Processing downstream

Another important problem in the production of chemicals by fermentation is that the products are obtained in diluted form in an aqueous soup that contains many components. Concentrating the solutions and separating the products from the other products of the fermentation broth is tedious and often the main cost factor. About 60 to 95 percent of the total cost is for product recovery. 

The last step in downstream processing is the final purification and conditioning of the product. Chemicals and proteins are often recrystallized and heat or freeze dried. They are stored and sold in hags or drums. Very sensitive products are not fully isolated hut sold as concentrates. Liquid products like ethanol or acetic acid are distilled and sold in tanks, drums, or bottles. 

After performing the bioconversion in an ionic liquid, the product needs to be recovered and the biocatalyst and the ionic liquid recycled. Relatively volatile products can be removed by evaporation. Alternatively, immiscible organic solvents can be used to extract the product, and the biocatalyst can be recycled as a suspension in the ionic liquid phase [58]. A more elegant, green method, which avoids the use of volatile organic solvents altogether, involves the use of supercritical carbon dioxide as the extractive phase [96, 147, 148]. 

The principle has been demonstrated with CaLB in simple model transesterifications as well as in the enantioselective acylation of 1-phenylethanol, in batchwise and continuous procedures. The high operational stability of CaLB, which contrasts with the generally rapid deactivation in pure scC02, is one of the attractive aspects of this approach. The reaction rate was approximately eight times better than that in pure scC02 under otherwise identical conditions [96]. 

The continuous reaction system could be combined with solid acid-catalyzed in situ racemization of the slow-reacting alcohol enantiomer [149]. The racemiza-tion catalyst and the lipase (Novozym 435) were coated with ionic liquid and kept physically separate in the reaction vessel. Another variation on this theme, which has yet to be used in combination with biocatalysis, involves the use of scC02 as an anti-solvent in a pressure-dependent miscibility switch [150]. 

6 Some commerdal suppliers Acros (www.acros.be), Covalent Associates (www.covalentassociates.com), IOLITEC (www.iolitec.de), Merck (www. ionicliquids-merck.de), Sachem (www. sacheminc.com), Sigma-Aldrich (www. sigmaaldrich.com), Solvent Innovation (www.solvent-innovation.de) and TCI (www.tciamerica.com). 

Eckstein, N. Kaftzik, Curr. Opin. Biotechnol. 2002,13, 565. 

Stoger et al. [23] also observed that between species, the amounts of scFv per unit fresh weight were in the same range, and did not correlate with the total protein content in the plant. For example, even though pea is a much more proteinaceous crop than rice, the amounts of antibody measured as a percentage of TSP were considerably lower. 

Within each species, individual promoters resulted in distinct, tissue-dependent accumulation patterns. The cauliflower mosaic virus (CaMV) 35S promoter, for example, led to high-level accumulation in callus and leaves whereas the maize ubiqui-tin-1 promoter was the best choice for producing recombinant proteins in cereal seeds even though it is not in itself seed-specific [23]. The lack of such comparative studies for proteins other than rAbs makes it difficult to generalize an optimal expression strategy for all proteins. Tables 7.1 and 7.2 list recombinant proteins expressed in plants and provide details of the production system, promoters and other regulatory elements used in each case. 

A minimalist extraction buffer is often recommended for protein purification [25], since most additives provide only a marginal improvement in yields, but will increase costs significantly. The removal of phenolics, for example by tangential-flow ultrafil-tration/diafiltration [25], is an important step that should be carried out as early as possible in the purification procedure, since these molecules can become covalently linked to amino acid side chains and can oxidize certain residual groups [123]. 

At the end of the fermentation, protein is separated from cell mass by filtration, typically with a rotary vacuum filter. The crude enzyme concentration is often lower than suitable for commercial applications, so the concentration of enzyme is increased by ultrafiltration. Most cellulase enzymes have a molecular weight of 25,000 to 75,000 and are retained by ultrafiltration membranes of 5000 molecular weight cutoff. The membranes permit the passage of low molecular weight salts, sugars and other impurities, and are sometimes operated in a diafiltration mode to increase the purity of the enzymes. The crude broth at this point is dark brown. 

Enzyme formulation is carried out at the fermentation plant, at a separate formulation facility, at the end-users site, or a combination of these. The enzyme is formulated as a liquid or a solid. 

For liquid formulations, stabiUzers such as sorbitol, glycerol, or propylene glycol are added to levels of 10% to 50% (w/w) to maintain the activity of the enzyme for up to several months at ambient conditions. Preservatives such as sodium benzoate, potassium sorbate, and sodium chloride are used to control microbial contamination. Buffers and surfactants such as Triton X-100 can also be added, although at lower levels than found in granulated cellulase enzymes due to the limits of tolerance of the proteins for these additives in Hquid solutions. 

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Process intermediates are generated which, because the downstream process is not operational, cannot be processed further. 

Because of the differences existing between the quality of different distillation cuts and those resulting from their downstream processing, it is useful to group them according to a major characteristic. That is, they are grouped into the three principal chemical families which constitute them paraffins, naphthenes and aromatics. From a molecular point of view, their chemical reactivities follow this order ... 

S. M. Wheelwright, Protein Purification Design and Scale-up of Downstream Processing, Hanser PubHshers, Munich, Germany, 1991, pp. 1—9, 61, 213—217. 

The most accurate flow rate control can be achieved by using the loss-in-weight method. The total amount of material required for a downstream process is first added to a tank or hopper scale. As the material is discharged, the loss-in-weight is monitored and used to modulate the discharge valve or gate to achieve the desired flow rate. 

