Enzymes are special types of proteins that act as catalysts to biological reactions. Compared to inorganic cataysts, enzymes can lower the activation energy of reactions better. Common industries that use enzymes are food and beverage, textile, detergent, and pharmaceuticals.

Enzymes have been widely used in several industries like food production and manufacture of commodities. Proteases remain the dominant enzyme type, because of their extensive usage in detergent and dairy products while celluloses and amylases are used in other industries such as textile and baking. The utility of enzymes will increase in the chemical, pulp and paper, textile, waste treatment, pharmaceutical and diagnostic industries. Other specialty applications include the use of enzymes in analytical applications, flavor production, protein modification, personal care products, DNA technology and fine chemical production. In the Philippines, most of the local industries rely on imported enzymes for basic, medical or industrial uses.Detergent Industry – Enzymes in this industry is treated as detergent additives. The major component is proteases, but other and very different hydrolases are introduced to provide various benefits, such as the efficient removal of specific stains. There are new enzymes which are more improved than the usual; proteases and amylases are being developed. Protease is for protein-stain removal while Amylase is for starch-stain removal. The most recent introduction of a new enzyme class into a detergent has been the addition of a mannanase — the result of a joint development between Procter and Gamble and Novozymes.Starch Conversion – A good example of starch conversion is the enzymatic conversion of starch to high fructose. Consecutive use of several enzymes is necessary for this process. Common example of enzyme used is α-amylase. Engineering efforts have also been undertaken to develop improved versions of the enzymes used later in the process (i.e. glucoamylase and glucose isomerise).Textile Applications – Enzymes play a big role for textile production. Some enzymes that are used are Cellulase for denim finishing and cotton softening, Amylase for de-sizing, Pectate lyase for scouring, Catalase for bleach termination, Laccase for bleaching and Peroxidase for excess dye removal. An alternative, enzyme-based process performed at much lower temperatures and using less water has now been developed based on a pectate lyase.Feed Industry -  Xylanases and β-glucanases are the common enzymes which function as feed additives and help in digestibility. The most recent advancement in feed enzymes have been aimed at the improvements of phytases. Better utilization of feed is achieved by using phytases. There are other approaches for the development of more effective enzymes. An example of improved enzyme is Aspergillus fumigates phytase.Food Industry -  Enzymes are applied to processed food products as processing agents upstream from the final product. Protease is used for milk clotting, Lipase for cheese flavour, Lactase for Lactose Removal, Pectin methyl esterase for Firming fruit-based products and Pectinase for Fruit-based products are just some of the most commonly used enzymes in food production. Several advances have been made in the optimization of enzymes for existing applications and in the use of recombinant protein production to produce enzymes that do not have side-effects.

Enzyme Application in the Textile Industry

Enzyme biotechnology for sustainable textiles

Abstract

Enzymes are used in a broad range of processes in the textile industry: scouring, bleach clean-up, desizing, denim abrasion and polishing. Enzymes are specifi c and fast in action and small amounts of enzyme often save large amounts of raw materials, chemicals, energy and/or water. This chapter describes enzyme use in the textile industry in the context of sustainable production and reports life cycle assessments (LCAs) on two enzyme applications: bioscouring and enzymatic bleach clean-up. The results show that resource use and impact on the environment can be reduced considerably when enzymes are implemented in the two processes.

Introduction

In many industries, enzymes are used as biological catalysts to replace harsh chemicals or perform reaction under milder conditions. In textile industry, enzymes not only make good economic sense by saving energy, water and chemicals by improving quality, they also give valuable environmental benefits. These benefits are very important in time as there is an increasing awareness in the issue of sustainable development and climate change.

