Methane is generally produced by methanogens, a group of over 50 different microorganisms belonging to Euryarchaeota. These microorganisms produce methane by the reduction of either CO2 or acetate, while some have the ability to use methylated compounds such as methanol and methylamine for methane production. Biomethane produced from waste from biodiesel and other biofuel plants can be used to generate energy for their operation. This may dramatically reduce the dependence of the biofuel plant on external energy inputs such as natural gas and coal. However, direct utilization of crude glycerol streams by microbes is not efficient because of high salt levels associated with biodiesel waste streams (glycerol). This problem may be overcome either by diluting the waste stream or by mixing it with other waste streams. Theoretically, 0.43 L methane is produced for every gram of pure glycerol. This yield will obviously go down as the glycerol content of the waste stream decreases. When crude glycerol from swine manure (1.2 g glycerol/g swine manure) was used as the sole carbon source, methane was produced at a yield of 232mL/g glycerol. However, when the glycerol/swine manure ratio was 4.6 or higher, the methane production was negligible, indicating that high glycerol levels are toxic to methanogens mainly due to high salt and methanol levels (Khanal 2008).

A cotton gin is basically a very sophisticated solid-solid separation technology. It not only removes the fiber from the seed, but also cleans the plant material from the fiber. The fiber is then baled into 220-kg bales that are enclosed in a plastic sleeve for protection. The bales are then stored in a warehouse at the gin and are shipped in tractor-trailer trucks to meet the demand schedule of the cotton buyer, typically a textile mill.

A cotton gin produces three main products:

1. Cotton fiber (bales)

2. Mote cotton (short fiber removed from seed and used for a variety of industrial applications)

3. Cotton seed (an important ingredient in dairy cattle feed)

The remaining material is referred to as cotton gin waste (CGW). Some of this material is fed to beef cattle, some is composted, and some is land applied. There are continuing efforts to find higher value uses for CGW, and some of these efforts may lead to its use to produce a biofuel.

The cotton gin not only stores the bales for continuous delivery to the textile industry, but it also stores seed for year-round delivery to the feed market. In this sense it functions as a preprocessing plant in the total logistic system—field to factory. The function of a preprocess­ing plant is to receive raw material and produce a more homogeneous product, or products, which meet customer quality standards and can be shipped more cost competitively to meet a year-round delivery schedule. This is the job of the cotton gin.

The cotton gin is an interesting model for the type of preprocessing plant that might be included in a biomass system. The raw biomass is brought in from the field and processed into products sold to three different markets. After ginning the products have at least twice the value ($/kg) as the raw biomass. This increase provides an opportunity to ship longer distances (because a truckload has a higher value), a benefit that could potentially be realized with a preprocessing plant in a biomass system. Raw biomass can be bulky, and thus has a low energy density per unit volume. If it can be transformed into a material with higher energy density, a point made earlier in this chapter, then this material can be hauled further to support a larger processing plant.

There is another lesson to be learned from the cotton industry. There is a compromise between the level of separation done with the mobile machine (cotton harvester) and the stationary machine (gin). In general, the annual hours of operation for a mobile machine is limited by field conditions (condition of crop, weather, and daylight) and these machines require a liquid fuel (diesel). Stationary installations can operate 24/7 and use electric power, thus avoiding, at least until electric cars become popular, a direct competition with the trans­portation sector for energy. System cost (harvest plus logistics) is often minimized when the mobile machine is simplified and more processing steps are allocated to the stationary machine. This is certainly the case with the cotton system. No one has advocated that the cotton harvester be modified to accomplish the ginning function and thus become a “mobile gin.”

Bin Wang and Hao Feng

Abstract

Typically, lignocellulosic biomass must be deconstructed into monosaccharides for efficient conversion into biofuel by fermenting microorganisms. Natural protection mechanisms of plants against foreign intrusions such as disease-causing bacteria and viruses create obstacles to biomass deconstruction and economical production of biofuel from biomass. Large-scale production of biofuel from lignocellulosic biomass, therefore, is still a challenge to both microbiologists and engineers. A principal problem associated with deconstruction of biomass by chemical, enzymatic, or biological process is the generation of microbial inhibitory chemi­cals. Presence of these inhibitory compounds in the lignocellulosic hydrolysates inhibits growth and biofuel production by fermenting microorganisms. An additional processing step called detoxification, therefore, has often been introduced to remove these microbial inhibi­tors from hydrolysates as a way of mitigating their negative effects on the fermenting micro­organisms. Efforts have been made over the years to develop effective detoxification processes with chemical, physical, or biological methods. This chapter, detoxification of lignocellulosic hydrolysates, will focus on the technical aspects of lignocellulosic hydrolysates detoxification approaches and their potential applications in biomass-to-fuel production.

