Download extraction and purification of hydrolytic enzymes from activated sludge

EXTRACTION AND PURIFICATION OF HYDROLYTIC ENZYMES
FROM ACTIVATED SLUDGE
NABARLATZ Debora a, STÜBER Frank a, FONT Josep a, FORTUNY Agustì b, FABREGAT
Azael a, BENGOA Christophe a
Departament d’Enginyeria Química, ETSEQ, Universitat Rovira i Virgili
Av. Països Catalans 26, 43007 Tarragona, Catalonia, SPAIN
b
Departament d’Enginyeria Química, EPSEVG, Universitat Politècnica de Catalunya
Av. Víctor Balaguer s/n, 08800 Vilanova i la Geltrú, Barcelona, Catalonia, SPAIN
Authors e-mail: [email protected], [email protected]
a
Abstract
The activated sludge generated during wastewater treatment is becoming an important
problem due to that its amount and complexity is increasing every year, and the legislation
regulates much more strictly the final sludge disposal. During the biological treatment of
wastewater, the microorganisms produce hydrolytic enzymes that are able to break complex
molecules (like lipids and proteins) into smaller ones that can pass the cell membrane and be
consumed by them. These enzymes are not free in the media, being bound to the cell surface
or immobilized on flocs by ionic and hydrophobic interactions. In order to extract these
enzymes several methods have been tested, like ultrasound and magnetic stirring
disintegration, combined with different types of additives (like resins or detergents) in order
to recover the enzymatic activity. Commercial hydrolytic enzymes have been used as
pretreatment of activated sludge to improve biogas production or to degrade contaminants
presents in water, so activated sludge is a promising source of this type of enzymes.
In the present work the extraction of two types of enzymes (protease and lipase) was carried
out by magnetic stirring disintegration and ultrasound disintegration, combining them with
different additives, like a cation exchange resin (CER), a non ionic detergent (Triton X-100)
and Tris buffer. It was found that the addition of buffer or CER does not improve significantly
the recovery of enzymatic activity. On the contrary, it was proven that the addition of Triton
X-100 has the major influence in the recovery of protease and lipase. It was found that the
addition of 2% v/v Triton X-100 is enough to recover 57 units protease/g VSS and 12200
units lipase/g VSS using magnetic stirring at 500 rpm and 5ºC during 1h. However, if
ultrasound treatment is applied using the same concentration of Triton X-100 (2% v/v), it was
possible to recover up to 52 units protease/g VSS, and 23500 units lipase/g VSS in just 20
min of treatment using 45% power (90 W) at 5ºC.
Preliminary experiments for the purification of the enzymes were carried out to isolate and
quantify their recovery. The purification steps include removal of Triton X-100, precipitation
of the protein with ammonium sulphate, dialysis to eliminate small molecules and salts, and
liophilization. In every step the total amount of protein and the enzymatic activity recovered
was measured to evaluate the efficiency of the purification procedure. Finally, it was found
that the disintegration method and the purification procedure has to be chosen and optimised
as function of the enzyme to be recovered, and as function of the final application of the
enzyme mixture.