Novozymes recently performed two life cycle assessment (LCA) studies at textile mills in China, one of the prime producing countries for cotton and textiles. One of the studies was of a process known as bioscouring for removing impurities from cotton yarn. This is an alternative to traditional scouring that involves a number of high-temperature steps with a large consumption of chemicals. The other process is known as bleach clean-up to remove excessive bleaching agent prior to dyeing. This enzymatic process required less water and less energy than the conventional process used in China. On the basis of a qualitative assessment, it might seem obvious to assume that enzymes contribute to sustainable development. But what are the hard facts about the environmental impact of the use of enzymes in the textile industry? It should not be forgotten that the production of enzymes is also associated with environmental burdens (Nielsen et al., 2007). The purpose of the LCA studies presented here is to assess and compare the environmental burdens created by enzyme production and distribution in comparisonwith the environmental burdens avoided in the processes at textile mills. Here are two concrete examples from China based on the specific conditions at two mills. It could be argued that these facts only apply to the two specific mills. Therefore sensitivity analyses have been performed to look at a variety of scenarios, such as the use of fuels other than coal to generate electricity or the use of further optimised processes. In all cases, enzymes gave clear environmental benefits and helped to reduce contributions to global warming.

Objective

The objective of the study of the two processes is to compare the process without the use of enzymes and with the use of enzymes. Life cycle assessment was used for the study.Environmental assessment of the enzymatic scouring of package cotton yarn for dark-shade dyeing as an alternative to conventional chemical scouring

The assessment took place at the Rongxin Fibre Co. Ltd yarn dyeing mill located in Haining industrial park, 90 km from Shanghai in the Zhejiang province of eastern China. Here conventional scouring was replaced by enzymatic scouring with Scourzyme® 301 L in 2007 and enzymatic scouring is now running in full-scale production.

Light-colour yarns where pre-bleaching is required constitute about 80% of the market and dark shades constitute about 20%. The study addresses the 20% part of the market because the potential of bioscouring is largest for this product group. The reason for this is that pectate lyase does not remove seeds and other seed fragments, which is a problem when dyeing in lighter shades unless a natural look is preferred. Conventional scouring and bioscouring can be performed on the same production line with the same production equipment. Therefore there is no significant capital investment associated with a switch between the two scouring methods. The study refers to production at a full-scale commercial yarn dyeing line at Rongxin yarn dyeing factory with an output of 50 000 kg of dyed yarn per day. Chemicals are delivered mainly by local producers. Water used in production is extracted from a nearby river and treated by means of filtration and softening. Steam is supplied by the coal-fired Haining Dong Shan combined heat and power plant located about 1 km from the factory, and electricity is obtained from the national grid. Heat is not recovered in the textile mill. Wastewater generated in the process is initially treated on site and then fed to a central treatment plant before it is fed into a nearby river.

Methodology

Scouring is performed in a bath where a liquor of water and chemicals/enzymes is pumped through the yarn. The liquor is heated with steam to an appropriate temperature, and pumped through the yarn with an electric pump. Water and chemicals/enzymes are mixed with each other in a separate tank next to the yarn bath.

Conventional scouring was performed in three steps: a scouring process followed by two rinsing processes. The scouring process was performed at 100 ºC with penetration agent, sodium hydroxide (NaOH) and hydrogen peroxide (H2O2). The penetration agent is a surfactant that facilitated the water’s entry into the yarn. Sodium hydroxide removed the unwanted impurities and hydrogen peroxide bleaches the yarn. Sodium hydroxide is not selective in the substrates that it attacks. In addition to the unwanted impurities, it also degrades other substances in the yarn, including the cellulose.

Removed impurities were washed out of the yarn in the two rinsing steps. The first rinsing step was performed at 95 ºC. The second rinsing step was performed at 50 ºC and acetic acid was added to reduce pH. The starting temperature of the two rinsing steps was higher than the starting temperature of the scouring process because the wet yarn and the dye machine contained a large amount of residual heat transferred from the high temperature scouring step. The entire scouring process took 3 hours including filling and emptying the bath.

Figure 1 Process diagram for conventional scouring. Upward slopes indicate a rise in temperature. The final downward arrows indicate emptying of the bath.

Bioscouring is performed in a single process at 60 ºC with Scourzyme 301 L and penetration agent. Scourzyme 301 L is an enzyme product containing a pectate lyase that degrades pectins into soluble compounds. No rinsing steps or neutralisation steps are required prior to the yarn dyeing. The penetration agents used for bioscouring can be the same as used in the conventional scouring process. Bioscouring takes 90 minutes including the fi lling and emptying of the bath. Water from conventional scouring and bioscouring, as well as from any rinsing steps, is discharged directly for wastewater treatment after use.