AFEX pretreatment (Dale 1986) is an alkaline pretreatment process that alters physicochemi­cal structure of lignocellulosic biomass at moderate temperatures (60-100°C) and high pres­sures (250-300psi). Like the alkali pretreatment, AFEX pretreatment results in increased wetting of the treated biomass (Sulbaran de Ferrer et al. 1997; Shishir et al. 2007), decrystal­lization of cellulose (Gollapalli et al. 2002), partial depolymerization of hemicellulose, cleav­age of lignin-carbohydrate complex (LCC) linkages (Shishir et al. 2007), and increase in surface area due to structural disruption.

In a typical AFEX pretreatment process using corn stover (Shishir et al. 2007), corn stover is adjusted to 60% moisture (kilogram water/kilogram dry biomass) before being transferred to a high-pressure Parr reactor, and liquid ammonia (1 kg of ammonia/kg of dry biomass) is slowly added to the reactor. The temperature is raised and maintained at 90°C for a period of 5 minutes. Following a 5)minute residence time, the pressure is quickly released. The sudden drop in reactor pressure results in fast vaporization of ammonia, causing an explosive decompression and disruption of the biomass. The pretreated corn stover is stored under a fume hood overnight, during which the residual ammonia is vaporized. Subsequently, the AFEX pretreated corn stover is either subjected to enzymatic hydrolysis or stored in a freezer until further use.

The AFEX pretreatment process has some unique characteristics that distinguish it from other pretreatment methods and they are as follows (Teymouri et al. 2005): (1) a significant amount of the ammonia used in the pretreatment is recovered and reused; (2) AFEX is basically a dry to dry process because there is no waste or liquid stream from the process; (3) pretreated biomass is stable for long periods and can be used with great solid loadings in enzymatic hydrolysis and fermentation processes; (4) AFEX process yields intact cel­lulose and hemicellulose polymers with little or no degradation; (5) no need for neutraliza­tion prior to the enzymatic hydrolysis of AFEX-pretreated biomass. The AFEX pretreatment process was used recently to pretreat DDGS, and subsequently fermented into ABE (Ezeji and Blaschek 2008a). In this investigation, various Clostridium species were tested for the ability to ferment AFEX-pretreated DDGS to ABE. The AFEX pretreated DDGS (15% total solids loading) generated 41.4g/L total sugars upon enzymatic hydrolysis comparable to that generated by hot water pretreated samples.

Cellulases

For the engineer seeking to improve upon the natural process of converting plant biomass to fermentable sugars, the key challenge is to make cell wall depolymerization a more rapid and less costly process. The cost of biomass ethanol production has been reduced dramatically over the past two decades, to the point where the fuel is now competitive for the blending market, but further processing cost-reduction opportunities have been identified that would make it competitive as a pure fuel without subsidies (Lynd et al. 1996). Because the cost of producing the enzymatic catalysts proposed in the SSF process is a critical issue, the available enzymatic activity must be maximized to effectively incorporate cellulases into these process schemes. This requirement can be met by ensur­ing that the enzymes used are obtainable at minimal cost and of the highest specific activity, the highest possible stability, and optimal in terms of pH and temperature tolerance.

While cellulosic biomass is produced at a rate of nearly 3 x 109 tons per year and represents 50% of all available biomaterial, the biologically mediated depolymerization of this resource has eluded clear, precise definition at the molecular level. Although the biological depoly­merization of native plant matter requires a suite of glycoside hydrolases aided by chemical or mechanical conditioning, in many ways this problem is primarily one that focuses on the enzymes that act on cellulose. Many workers in the field agree that cellulose decrystallization and depolymerization are indeed the rate-limiting steps in biomass conversion (Himmel et al. 2007).

