Biocatalyst recovery

To improve biocatalyst recovery and ease of recycle, a research group reported immobilization of Gordona strain CYKS1 on celite beads [197], A cell loading of 1.5 mg cell/g celite was obtained. Use of immobilized biocatalyst at 50% v/v relative to aqueous buffer volume probably results in higher cell density however, the specific rate of desulfurization appeared to decrease. The reports did not have sufficient data to correctly... 

A procedure for immobilization of a P. stutzeri UP-1 strain using sodium alginate was reported [133], This strain does not perform sulfur-specific desulfurization, but degrades DBT via the Kodama pathway. Nevertheless, the report discussed immobilization of the biocatalyst cells in alginate beads with successful biocatalyst recovery and regeneration for a period of 600 h. However, the immobilized biocatalyst did decrease in specific activity, although the extent of loss was not discussed. The biocatalyst was separated after every 100 h of treatment, washed with saline and a boric acid solution and reused in subsequent experiment. The non-immobilized cells were shown to loose activity gradually with complete loss of activity after four repeat runs of 20 hour each. The report does not mention any control runs, which leaves the question of DBT disappearance via adsorption on immobilized beads unanswered and likewise the claim of a better immobilized biocatalyst. 

Batch No. Esterification (%) Stanol/Sterol Esterification Ratio Biocatalyst Recovery ... 

Biocatalyst recovery and re-use or continuous operation, as well as integrated product recovery, are main assets for the economic viability of an industrial process. Much work has been carried out recently, mainly focusing on the transformation of oils and fats (Table 8). 

The transition between the soluble and insoluble state of stimuli-responsive polymers has been used to develop reversibly soluble biocatalysts. A reversibly soluble biocatalyst catalyzes an enzymatic reaction in a soluble state and hence could be used in reactions of insoluble or poorly soluble substrates/products. As soon as the reaction is completed and the products are separated, the conditions (pH, temperature) are changed to promote precipitation of the biocatalyst. The precipitated biocatalyst is separated and can be used in the next cycle after dissolution. The reversibly soluble biocatalyst acquires the advantages of immobilized enzymes (ease of separation from the reaction mixture after the reaction is completed and the possibility for biocatalyst recovery and repeated use in many reaction cycles) but at the same time overcomes the disadvantages of enzymes immobihzed onto solid matrices such as diflfusional limitations and the impossibility of using them in reactions of insoluble substrates or products. 

Perhaps the first decision to be made in process development is the difficult decision of whether the enzymes to be used should be used in an integrated format. Such a question does not arise with conventional single biocatalytic steps but is highly important in multienzyme processes. One of the key criteria here is whether the enzymes can be operated together without compromise to any of the individual enzyme s activity or stability. An interaction matrix (see Section 10.6) can be used to assist such decision making. In cases where the cost of one or more of the enzyme(s) is not critical, it will be possible to combine in a one-pot operation. In other cases, where the cost of an individual enzyme becomes critical, then it may be necessary to separate the catalysts, such that each can operate under optimal conditions. Likewise, selection of the biocatalyst format (immobilized enzyme, whole cell, cell-free extract, soluble enzyme, or combinations thereof) in combination with the basic reactor type (packed bed, stirred tank, or combinations thereof) and biocatalyst recovery (mesh, microfiltration, ultrafiltration, or combinations thereof) will determine the structure of the process flowsheet and therefore is an early consideration in the development of any bioprocess. The criterion for selection of the final type of biocatalyst and reactor combination is primarily economic and may best be evaluated by the four metrics in common use to assess the economic feasibility of biocatalytic processes [29] ... 

When ionic liquids are used as replacements for organic solvents in processes with nonvolatile products, downstream processing may become complicated. This may apply to many biotransformations in which the better selectivity of the biocatalyst is used to transform more complex molecules. In such cases, product isolation can be achieved by, for example, extraction with supercritical CO2 [50]. Recently, membrane processes such as pervaporation and nanofiltration have been used. The use of pervaporation for less volatile compounds such as phenylethanol has been reported by Crespo and co-workers [51]. We have developed a separation process based on nanofiltration [52, 53] which is especially well suited for isolation of nonvolatile compounds such as carbohydrates or charged compounds. It may also be used for easy recovery and/or purification of ionic liquids. 

The interest and success of the enzyme-catalyzed reactions in this kind of media is due to several advantages such as (i) solubilization of hydrophobic substrates (ii) ease of recovery of some products (iii) catalysis of reactions that are unfavorable in water (e.g. reversal of hydrolysis reactions in favor of synthesis) (iv) ease of recovery of insoluble biocatalysts (v) increased biocatalyst thermostability (vi) suppression of water-induced side reactions. Furthermore, as already said, enzyme selectivity can be markedly influenced, and even reversed, by the solvent. 

