(8b) Thermophilic Anaerobic Digestion of Mixed Substrates: The Effect of Commercial Enzymes Addition in the Efficiency of the Process

The aim of this work was to assess the effects that the addition of commercially available enzymes have on the anaerobic digestion of olive mill wastewater (OMW), olive mill solid waste (OMSW), winery wastes (WW), distillery wastes (DW), dairy cattle manure (CM), slaughterhouse wastes (SHW), animal white fat (WF) and sterilized mass – rendering product (SM). All experiments took place under thermophilic conditions because of their superiority when compared to the mesophilic conditions for bio-converting substrates into bio-methane but also due to the trend of industry to move toward thermophilic temperatures, especially when the generated hot water cannot be utilized for any other uses or when superior effluent quality is required or enforced. For this purpose four commercially available enzymes namely cellulose, lipase, xylase and protease, were used. Two sets of experiments were undertaken in batch systems.

Fuel gases that are generated by microorganisms have attracted significant scientific and financial interest in recent years as renewable alternatives to fossil fuels. This is due to the great potential that these methods are offering and their applicability on a number of different substrates. Furthermore, these biological methods can be developed in such a way as to allow the bio-conversion of substrates into biogas. The latter  can be considered as the target product, but at the same time, zero or negative value biomass can be treated improving in this way the financial performance of the industrial facility through gate fees, carbon credits and higher gas generation. The most commonly used gases derived by microorganism fermentation are bio-hydrogen and biogas. However, bio-methanation, also known as anaerobic digestion (AD), is by far the most commonly applied method for the production of fuel gas mainly due to its greater flexibility and applicability when compared to bio–hydrogen production. AD has been employed for the valorization of a plethora of different substrates including farm, municipal and industrial wastes as well as energy crops and agricultural byproducts.

Anaerobic digestion is a four-stage process commencing with the hydrolysis of the organic macromolecules, followed by acidogenesis, acetogenesis and methanogenesis where methane is being formed. Although anaerobic digestion is a well proven process it suffers  by a number of inhibiting factors (volatile fatty acid accumulation, ammonia and C/N imbalances) and difficulties of the process to hydrolyze and subsequently utilize, efficiently, a number of natural macromolecules (cellulose, semi cellulose, lignin and fat). While the problems created by the inhibitors can be overcome with proper management, inefficient digestion of some macromolecules can only be overcome by process technology updates in order to improve efficiency and effectiveness.

In order to improve hydrolysis, which in mixed substrates is usually the rate limiting stage, different methods and technologies have been proposed including steam explosion and alkali pretreatment. Enzymatic pretreatment is a valid alternative. Enzymes are used by anaerobic microorganisms in order to disintegrate the macromolecules and in this way generate energy and ready to utilize substrate. By increasing the concentration of the specialized enzymes available in the mixed liquor, theoretically the hydrolysis rate can be improved even in substrates that microorganisms are only partly able to exploit. Nonetheless, enzymatic pretreatment has rarely been studied, mainly due to the high cost of the process. On the other hand, as cost of production of enzymes is going down due to the development of metabolic engineering and biotechnology, enzymatic pretreatment presents significant prospects and is expected to be developed amply in the forthcoming years.

The different substrates used in this work present significant heterogeneity as an effect of the different production processes they are byproducts and/or waste of. The highest TS levels (99,5%) was that of WF followed by the SM (96,3%). In contrast OMW was the substrate with the lowest TS levels among those examined, while at the same time it had the highest VS content. Regarding theoretical methane production, WF, showed the highest value due to the high lipids content followed by SHW, while WW and DW had the lowest values, at levels lower than half that of WF. Based on the generated data, the effect that the examined enzymes have on anaerobic digestion of mixed substrates can be divided into three categories: (a) no effect or neglectable effect, as is the case of olive mill waste and distillery wastes; (b) positive effect with methane production taking place faster and organic matter getting exhausted faster. This category includes the white fat and the olive mill solid wastes; (c) negative effect with methane production taking place slower and cumulative methane production being lower when compared to the methane generated by the batches where no external enzymes were added. This category includes cattle manure and sterilized mass.

The highest methane production was achieved by WF, reaching levels very close to the theoretical maxima. The addition of lipase to this substrate significantly improved production throughout the experimental period. During the first 5 days of the experiment methane generation by WF was 96% higher compared to the control batches and on day 12 more than 76% of the theoretical methane production had already been recovered. This is higher by 24% when compared to the untreated batches. On the other hand on day 30 the difference in cumulative production between the treated and untreated batches was less than 9%. This behavior shows that while with the addition of lipase the retention time of the substrate in the reactor can be significantly reduced, lipase addition can have only a limited effect on the process when considering a SRT of 30 days,.

The SMW was the second substrate, which benefitted by the addition of enzymes. Methane recovery by enzymatic hydrolysis improved with the addition of cellulose and xylanase on this substrate by 41% on day 5, 13% on day 12 while on day 30 production was marginally (3%) higher for the untreated substrate. Nonetheless, in both cases methane production was lower than 50% compared to the theoretical value.  This shows that while the addition of enzymes can improve the rate of the process, it does not show improvements in the overall effectiveness of the process.

In contrast to what was observed with WF and MSW, the addition of (a) xylanase and lipase and (b) xylanase alone in OMW and DW respectively seems to have insignificant effects on biomethane production. On the other hand the addition of enzymes affected negatively biomethanation of SM, CM and SHW with reduced yields of -30, -61 and -8% for the three substrates when compared to the yields achieved with untreated samples. The addition of cellulase had the worst effects with the specific methane production of CM failing to pass above the 100mLCH4/gVS mark. On the other hand the untreated substrate achieved a production of 147mLCH4/gVS, which while at the low end, is still within the reported values. Furthermore VS reduction calculated from the theoretical methane production achieved for CM were 21.4% and 34.5% for the pretreated and untreated samples respectively.

Enzymes are characterized for their selectivity toward the substrate and the metabolic pathway that they are activate. In this study addition of enzymes significantly improved the rate of the process in specific substrates while at the same time reduced the required reactor retention time. The latter is of primary importance financially as the system can be downsized and thus improve its throughput. In addition, post bio-methanation greenhouse gas release into the environment during storage is minimized. On the other hand addition of two enzymes (especially protease) affected negatively the process probably through ammonia inhibition. Further work is required to pair specific enzymes to substrates and in this way produce specialized enzymatic cocktails that will be able to result in improvements in the overall performance of anaerobic digestion systems while at the same time minimize the negative side-effects toward anaerobic microorganisms.