Hemicellulose removal by dilute acid treatment is a classical means of rendering biomass more amenable to cellulase action (Grohmann et al. 1985). Kong and coworkers (Kong et al. 1993) also showed that biomass with reduced acetylation responded significantly more favor­ably than native biomass to cellulase action. Biomass with reduced lignin content, or perhaps altered chemistry, appears to be more readily hydrolyzed by cellulases (Vinzant et al. 1997; Kristensen et al. 2007). The structural and reactive chemical features of the substrate (primar­ily defined as acetyl and lignin contents) can be pictured as controlling the accessibility of enzyme to cellulose; the degree of cellulose crystallinity can be visualized as controlling the hydrolytic rate (Jeoh et al. 2007).

The definitive enzymatic degradation of cellulose to glucose in fungi and most bacteria is generally accomplished by the synergistic action of three distinct classes of enzymes:

• The “endo-1,4-P-glucanases” or 1,4-P-D-glucan 4-glucanohydrolases (EC 3.2.1.4), which act randomly on soluble and insoluble 1,4-P-glucan substrates and are commonly measured by detecting the reducing groups released from carboxymethylcellulose (CMC). A relatively new subset of this class of cellulase, called “processive endoglu — canases,” has recently been classified (Reverbel-Leroy et al. 1997; Wilson et al. 1998).

• The “exo-1,4-P-D-glucanases,” including both the 1,4-P-D-glucan glucohydrolases (EC 3.2.1.74), which liberate D-glucose from 1,4-P-D-glucans and hydrolyze D-cellobiose slowly, and 1,4-P-D-glucan cellobiohydrolase (EC 3.2.1.91), which liberates D-cellobiose from 1,4-P-glucans.

• The “P-D-glucosidases” or P-D-glucoside glucohydrolases (EC 3.2.1.21), which act to release D-glucose units from cellobiose and soluble cellodextrins, as well as an array of glycosides.

Cross-synergism between endo — and exo-acting enzymes isolated from the same or differ­ent species, genera, or microbial families has been demonstrated many times (Wood and McCrae 1979; Coughlan et al. 1987; Eveleigh 1987). Exo-exo synergism was first reported in 1980 (Fagerstam and Pettersson 1980). It is currently believed that exo-endo synergism is explained best in terms of providing new sites of attack for the exoglucanases. The latter enzymes normally find available cellodextrin “ends” at the reducing and nonreducing termini of cellulose microfibrils. Random internal cleavage of surface cellulose chains by endoglu — canases provides numerous additional sites for attack by cellobiohydrolases. Therefore, each hydrolytic event by an endoglucanase yields both a new reducing and a new nonreducing site. Thus, logical consideration of catalyst efficiency dictates the presence of exoglucanases specific for reducing termini and nonreducing termini. The principle of interspecies inter­changeability of cellulase components is now the cornerstone of recombinant cellulase system design and construction. If indeed cellulase component enzymes are truly generalized in both structure and function, components can be selected and combined from a wide array of source organisms to form novel enzyme cocktails. For example, Trichoderma reesei cellobiohydro — lase I (CBH I) has been shown to be a powerful element in multi-enzyme mixtures using either fungal or bacterial endoglucanases (Baker et al. 1998).

Large square bales are made with tractor pulled balers. Large square bales are currently made either in dimensions 1.2 m x 1.2m x 2.4m (4′ x4’x 8′) or 0.9m x 1.2m x 2.4m (3′ x4’x 8′). A bale accumulator is pulled behind the baler that collects the bales in groups of four and leaves them on the field. At a later date when available, an automatic bale collector travels through the field and collects the bales. The automatic bale collector travels to the side of the road and unloads the bales into a stack. If the automatic bale collector is not available bales may be collected using a flat bed truck equipped with a front-end bale loader. A loader is needed at the storage site to unload the truck and stack the bales. The stack is tarped using a forklift and manual labor.

Loafing

Mowing, conditioning, and raking operations are identical to those for baling. When biomass is dry, a loafer picks the biomass from windrow and makes large stacks of about 2.4-m wide, up to 6-m long and 3.6-m high (SAF 1979; FMO 1987). The roof of the stacker acts as a press pushing the material down to increase the density of the biomass. Once filled, loafer transports the biomass to storage area and unloads the stack. The top of the stack gets the dome shape of the stacker roof and thus easily sheds water. The loafer has been used for hay and for corn stover. It was used for experimental wheat straw in Idaho. To the knowledge of the authors, the loafer has not been used for switchgrass, so its practical performance is not known at this time.