The above two processes employ isolated enzymes - penicillin G acylase and thermolysin, respectively - and the key to their success was an efficient production of the enzyme. In the past this was often an insurmountable obstacle to commercialization, but the advent of recombinant DNA technology has changed this situation dramatically. Using this workhorse of modern biotechnology most enzymes can be expressed in a suitable microbial host, which enables their efficient production. As with chemical catalysts another key to success often is the development of a suitable immobilization method, which allows for efficient recovery and recycling of the biocatalyst. 

Another important argument for the use of the organic solvent is the reverse hydrolytic reactions that become feasible [61,75]. The inhibition of the biocatalyst can be reduced, since the substrate is initially concentrated in the organic phase and inhibitory products can be removed from the aqueous phase. This transfer can shift the apparent reaction equilibrium [28,62] and facilitates the product recovery from the organic phase [20,29,33]. A wide range of organic solvents can be used in bioreactors, such as alkanes, alkenes, esters, alcohols, ethers, perfluorocarbons, etc. (Table 1). 

One of the most important advantages of the bio-based processes is operation under mild conditions however, this also poses a problem for its integration into conventional refining processes. Another issue is raised by the water solubility of the biocatalysts and the biocatalyst miscibility in oil. The development of new reactor designs, product or by-product recovery schemes and oil-water separation systems is, therefore, quite important in enabling commercialization. Emulsification is thus a necessary step in the process however, it should be noted that highly emulsified oil can pose significant downstream separation problems. 

Secondary recovery of biocatalyst from the aqueous phase (via filtration, etc.) ... 

Desulfurization of other diesel feedstocks from Total Raffinage was also reported by EBC. In these studies, different engineered biocatalysts were used. Two different middle distillate fractions, one containing 1850 ppm sulfur and other containing 650 ppm sulfur, were tested. R. erythropolis sp. RA-18 was used in one experiment and was reported to desulfurize the diesel from 1850 to < 1200ppm sulfur within 24 hours. On the other hand, it removed sulfur from a middle distillate with 650ppm sulfur to below 200 ppm sulfur [222], Various Pseudomonas strains were also tested in this study and reported to remove less amounts of sulfur. A favorable characteristic of the Pseudomonas strains is their inability to form stable emulsions, which can be useful trait for product recovery. 

Further research is also needed in this area. Particularly, (a) to create a new generation of cheap enzymes for hydrolysis of cellulose and lignocellulose to fermentable sugars (able to complete the biomass hydrolysis during fermentation) (b) to develop improved biocatalysts that allow us to simplify the process and reduce energy input and (c) to improve separation and recovery. 

Figure 15.9 shows that this approach worked quite nicely the substrate was added to a total concentration of 2.5 g/L, but neither substrate nor product accumulated in the bioreactor medium. Without a product recovery loop the product concentration (3-phenylcatechol) did not exceed 0.4 g/L, because of biocatalyst deactivation (results not shown). With the loop, 2-phenylphenol and 3-phenylcatechol concentrations remained below 0.1 g/L. Therefore, cell viability and biocatalytic activity were maintained, as indicated by the constantly low dissolved oxygen tension in the aerated reactor. As a result product yields (based on the 3-phenylcatechol eluted from the product sink) increased by one order of magnitude." ... 

The use of enzymes as biocatalysts for the synthesis of water-soluble conducting polymers is simple, environmentally benign, and gives yields of over 90% due to the high efficiency of the enzyme catalyst. Since the use of an enzyme solution does not allow the recovery and reuse of the expensive enzyme, well-established strategies of enzyme immobilization onto solid supports have been applied to HRP [22-30]. A recent work reported an alternative method that allows the recycle and reuse of HRP in the biocatalytic synthesis of ICPs. The method is based on the use of a biphasic catalytic system in which the enzyme is encapsulated by simple solubilization into an IL. The main strategy consisted of encapsulating the HRP in room-temperature IPs insoluble in water, and the other components of the reaction... 

Frequently however, enzymes found in the fermentation broth at the time of harvesting are quite different from the state in which they can be used as industrial biocatalysts, reagents in clinical chemistry or as therapeutic agents, The recovery of an enzyme from microbial culture, its concentration and purification will require careful and effective sequential operations, which ate called downstream processing (Wheelwright, 1989). 

In general the use of immobilized biocatalysts makes continuous processes possible. This facilitates process control, which optimizes product yield and quality. Other advantages of immobilized biocatalysts are 1) that they do not become mixed with the product, which makes product recovery easier and 2) that the biocatalyst, in the case of enzymes, usually becomes more stable. 

Describe in your own words the advantages and disadvantages of using whole cells compared to isolated enzymes as biocatalysts, with respect to catalyst immobilization, catalyst recovery, ease of use, and product selectivity and purification. 

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