Figure 2 Process diagram for bioscouring

Results

The results of the environmental impact assessment are shown in Fig. 3. Figure 3 shows that resource consumption and environmental impacts induced by enzyme production generally are very small compared with the savings. The reason is that a small amount of enzyme saves large amounts of chemicals, energy and water in the scouring process. Transport of the enzyme from Denmark to China does not add significantly to the environmental impact of Scourzyme 301 L even though the transportation distance is rather long. The explanation is that the quantity of enzyme used is small and that ocean freight is energy efficient.

Figure 3 Added and saved resource consumption and environmental impacts when switching from conventional scouring to bioscouring. All data are per tonne of yarn (dry weight).

The main factors behind the saved contributions to global warming are shown in Fig. 4, which demonstrates that the heat saving in the bioscouring process is the main factor behind the reduced contribution to global warming, followed by electricity and yarn savings. Savings of water and chemicals in the process and the transport of chemicals from the manufacturers to Rongxin are less important. Reduced water consumption in the scouring process explains most of the water savings in Fig. 3. Water savings resulting from the reduced consumption of chemicals and yarn (primarily from the irrigation of cotton fields) are of minor importance. The saving of agricultural land observed in Fig. 3 can be entirely explained by the reduced cotton production needed as a result of the use of enzymatic scouring.

Figure 4 Main factors behind saved contributions to global warming.

Environmental assessment of enzymatic bleach clean-up of light-coloured package yarn and knitted fabrics as an alternative to rinsing with hot water

Fabric and yarn need to be dyed evenly and reproducibly to meet the quality requirements of customers. For this reason, fabrics and yarns intended for fi nal products in light colours are often bleached prior to the dyeing process. Bleaching is usually performed with hydrogen peroxide and the excess bleaching agent must be carefully removed prior to dyeing to avoid breakdown of the dye during the dyeing process. Bleach removal has traditionally been performed by rinsing in several baths with water or by adding a reducing agent such as sodium thiosulphate. Both of these traditional processes use considerable amounts of water and energy for heating the water. Enzymes are now used as an alternative by many textile mills that dye yarn and fabrics. Enzymatic bleach clean-up is based on a catalase, which catalyses the conversion of hydrogen peroxide into water and oxygen. The LCA study took place at a textile mill in the Esquel Group located in Guangdong in South East China where bleach removal based on rinsing with hot water was replaced by enzymatic bleach clean-up with Novozymes’ Terminox Ultra® 50 L in 2002. The enzymatic process has been running in full-scale operation since then.

Methodology

Bleach clean-up of package yarn is performed in a series of batch processes in water mixed with chemicals/enzymes at appropriate temperatures. Liquid is continuously pumped through the yarn rolls or the knitted fabrics to establish the necessary contact with the cotton.

Baths are heated with steam, and the pumping of water is facilitated by electric pumps. The liquor ratio is 10 tonnes of water per tonne of yarn.

Reference for bleach removal. The traditional way for the mill to remove bleach is a three-step process composed of one hot rinse and two cold rinses with pH adjustment in the second cold rinse. The output from the process is bleached yarn or fabric without the presence of hydrogen peroxide. The yarn or fabric is then dyed in a new bath.

Figure 5 Process diagram for the traditional way of removing bleach by rinsing with hot water. Upward slopes indicate a rise in temperature. The final downward arrows indicate emptying of the bath.

Enzymatic bleach clean-up. Enzymatic bleach clean-up is performed in a two-step process composed of one cold rinse and a cold hydrogen peroxide decomposition step where the enzyme removes the hydrogen peroxide. Acetic acid is used for pH adjustment. The enzyme works at high speed and the bath is free of hydrogen peroxide after 25 minutes. Then dyeing is performed in the same bath with the same water.

Figure 6 Process diagram for enzymatic bleach clean-up.

Results

The results of the environmental impact assessment are given in Fig. 7 which shows that the consumption of resources and environmental impacts caused by enzyme production are generally very small compared with the savings, except in the case of agricultural land use. The explanation is that a small amount of enzyme saves large amounts of energy and water in the bleach clean-up process. Just as in the case of scouring with enzymes, transport of the enzymes from the enzyme factory in Denmark to China does not add significantly to the environmental impact of using enzymes.