Dry Chop

In this system a forage harvester picks up the dry biomass from windrow and chops it into smaller pieces (2.5-5.0cm). The chopped biomass is blown into a forage wagon traveling along side of the forage harvester. Once filled, the forage wagon is pulled to the side of the farm and unloaded. A piler (inclined belt conveyor) is used to pile up the material in the form of a large cone.

Wet Chop

In this system, a forage harvester picks up the dry or wet biomass from the windrow. The chopped biomass is blown into a forage wagon that travels along side of the harvester. Once filled, the wagon is pulled to a silage pit where biomass is compacted to produce silage (Luginbuhl et al. 2002). For silaging dry corn stover, water is added to create silaging mois­ture content. To the knowledge of the authors, no literature is available for silaging dry switchgrass. Work is in progress for silaging corn stalks and wheat straw.

Most ethanol is currently produced from corn using a dry-milling process. Conventional dry — mill ethanol production is a relatively simple procedure. Corn is cleaned, tempered with steam, ground, and wetted to a free flowing “mash.” The mash is superheated in a cooker where acid and/or enzymes are added to solubilize starch. Water is added to adjust solids concentration and temperature along with saccharifying enzymes. The mash is placed in a fermenter, and yeast is added to convert sugars to alcohol. When the fermentation is complete,

alcohol is removed by distillation and the residual “still bottoms” are recovered for animal feed. In such a plant, the only two products are ethanol and DDGS. Some plants now separate the germ before “mashing.” Food-grade corn oil can then be extracted from the germ, thus creating another salable product. Figure 8.1 provides a basic process flow diagram that describes the dry — milling process.

The rising demand and cost of corn has challenged the economic feasibility of conventional dry-mill processing in recent years. Conversion and retrofitting existing dry-mill ethanol plants such that they can process cellulosic biomass materials is becoming a topic of consid­erable interest. Most ethanol plants have been located in relatively rural settings. Therefore, access to relatively low-cost land is frequently available to allow for expansion of processing equipment as well as additional options for storing feedstocks and products. The DDGS produced in the dry-mill process has considerable value as livestock feed. However, practical methods for improving the value of this material and extracting additional marketable prod­ucts from the DDGS could greatly improve the economic viability of dry-mill ethanol plants.

This chapter discusses relatively nondisruptive methods for incorporating cellulosic feedstocks into existing dry-mill ethanol plants using existing off-the-shelf technology. Additionally, practical techniques for extracting marketable oil suitable for food, feed, or conversion to biodiesel will be discussed. By expanding both the feedstock options and the marketable products that can be produced at these facilities, dry-mill ethanol plants can improve their long-term competitiveness and viability.

Jonsson et al. (1998) studied the detoxification effect of laccase, phenol oxidase, and lignin peroxidase on hydrolysates. S. cerevisiae was used for subsequent ethanol fermentation. The results showed more rapid consumption of glucose and a higher ethanol productivity for samples treated with laccase than for untreated samples. Treatment of hydrolysates with lignin peroxidase also resulted in improved fermentability. Analyses by GC-MS indicated that the mechanism of laccase detoxification involved removal of monoaromatic phenolic compounds present in the hydrolysate (Jonsson et al. 1998).

Chandel et al. (2007) conducted laccase detoxification tests using sugarcane bagasse hydro­lysates produced by 2.5% (v/v) HCl, which contained 30.39 g/L of total reducing sugars along with various fermentation inhibitors such as furans, phenolics, and acetic acid. The laccase reduced total phenolics by 77.5% without affecting furans and acetic acid content in the hydrolysate. In comparison, the anion-exchange resin brought about a maximum reduction of 63.4% in furans and 75.8% in total phenolics, while the treatment with activated charcoal caused 38.7% and 57.5% reduction in furans and total phenolics, respectively. Fermentation of these hydrolysates with C. shehatae NCIM 3501 showed maximum ethanol yield (0.48 g/g) from ion exchange-treated hydrolysates, followed by activated charcoal (0.42 g/g), laccase (0.37g/g), overliming (0.30g/g), and neutralized hydrolysates (0.22g/g).

