(623bh) Cellulase Recycling and Reuse: Comparison of Immobilization, Ultrafiltration and Re-Adsorption

    Until now, the enzyme cost is still the dominating economic barrier to produce ethanol from lignocellulosic biomass. Therefore, lower enzyme cost is being pursued for the industrialization of cellulosic bioethanol. The recycling of enzymes has been explored as an effective way of reducing the high cost of enzymes [1, 2]. After enzymatic hydrolysis of lignocellulosic substrates, some enzymes remain free in the hydrolysate, while others are bound to the residual substrates. In our lab, we compare several strategies developed to recover and reuse the enzymes, including ultrafiltration, readsorption onto fresh substrates, and enzyme immobilization.

    (1) Immobilization. Immobilization of enzymes is an important approach for enzyme recycling. Among cellulases, b-glucosidase is frequently used to supplement cellulase preparations for hydrolysis of cellobiose to glucose. Compared to free cellobiase, immobilized cellobiase had improved thermal stability and mechanical strength. It could be reused several times in a continuous reaction packed column. Moreover, immobilized cellobiase is readily separated from the substrate and products, which can simplify subsequent purification processes. However, few studies have used lignocellulosic biomass as substrates and none have been done at high solids loadings. The use of immobilized cellulases for lignocellulose hydrolysis is still a technically difficult task because immobilization may hinder enzyme access to insoluble cellulose. Multi-enzymes complex that are required for hydrolysis of lignocellulose also increase the difficulty in immobilization process.

    (2) Ultrafiltration. Ultrafiltration has been proven to be capable of recovering cellulases and b-glucosidase, and it has the capacity to continuously remove sugars and other small compounds that may potentially inhibit enzyme activity. For this reason, a membrane bioreactor (MBR) may provide some advantages in performing enzymatic hydrolysis. The MBR combines product inhibitor removal and cellulase reuse in one device, which facilitates operation and reduces equipment costs by eliminating an extra reactor. Another advantage of MBR is that the enzymatic hydrolysis rate increases because product inhibition is lowered compared with that in a conventional batch reactor. In the fed-batch MBR, fresh substrate was supplemented five times at the rate of 10 g cellulose per liter each time, resulting in a total cellulose loading of 7%. After 60 h, the enzyme utilization efficiency was 3.13-fold of MBR. An additional process, like reverse osmosis, is required on the downstream processes to concentrate the sugar products.

    (3) Re-adsorption. Cellulases have relatively high stability and high affinity for cellulose. After hydrolysis, most cellulases are free in the hydrolysate. Therefore, free cellulases can be recovered for further use by readsorption onto fresh substrates and subsequent microfiltration. One of the most important factors that limit enzyme recovering in the readsorption process is the adsorption of cellulases to lignin. To reduce the nonproductive adsorption, nonionic surfactants, such as Tween 80, were added to increase free enzymes in the supernatant. In this case, the enzymes (e.g. Spezyme CP) can be used in four successive rounds of enzyme recycling, achieving an above 80% hydrolysis yield after the fourth round. Compared to ultrafiltration, enzyme recycling by readsorption is unable to recover b-glucosidase because it does not typically bind to the cellulosic substrate. Therefore, the supplementation of fresh b-glucosidase is commonly necessary to subsequent rounds of hydrolysis.

Acknowledgement

The authors acknowledge the financial supports received from the Program for New Century Excellent Talents in Chinese University (NCET-08-0386), the Natural Science Foundation of China (20976130, 20806057).

 References

  [1]    Steele E, Raj S, Nghiem J, Stowers M (2005) Enzyme recovery and recycling following hydrolysis of ammonia fiber explosion-treated corn stover. Appl Biochem Biotechnol 121:901-910.

  [2]    Tu MB, Chandra RP, Saddler JN (2007) Evaluating the distribution of cellulases and the recycling of free cellulases during the hydrolysis of lignocellulosic substrates. Biotechnol Prog 23:398-406.