Figure 7 Added and saved resource consumptions and environmental impacts when switching from the traditional removal of bleach based on rinsing with hot water to enzymatic bleach clean-up. All data are per tonne of package yarn or knitted fabric (dry weight).

The main factors behind the saved contributions to global warming are indicated in Fig. 8 which shows that the heat saving is by far the most important factor behind a reduced contribution to global warming.

Figure 8 Main factors behind saved contributions to global warming.

Toxicity has not been included in the quantitative assessment for the same reasons as explained for scouring. For the same reasons as before, it is considered very likely that the toxicity impact reflects the other impact categories in Fig. 7 and that the use of enzymes reduces the toxicity impact on the environment.

Conclusions about the two studies

The use of enzymes in scouring and bleach clean-up as alternatives to chemical treatment and rinsing with hot water, respectively, led to considerable environmental improvements at the two production lines at textile mills in China. The explanation is that a small amount of enzyme saves considerable amounts of energy and water in both cases and also chemicals in the case of scouring. Sensitivity analyses indicate that the general conclusion of the assessment holds up under different energy supply scenarios although the sizes of the reductions in environmental impacts are subject to much variation and uncertainty. The impact of the transport of enzymes from the manufacturer to the final user is insignificant even though the transportation distance is long. The main findings of the study are therefore applicable to other textile mills with similar production systems elsewhere in the world.

The magnitude of the environmental improvements obtained by replacing the existing production methods with the enzymatic technologies are highly dependent on the type of fuel used and the actual production conditions. An estimation of environmental improvements at other factories must therefore rely on specific information on production processes and energy supply systems. The study has not addressed the removal of bleach with a reducing agent and further environmental assessments are required before any conclusions can be made about this method.

Silk Industry

History of silk

Sericulture or silk production has a long and colorful history unknown to most people. For centuries the West knew very little about silk and the people who made it. For more than 2000 years the Chinese kept their secret and was the most zealously guarded secret in history. History of silk began in the 27th century BCE. The key to understanding the great mystery and magic of silk, lies with one species: the blind, flightless moth, Bombyx mori. It lays 500 or more eggs in four to six days and dies soon after. From one once of eggs come about 30,000 worms which eat a tom of mulberry leaves and produce twelve pounds of raw silk, but this silk worm is unique in China.

Production

Producing silk is a lengthy process and demands constant close attention. To produce high quality silk, there are two conditions which need to be fulfilled – preventing the moth from hatching out and perfecting the diet on which the silkworms should feed. Chinese developed secret ways for both.For  the  commercial  production  of  silk  silkworms  are  cultivated  and  fed  with  mulberry leaves. The eggs are hatched by artificial means such as an incubator, and in the olden times, the people carried it close to their bodies so that it would remain warm. Silkworms that feed on smaller, domestic tree leaves produce the finer silk, while the coarser silk is produced by silkworms that have fed on oak leaves. From the time they hatch to the time they start to spin cocoons, they are very carefully tended to. Noise is believed to affect the process, thus the cultivators try not to startle the silkworms.Once the cocoons are ready then cultivators gather the cocoons and the chrysales are killed by heating and drying the cocoons. In the olden days, they were packed with leaves and salt in a  jar,  and  then  buried  in  the  ground,  or  else  other  insects  might  bite holes  in  it.  Modern machines and modern methods can be used to produce silk but the old-fashioned hand-reels and looms can also produce equally beautiful silk. In the process of silk many steps runs in cascade manner in order to have a complete process of silk production. Many process like removal of raw silk, washing, removal of impurities and dying and drying are bit essential but removal of wax and other unwanted protein form raw silk is most important step which make difference in the normal and quality silk known as degumming.Silk todayWorld silk production has approximately doubled during the last 30 years in spite of man-made fibers replacing silk for some uses. China and Japan during this period have been the two main producers, together manufacturing more than 50% of the world production each year. During the last 1970’s China, the country that first developed sericulture thousand years ago dramatically increased its silk production and has again become the world’s leading producer of silk.

Silk industry in the Philippines

At present, the development of the silk industry is spearheaded by the Fiber Industry Development Authority (FIDA), with the joint cooperation of PTRI, the Sericulture Research and Development Institute (SRDI) of the Don Mariano Marcos Memorial State University (DMMMSU), the University of the Philippines at Los Baños (UPLB) and other state universities and colleges (SUCs).