Adaptation of Microorganisms

Microorganisms have the ability to adapt to perturbations of the surrounding environment to grow (Dinh et al. 2008). Utilizing the microorganism of a previous experiment as the inocu­lum of the next one, the adaptation of a microorganism to the hydrolysate is another biological method for improving the fermentation of lignocellulosic hydrolysate media (Mussatto and Roberto 2004). To analyze the adaptation process of S. cerevisiae to a high ethanol concen­tration, Dinh et al. (2008) performed repetitive cultivations with a stepwise increase in the ethanol concentration in the culture medium. They found that the mother cells of the adapted yeast were significantly larger than those of the non-adapted strains and that the content of palmitic acid in the ethanol-adapted strains was lower than that in the non-adapted strain in media containing ethanol.

Martm et al. (2007) adapted a xylose-utilizing genetically engineered strain of S. cerevisiae with sugarcane bagasse hydrolysates by 353-hour cultivation using a medium with increasing concentrations of phenols (from 1.5 to 2.3 g/L), furfural (from 0.7 to 3.4 g/L), and aliphatic acids (from 2.5 to 8.7 g/L). The performance of the adapted strain was compared with the parental strain: the ethanol yield after 24 h of fermentation of the bagasse hydrolysate with inhibitors (phenols: 1.4 g/L, furfural: 2.2 g/L, apliphatic acids: 5.0 g/L) increased from 0.18 g/g of total sugar with the non-adapted strain to 0.38 g/g with the adapted strain. The specific ethanol productivity increased from 1.15 g ethanol per gram initial biomass per hour with the non-adapted strain to 2.55g/g/h with the adapted strain.

Agbogbo et al. (2008) investigated the effect of adaptation of P. stipitis in acid-pretreated CSH without detoxification for ethanol fermentation. Fermentation results showed that the solid agar adaptation improved both the sugar consumption rate and the rate of ethanol pro­duction. Liquid and solid agar adaptation increased the sugar consumption from 64 to 72% after 96 hours of fermentation at 100 rpm. The ethanol concentration (g/L) was increased from unadapted 16.3 ± 0.51 to 18.4 ± 0.20 (liquid adapted) and 19.4 ± 0.12 (solid adapted). The solid agar-adapted stains started using xylose after 96 hours of fermentation while wild strains did not consume xylose. However, when rotation speed in the flask was increased to 150 rpm, 92% of the total sugar was consumed within 72 hours of fermentation.

Conclusion

The presence of inhibitors in lignocellulosic hydrolysates directly influences biofuel fermen­tation. Due to a lack of understanding about the synergistic interactions among inhibitors and the mechanisms of these interactions, highly inhibitor-resistant microorganisms might not be expected in the short term. Problems associated with biomass hydrolysates, however, may be resolved by the development of inhibitor-tolerant strains using genetic modification and metabolic engineering. From an economic standpoint, the ultimate goal is to develop a decon­struction process without detoxification. The main features of a number of detoxification methods are summarized in this chapter. Some of them are relatively new, while others have existed for decades but need some improvements for optimal performance. Among the methods, the biological detoxification methods are promising. With the isolation and develop­ment of some inhibitors degrading microorganisms and mutants, there exist some prospects of SSF of biomass to biofuel or combining a biological detoxification step with the SSF process.

Acknowledgem ent

This work was financially supported by Energy Biosciences Institute.

As stated above, a successful economic outcome for anaerobic digesters may be dependent on co-digestion of, for example, animal manures with other organic feedstocks (only when other feedstocks are available locally). In addition to crops and crop residues, the use of food and organic industrial wastes offers excellent possibilities to develop waste management opportunities for these wastes and to avoid increasingly costly and limited landfill disposal. Manure co-digestion increases the buffering capacity of substrate mixtures and adds nutrients that can increase methane yields and substrate affinities compared with digestion of one substrate (Mladenovska and Ahring 2000; Mladenovska et al. 2003). Despite the perceived benefits of co — digestion, the overall and specific knowledge of adding organic feedstocks relative to single substrates is very limited (both the synergistic and antagonistic effects). Therefore, we initiated a study to evaluate the potential of co-digestion of dairy manure with a variety of organic substrates. The results of ~175 individual biochemical methane potentials (BMPs) on more than 30 different substrates showed that substrates rich in lipids and/or carbohydrates with a high VS content are good candidates for co-digestion with dairy manure (Figure 4.1; Labatut and Scott 2008). A critical parameter to estimate the BMP of a myriad of substrates is the substrate biodegradability and we suggest that future research needs to develop models to estimate the methane potential of the numerous farm waste options based on biodegradability of substrates.