Phosphorus Pentoxide. Phosphoms compounds form PF or POF compounds in the furnace. Some may be hydroly2ed to higher boiling forms in downstream process operation. Some of the phosphoms compounds do appear in the final product. This is objectionable to some users. 

Ironmaking refers to those processes which reduce iron oxides to iron. By the nature of the processes, the iron produced usually contains carbon and/or other impurities which are removed in downstream processing. There are three principal categories of ironmaking processes, in order of commercial importance blast furnace, direct reduction, and direct smelting. 

The level of technical service support provided for a given product generally tracks in large part where the suppHer considers thek product to be located within the spectmm of commodity to specialty chemicals. Technical service support levels for pure chemicals usually provided in large quantities for specific synthetic or processing needs, eg, ammonia (qv), sulfuric acid (see SuLFURic ACID AND SULFURTRIOXIDe), formaldehyde (qv), oxygen (qv), and so forth, are considerably less than for more complex materials or blends of materials provided for multistep downstream processes. Examples of the latter are many polymers, colorants, flocculants, impact modifiers, associative thickeners, etc. For the former materials, providing specifications of purity and physical properties often comprises the full extent of technical service requked or expected by customers. These materials are termed undifferentiated chemicals (9),... 

Downstream Processing. In addition to extraction, various downstream operations are often carried out on the BTX product to produce products in proportions to fit the market demand. A typical aromatics processing scheme is shown in Eigure 8 in which ben2ene, xylene, and o-xylene are the products. 

After the cmde BTX is formed, by reforming in this case, a heart cut is sent to extraction. Actually, the xylenes and heavier components are often sent to downstream processes without extraction. The toluene produced is converted to ben2ene, a more valuable petrochemical, by mnning it through a hydrodealkylation unit. This catalytic unit operates at 540—810°C with an excess of hydrogen. Another option is to disproportionate toluene or toluene plus aromatics to a mixture of ben2ene and xylenes using a process such as UOP s Tatoray or Mobil s Selective Toluene Disproportionation Process (STDP) (36). 

Most of these methods are commonly employed in the downstream processing of the desired ceU culture technology product. Hence, most of the time it is only necessary to demonstrate that the designed process is reducing the putative risk factors to acceptable levels. Validation methods employed for risk reduction are discussed in the Hterature (25). 

The hberation of a valuable constituent does not necessarily translate direclly into recoveiy in downstream processes. For example, flotation tends to be more efficient in intermediate sizes than at coarse or fine sizes [Mclvor and Finch, Minerals Engineeiing, 4(1), 9-23 (1991)]. For coarser sizes, failure to liberate may be the hmitation finer sizes that are liberated may still be carried through by the water flow. A conclusion is that overgrinding should be avoided by judicious use of size classifiers with recycle grinding. 

FIG. 22-84 General stages in downstream processing for protein production indicating representative types of operations used at each stage. 

In the development of new products, optimization of the fermentation medium for titer only often ignores the consequences of the medium properties on subsequent downstream processing steps such as filtration and chromatography. It is imperative, therefore, that there be effective communication and understanding between workers on the upstream and downstream phases of the produc t development if rational trade-offs are to be made to ensure overall optimahty of the process. One example is to make the conscious decision, in collaboration with those responsible for the downstream operations, whether to produce a protein in an unfolded form or in its native folded form the purification of the aggregated unfolded proteins is simpler than that of the native protein, but the refolding process itself to obtain the product in its final form may lack scalabihty. 

FIG. 22-86 Process scheme for protein extraction in aqueous two-phase systems for the downstream processing of intracellular proteins, incorporating PEG and salt recycling. RepHnted from Kelly and Hatton in Stephanopoulos (ed), op. cit. adapted from Qre-oe and Kula, op. cit.]... 

FIG. 22-87 Schematic illustration of the chromatographic methods most commonly used in downstream processing for protein recovery... 

Traditionally, the upstream fermentation and cell culture processes have been viewed as being distinct from the subsequent downstream processing and purification steps, and the two different sets of processes nave been optimized individually. In some instances, careful consideration of the conditions used in the fermentation process, or manipulation of the genetic makeup of the host, can simplify and even... 

Consider downstream processing that does not require that the intermediate be stripped to dryness... 

It is not a bad idea for the process engineer to familiarize himself with compressor surge controls. The interaction of the compressor surge controls with downstream process control valves can become a problem area later, and this study phase is not too early to put such items on a checklist. An LNG plant example comes to mind where such an operating problem existed. 

Refining crude oil into useful petroleum products can be separated into two phases and a number of supporting operations. The first phase is desalting of crude oil and the subsequent distillation into its various components or "fractions." The second phase is made up of three different types of "downstream" processes combining, breaking, and reshaping. 

The following section provides general information on the major products and processes used to manufacture them from crude oil. The basic refinery operations have already been described. Emphasis is now given to the downstream processes which are used in transforming distillates into a multitude of consumer products. Not all products and processes are discussed, and indeed, only highlights are provided on those discussed. 

Product separation for main fractionators is also often called black oil separation. Main fractionators are typically used for such operations as preflash separation, atmospheric crude, gas oil crude, vacuum preflash crude, vacuum crude, visbreaking, coking, and fluid catalytic cracking. In all these services the object is to recover clean, boiling range components from a black multicomponent mixture. But main fractionators are also used in hydrocracker downstream processing. This operation has a clean feed. Nevertheless, whenever you hear the term black oil, understand that what is really meant is main fractionator processing. 

Note that the use of metal salts for coagulation may increase the quantity of dissolved solids. One must consider the downstream impact of these dissolved solids. In addition, the impact of carryover of suspended Al. .. and Fe... compounds and their related effect on downstream processes must be considered. 

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