Evaluating limiting steps of anaerobic degradation of food waste ...

Q IWA Publishing 2008 Water Science & Technology—WST | 57.3 | 2008

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Evaluating limiting steps of anaerobic degradation of food waste based on methane production tests Luis Ortega, Ce´line Husser, Suzelle Barrington and Serge R. Guiot

ABSTRACT This research adapted a batch test for biochemical methane production (BMP) to follow the degradation of complex compounds such as proteins and vegetable oils. The test measured the transformation of albumin and olive oil into methane under mesophilic and thermophilic conditions and assess limiting step in the overall degradation process. The thermophilic sludge

Luis Ortega Suzelle Barrington Department of Bioresource Engineering, McGill University, 21 111 Lakeshore Road, Ste-Anne-de-Bellevue (Quebec), H9X 3V9, Canada E-mail: [email protected]

used for the BMP tests was adapted during ten month from mesophilic sludge while being fed food waste. As compared to acetic acid, the specific rate of transformation of albumin and olive oil into methane reached 22 and 51%, respectively, under mesophilic conditions. Acetoclastic methanogenesis was not the limiting step in the presence of albumin or olive oil (and its monomer-like molecules such as amino acids, glycerol and oleic acid). Rather, the degradation of albumin was restricted by the presence of proteins. The thermophilically adapted sludge showed good proteolytic activity, but its acetoclastic methanogens were unable to degrade olive oil,

Ce´line Husser Serge R. Guiot National Research Council of Canada, Biotechnology Research Institute, 6100 Royalmount Avenue, Montreal (Quebec), H4P 2R2, Canada. E-mail: [email protected] [email protected] [email protected]

because of the inhibitory effect of oleic acid. Key words

| albumin, amino acids, anaerobic digestion, oleic acid, olive oil, thermophilic

INTRODUCTION The anaerobic degradation of organic mater is a complex

governed by the presence of complex organic matter and

process requiring the coordinated interaction of several

by the presence of the microorganisms capable of

groups of microorganisms: hydrolytic and acidogenic

breaking down such compounds. The objective of this

fermentative bacteria, syntrophic acetogenic bacteria,

study was to investigate the degradation of albumin and

and

methanogenic

olive oil in serum bottles under mesophilic and thermo-

archaea. Acetoclastic methanogenesis accounts for 70%

philic conditions using an easy to follow, methane-

of the organic matter transformation into methane

producing based methodology, such as the modified

(Gujer & Zehnder 1983). Normally, the anaerobic degra-

biochemical methane potential (BMP) assay. First, the

dation rate for a highly degradable substrate is limited by

BMP test evaluated degradation rates under non-limiting

the slow activity of acetoclastic methanogens, since

conditions using acetic acid as substrate. Then, the BMP

their growing rate is several times slower than that

test evaluated the degradation rate of albumin and olive

observed for bacterial populations (Ferry 1993). For

oil and their transformation into methane and carbon

example, in food waste containing degradable macro-

dioxide. The rate of hydrolysis was evaluated along with

molecules such as proteins, carbohydrates, and lipids,

the methanization rate for the original compounds

the

towards

and their monomer (a mixture of alanine and glycine

acetic

for albumin and oleic acid and glycerol in the case of

acetoclastic

limiting

hydrolysis

and

hydrogenophilic

degradation

instead

of

step the

is

displaced

degradation

of

acid. Thus, both methanization rate and potential are doi: 10.2166/wst.2008.060

olive oil).

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Luis Ortega et al. | Anaerobic degradation of food waste

METHODOLOGY Sludge The tests were carried out using a mixture of mesophilic granular sludge taken from industrial up-flow anaerobic

Water Science & Technology—WST | 57.3 | 2008

Cornacchio et al. 1986) and the methane production was measured from three of the quadruplets to be expressed in mmol of CH4 per gVSS of sludge per day. The fourth quadruplet was used to follow the evolution of acetic acid concentration.

sludge blanket reactors treating wastewater from a cheese plant and an apple-juice plant. The sludge was adapted under thermophilic conditions for ten months, while being fed a food waste characterized previously (Ortega et al. 2007).

