Isolation, production and optimization of endogenous alkaline protease from in-situ sludge and its evaluation as sludge hydrolysis enhancer

Bioconversion (e.g. anaerobic fermentation and compost) is the common recycling method of waste activated sludge (WAS) and its hydrolysis, as the rate-limiting step of fermentation, could be accelerated by protease. However, the commercial protease was unstable in sludge environment, which increased the cost. Herein, an endogenous alkaline protease stable in sludge environment was screened in this study and its suitability for treating the sludge was studied. The optimal production medium was determined by Response Surface Methodology as starch 20 g/L, KH2PO4 4 g/L, MgSO4·7H2O 1 g/L, sodium carboxy-methyl-cellulose 4 g/L, casein 4 g/L and initial pH 11.3, which elevated the yield of protease up to 15 times (713.46 U/mL) compared with the basal medium. The obtained protease was active and stable at 35 °C–50 °C and pH 7.0–11.0. Furthermore, it was highly tolerant to sludge environment and maintained high efficiency of sludge hydrolysis for a long time. Thus, the obtained protease significantly hydrolyzed WAS and improved its bioavailability. Overall, this work provided a new insight for enzymatic treatment of WAS by isolating the endogenous and stable protease in sludge environment, which would promote the resource utilization of WAS by further bioconversion.

Enzymatic Hydrolysis of Softwood Derived Paper Sludge by an In Vitro Recombinant Cellulase Cocktail for the Production of Fermentable Sugars

Paper sludge is an attractive biomass feedstock for bioconversion to ethanol due to its low cost and the lack of pretreatment required for its bioprocessing. This study assessed the use of a recombinant cellulase cocktail (mono-components: S. cerevisiae-derived PcBGL1B (BGL), TeCel7A (CBHI), ClCel6A (CBHII) and TrCel5A (EGII) mono-component cellulase enzymes) for the efficient saccharification of softwood-derived paper sludge to produce fermentable sugars. The paper sludge mainly contained 74.3% moisture and 89.7% (per dry mass (DM)) glucan with a crystallinity index of 91.5%. The optimal protein ratio for paper sludge hydrolysis was observed at 9.4: 30.2: 30.2: 30.2% for BGL: CBHI: CBHII: EGII. At a protein loading of 7.5 mg/g DW paper sludge, the yield from hydrolysis was approximately 80%, based on glucan, with scanning electron microscopy micrographs indicating a significant alteration in the microfibril size (length reduced from ≥ 2 mm to 93 µm) of the paper sludge. The paper sludge hydrolysis potential of the Opt CelMix (formulated cellulase cocktail) was similar to the commercial Cellic CTec2® and Celluclast® 1.5 L cellulase preparations and better than Viscozyme® L. Low enzyme loadings (15 mg/g paper sludge) of the Opt CelMix and solid loadings ranging between 1 to 10% (w/v) rendered over 80% glucan conversion. The high glucose yields attained on the paper sludge by the low enzyme loading of the Opt CelMix demonstrated the value of enzyme cocktail optimisation on specific substrates for efficient cellulose conversion to fermentable sugars.

Effect of different hydrolytic enzymes pretreatment for improving the hydrolysis and biodegradability of waste activated sludge

In this study, the effects of lysozyme, protease and α-amylase pretreatments for improving the hydrolysis and biodegradability of waste activated sludge (WAS) were investigated. The results showed that lysozyme was more effective in increasing the soluble chemical oxygen demand (SCOD) concentration in the liquid phase of sludge and improving the release of protein and carbohydrate from sludge flocculation to enhance sludge hydrolysis. After 8 h hydrolysis, the net SCOD increase in a reactor with lysozyme was 2.23 times and 2.15 times that of the reactors with protease and α-amylase, respectively. Meanwhile, lysozyme and protease could improve the lysis of microorganism cells and the dissolution of extracellular polymeric substances (EPS) to a certain extent, and lysozyme was more effective. Furthermore, the compositional characteristics of dissolved organic matter (DOM) and EPS were analyzed by EEM fluorescence spectroscopy and fluorescence regional integration (FRI) analysis. Tryptophan-like protein was the main component of sludge, which accounted for 31% and 38% of DOM and EPS, respectively. Lysozyme could decrease the percentage of non-biodegradable materials in sludge, such as humic acid-like substances and fulvic acid-like substances, so it could improve the biodegradability of sludge. This study can provide valuable information for future studies about hydrolytic enzyme pretreatments for WAS disposal.

Preliminary Investigation of Primary Sludge Hydrolysis

One of reasons of non-effective biological nutrient removal from wastewater is lack of readily biodegradable organic matter. This problem could be solved by application of sludge hydrolysis process. The conditions for hydrolysis of primary sludge could be created performing the recirculation of the primary sludge and ensuring the required sludge retention time. In the period of preliminary investigation, the following conditions were created in the primary sedimen-tation tank of Vitebsk WWTP: average sludge recirculation was 4.8 % of the inlet flowrate to the sedimentation tank and average SRT was 5 days. Obtained results showed that hydrolysis process allowed improving the ratio between organic matter and nutrients in wastewater.