B6, B12, biotin, niacin and pantothenic acid. The table below gives a brief description of the role of some vitamins required by the microbes (Burgess et al., 1999):
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Table 5: Role of Some Vitamins in microbiological processes
Vitamin Role
K Plays a part in respiration
B1 Used for carbohydrate metabolism and cell growth
B2 Required for growth
B6 Growth factor, also required for other metabolic processes
B12 Required for growth
Biotin Required for metabolic activity
Niacin Growth factor. Takes part in oxidative phosphorylation and in the production of cozymase.
Pantothenic Acid
Growth factor in initial cell growth, fermentation, propagation, respiration and glycogenesis.
Other vitamins such as A, D,E and P do not have defined roles but additions of these have resulted in superior degradation, decreased biomass production and reduced digester disturbances (Burgess et al., 1999).