Analytical methods To calculate the amount of substrate supplied to the bottles, all substrates were analyzed for COD and to calculate the amount of inoculation, the sludge was analyzed for VSS (g l21). All analyses were carried out

Substrates

according to Standard Methods (1995). Acetic acid and biogas composition were measured as previously described

The substrates selected to run the BMP tests corresponded

(Ortega et al. 2007).

to specific steps in the methanization process via acetogenesis: 1) Acetic acid (CH3COOH) transformed into methane by acetoclastic methanogens; 2) Albumin hydrolysed into

RESULTS AND DISCUSSION

peptides, then amino acids, and then acetic acid (Ramsay & Pullammanappallil 2001); 3) Alanine (C3H7O2N) and

Acetoclastic methanogenesis under mesophilic

Glycine(C2H5O2N) transformed into acetic acid (Ramsay

conditions

& Pullammanappallil 2001); 4) Olive oil containing triglycerides transformed by the hydrolytic and fermentative bacteria into long chain volatile fatty acids (VFA) and glycerol (Rawn 1990); 5) Oleic acid (C18H34O2) transformed into acetate throughout b-oxidation (Rawn 1990); and 6) Glycerol (C3H8O3) a possible (after oil hydrolysis) methanogenic substrate via its transformation into acetic acid. Oleic acid determines the production of methane resulting from the degradation of unsaturated long chain VFA since oleic acid accounts for 85% of the mass of olive oil (Michigan State University—Department of Chemistry 2007).

The first experiments using mesophilic conditions and acetic acid as substrate represents the last step in the anaerobic degradation of organic matter into methane. It is instrumental to know if acetoclastic methanogenesis is the most limiting step. The bottles contained 0.06, 0.15 and 0.21 M acetic acid, which corresponded to 3.4, 8.9 and 12.8 g acetic acid l21, respectively or low, intermediate, and high concentrations of the acid. The degradation of 0.06 and 0.15 M of acetic acid into methane under mesophilic conditions started almost immediately without lag phase (Figure 1), but with 0.21 M of acetic acid, the lag phase lasted 2.6 days. A methane production plateau was first

Methodology The BMP tests were conducted in quadruplutes, where a specific amount of substrate and sludge was added to each bottle and incubated at 35 (M) or 558C (T), for as long as required to reach a plateau in methane production. In all cases, quadruplute control bottles were also tested, where these bottles only received the sludge but no substrate. The BMP tests were carried out using 120ml serum bottles (Owen et al. 1979; Shelton & Tiedje 1984;

Figure 1

|

Mesophilic BMP activity with 0.06, 0.15 and 0.21 M of acetic acid.


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reached with 0.06 M acetic acid after 4 days while it took 9 and 13 days to reach this plateau with 0.15 and 0.21 M of acetic acid, respectively. The biomass-specific rate for methane production and yield (Table 1) indicated that the high and low concentrations of 0.21 and 0.06 M of acetic acid had no inhibitory and limiting effects, respectively. These results were used to evaluate the methanization of albumin, olive oil, glycerol, and oleic acid.

Mesophilic and thermophilic BMP test for albumin and Figure 2

amino acids For mesophilic (M) and thermophilic (T) conditions, the cumulative methane production with albumin and amino acids (a mixture of alanine and glycine) are compared to the

|

Mesophilic (M) and thermophilic (T) BMP test for albumin and amino acids.

(T) conditions are presented along with the non-limiting BMP results for acetic acid under thermophilic conditions (Figure 3). All tests were conducted using 4.5 g

non-limiting BMP profile for 0.15 M of acetic acid under

CODSubstrate l21. Under mesophilic conditions, olive oil and

thermophilic conditions (Figure 2). All tests were con-

glycerol produced 89 and 62% of the methane obtained

21

ducted using 4.5 g CODSubstrate l

with 0.15 M of acetic acid under mesophilic conditions.

.

Under mesophilic conditions, albumin had a low BMP

Because of a similar BMP activity of 1.3 ^ 0.2 mmolCH4 -

21

) as compared to that with

gVSS21 d21 for olive oil and glycerol, the degradation of

0.15 M of acetic acid (40 mmol CH4 gVSS21). But under

olive oil was not limited by the hydrolysis step under

thermophilic conditions, its BMP activity was almost

mesophilic conditions. The degradation of olive oil at 558C

equivalent. The cumulative methane production with

was much slower (2 0.04 ^ 0.1 mmolCH4 gVSS21 d21) and

amino acids under thermophilic conditions (43.8 mmol

could not be attributed to solubility or hydrolysis, but rather

potential (20 mmol CH4 gVSS

CH4 gVSS

) exceeded that of acetic acid (42.6 mmol

to the low degradability of the oil and the release of long

21

), despite a slower initial rate explained by the

chain volatile fatty acids (mostly oleic acid) into the media.

initial transformation of amino acids into acetic acid. The

Under mesophilic conditions, oleic acid did not produce

lower albumin (protein) BMP activity, as compared to that

more methane than the control. Oleic acid clearly had an

of the amino acids indicated that protein hydrolysis could

initial inhibitory effect on thermophilic methanogenic

limit the overall anaerobic degradation rate.

microorganisms, but this effect slowly disappeared with

CH4 gVSS

21

time especially after 30 days. Mesophilic and thermophilic BMP test for olive oil, glycerol and oleic acid