From a logistic and economic perspective, the availability of food wastes and distance from source to farm anaerobic digester is critical because of transportation costs and energy con­sumption. The development of geographical information systems (GIS) offers a useful way to connect food waste sources to existing digesters (Ma et al. 2005). We developed a web site (http://wastetoenergy. bee. cornell. edu/) to identify the location of dairy farms and sources of food waste from food processors, restaurants (including fast food), universities and col­leges, K-12 public schools, supermarkets, correctional facilities, hospitals, and nursing homes. This tool has proven effective to link the mutual interests of the farmer and food waste sources.

Lignocellulose-degrading microbes are found all over our planet, as free-living organisms and in the microbiomes of invertebrates and vertebrates. Genome sequencing of lignocellu- lose-degrading microbes is being used to reveal the relevant molecular components for optimal cellulose degradation in these microorganisms via sequence similarities to CAZyme components from other organisms. To date, there are at least 25 different microbes for which
genome projects are either in progress or completed (www. genomesonline. org/). Interestingly, based on the genome projects, there appears to be many different paradigms for lignocellu — lose-degradation, and each of these bacteria seem to have evolved organism-specific modes of plant cell wall deconstruction, which, while very efficient, are distinctly different.

This has led to comparative genome efforts, one of which is expression profiling. The idea is to monitor changes in gene expression in response to exposure to different plant cell wall substrates. This combined genomic and proteomic approach is needed to under­stand the regulation and assembly of this remarkable cadre of CAZyme and cellulosome components, and to identify those CAZymes that have maximal degradative capacity against a given substrate. To date, this approach has been used for two cellulosome — containing organisms, Clostridium thermocellum (Brown et al. 2007) and Ruminococcus flavefaciens (Berg et al. 2006). These functional and proteomic approaches can define candidate enzymes and are necessary for maximal lignocellulose degradation. Such func­tional and comparative genomics approaches are essential for defining how lignocellulose sources affect microbial-borne gene families and the rate and extent of lignocellulose degradation.

In the next few years, the powerful approach of comparative genomics will enable rapid advances. This is primarily due to the recent development of nexfigeneration sequencing technologies, which have dramatically reduced the time, cost, and labor for genome sequenc­ing projects. Pyrosequencing (also called 454 sequencing) was originally developed in the mid 1990s (Ronaghi et al. 1996, 1998) and has been continuously developed since then, and has become widely used in genome sequencing projects. The elimination of cloning vectors and their associated biases in terms of the clonability of certain DNA fragments is a major advantage in using this system (Hyman 1988; Ronaghi et al. 1996, 1998; Margulies et al. 2005) . This sequencing technology also readily reads through secondary structures, and has the capacity to produce very large amounts of sequence. Current esti­mates from the latest version called “Titanium” suggest that read lengths with an average length of 400 bp and a five) fold throughput increase to 400-600 million bp per run for approximately $12,000.

Other next generation of sequencing technologies also include the Solexa/Illumina 1G Genome Analysis System and Applied Biosystems SOLiD Sequencing. While presently average read lengths are much shorter than those obtained from the traditional methods, a far higher number of sequence reads can be produced in a single day or on a single run by these technologies. It is not unreasonable to predict that these next-generation technologies will eventually generate as good as or even longer read lengths than some of the traditional methods. Furthermore, there are additional “next-generation” technologies that will be released in the near term, including those from Helicos (www. helicosbio. com) and Complete Genomics (www. completegenomics. com).

These cost-effective next-generation sequence technologies will allow the generation of huge reference genome databases where one will now sequence up to 10 isolates of a micro­bial species for use in comparative studies. This approach was recently pioneered for micro­organisms from the human gastrointestinal tract and their glycoside hydrolase components (Lozupone et al. 2008). Analysis of 67 microbial genomes from the human gastrointestinal revealed that the CAZyme repertories found in these microbes had converged due to hori­zontal gene transfer, with limited evolution of the gene families. This implies that the envi­ronment can drive the adaptation of gene families. In this case, the plant cell wall material in the biome was the environment, and therefore the genes and enzymes needed for maximal degradation were the targets for optimization.