These results were confirmed by the acetic acid profile obtained from the analysis of the liquid phase of a fourth bottle included in the BMP test (data not shown). With

The cumulative methane production with olive oil, glycerol,

olive oil, the production of acetic acid never exceeded that

and oleic acid, under both mesophilic (M) and thermophilic

of the control, indicating no oil degradation. Although no

Table 1

|

Mesophilic BMP activity with 0.06, 0.15, and 0.21 M of acetic acid

Acetic acid concentration (M)

0.06

0.15

0.21

Initial rate (mmol CH4 gVSS21 d21)

2.3 ^ 0.9

2.6 ^ 0.6

3 ^ 0.05

Yield (mmol CH4 mmol21 Acetic acid)

0.94 ^ 0.03

0.87 ^ 0.01

0.92 ^ 0.05

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acetoclastic methanogenes slowly disappeared with time, as other groups of microorganisms transformed the long chain volatile acids.

ACKNOWLEDGEMENTS The technical assistance of Alain Corriveau and Ste´phane Deschamps is gratefully acknowledged. National Research Council of Canada supported this research. Additional support was granted by the Mexican Council of Science and Figure 3

|

Mesophilic (M) and thermophilic ( T ) BMP test for olive oil, glycerol and oleic acid.

inhibitory effect could be assumed per se, a gradually

Technology (CONACYT) and the Natural Science and Engineering Research of Canada, towards a graduate scholarship for Mr. L. Ortega.

diminishing inhibition is related to the release of long chain volatile acids, especially oleic acid. Oleic acid was completely transformed into acetic acid (0.03 mol acetic acid gVSS

21

) only after 49 days, followed by a drop in acetic

acid levels. Thus, the limiting step of the degradation of olive oil is not related to the hydrolysis of the triglyceride molecule, but to the inhibitory effect of long chain volatile fatty acids on the acetoclastic methanogens. The syntrophic microorganisms also suffer a lag phase before degrading oleic acid and unsaturated volatile fatty acid. In those cases, the b – oxidation process requires three additional enzymes: cis-D 3 Enoyl-CoA isomerase, Enoyl-CoA hydratase, and S-3-hyroxyacyl-CoA epimerase (Rawn 1990).

CONCLUSIONS The experiments conducted in this research showed that the BMP methodology developed can be used to determine the limiting steps in the degradation of macromolecules such as albumin and olive oil. The BMP tests also demonstrated that the limiting step in the degradation of albumin was related to the hydrolysis of the protein into peptides, since the mixture of amino acids produced methane more quickly and efficiently. As for olive oil, the slow methane production resulted from the release of long chain volatile acids (oleic acid) following oil hydrolysis which appeared to inhibit the next steps. However, this inhibitory effect on the thermophilic

REFERENCES Cornacchio, L., Hall, E. R. & Trevors, J. T. 1986 Modified serum bottle testing producers for industrial wastewaters. Technology Transfer Workshop On Laboratory Scale Anaerobic Treatability Testing Technique. Environment Canada Wastewater Technology Centre, Downsview ON, Canada. Ferry James, G. 1993 Methanogenesis: Ecology, Physiology, Biochemistry and Genetics. Chapman & Hall, New York NY, USA. Gujer, W. & Zehnder, J. B. 1983 Conversion process in anaerobic digestion. Wat. Sci. Tech. 15 (8 –9), 127 –167. Michigan State University—Department of Chemistry. 2007 Lipids. http://www.cem.msu.edu/, reusch/VirtualText/lipids.htm (accessed March 29, 2007). Ortega, L., Barrington, S. & Guiot, R. S. 2007. Thermophilic adaptation of a mesophilic anaerobic sludge for food waste treatment. J. Environ. Manag. (in press; doi: 10.1016/ j.envman.2007.03.032). Owen, W. F., Stuckey, D. C., Healy, J. B., Young, Y. & McCarty, P. L. 1979 Bioassay for monitoring biochemical methane potential and anaerobic toxicity. Wat. Res. 13, 485– 492. Ramsay, I. R. & Pullammanappallil, C. P. 2001 Protein degradation during anaerobic wastewater treatment: derivation of stoichiometry. Biodegradation. 12, 247 –257. Rawn, J. D. 1990 Traite´ de Biochimie. E´ditions de Renouveau Pe´dagogique. Belgium. Shelton, D. R. & Tiedje, J. M. 1984 General method for determining anaerobic biodegradation potential. Appl. Environ. Microbiol. 47(4), 850 –857. Standard Methods for the Examination of Water and Wastewater 1995, 19th Ed., American Public Health Association/American Water works Association/ Water Environmental Federation, Washington DC, USA.