The Organization for Industrial, Spiritual and Cultural Advancement-International started in 1999 by R. Shigemi Watanabe. Initially, the project is implemented to improve the economic condition of the local farmers who are dependent on the declining sugar industry. It is designed to create a source of livelihood among the locals living in mountainous areas through the cocoon production and secure a stable source of income for the women in rural areas through weaving and/or manual extraction of spun silk from the silk waste. It is found in Negros Occidental and Negros OrientalDegumming

Silk degumming process is a fundamental finishing process for silk yarn and silk fabric. The objective of degumming is remove the substrate such as silk gum (sericin), wax and some impurities from silk fibers. The principle of degumming process is breaking the peptide linkage of amino acid in sericin structure into a small molecule, which is soluble in water. The  methods  used  for  the  degumming  are  the  hydrolysis  reaction  performed  by  acid and alkaline, but these methods are not eco-friendly and same time they have a big problem on the surface area of silk. Hence the proteolytic enzyme have been used to solve this problem and shown promising results not only in the production level but also quality of silk. Since protease based degumming is eco-friendly which will be an additional advantage. Though the conventional protease are quite efficient for degumming but having some disadvantage like thermal and chemical stability which is one drawback has to solved, also alkaline protease can hamper the quality and physical appearance of silk as silk is quite sensitive to alkali and alkaline  protease.  The thermostable protease basically forms Geobacillus genus has been used for the enzymatic degumming of silk which are quite resistant to various chemicals and temperature.  In  this  article  we  have  summarized comparative  analysis  of  detergent, conventional protease and thermostable protease for efficient degumming.The principle behind silk degumming is increasing the silk gum solubility by breaking the peptide linkages of sericin structure into small molecule such as amino acids and its oligomer with hydrolysis reaction. Silk degumming can be performed by numerous methods such as using alkaline and synthetic detergent, though it can be harmful to the silk because of silk’s poor resistance to alkaline. In present the proteolytic enzyme is used to solve this problem, though it has disadvantages and high costs.Two process have emerged in the recent years, “H.T.-HP. Degumming” and “Enzyme Degumming”. The High Temperature-High Pressure degumming requires special pressured equipment and is energy intensive process. The Enzymatic degumming is emerging as eco-friendly fiber-gentle process where proteolytic enzymes that are effective under alkaline, neutral as well as acidic conditions are being used.

Soap Degumming

Soap has been used in the process of silk degumming with synthetic detergents implies the partial or total replacement of soap with synthetic non-ionic surfactants. To achieve the better yield and quality soaps have been combine with an oxidizing or reducing bleaching and, in some cases, even with dyeing, thus improving water and energy saving. Generally, alkali and detergent mixtures are used at temperatures around 95 °C - 98 °C. Such  a treatment  is  suited  to  continuous  processing.The  detergent  based  silk  degumming  leads  to  the  production  of  low  quality  of  silk as detergent interfere with the mechanical properties of silk fibers. The fibroin, main part of

silk protein  provides  tensile  strength  to  silk  fibers  which  is  very  sensitive  to  soap, alkali  and alkaline protease. Another complication with detergent based silk degumming as chemicals used for process are not eco-friendly hence long term effects on the surrounding. Generally in the detergent based silk degumming soap derived from higher fatty acid has been used which still remains in the silk even after many washing.

Microbial protease in silk production

The conventional detergents are not the choice for silk degumming as these chemical will interfere the basic physical and chemical properties of silk leading to the complication in the quality of silk. Though chemical based detergents have been used for long time but same time silk produced by these methods always has shorter shelf life and various other complications like less tensile strength, more hygroscopic etc.Proteases  cover  the  60%  of  total  enzyme  market  and  amongst  the  most  valuable commercial enzyme. Alkaline proteases hold a great potential for application in the detergent and leather industries and there is an ever increasing trend to develop environment friendly technologies. Plants, animals and microbes are the main sources for protease production. The preferred sources of proteases are microbes because of their rapid growth and the ease with  which  they  can  be  genetically  manipulated  to  generate new  enzymes  with  altered properties  and  are  currently  being  utilized  by  the  detergent industry  eg.  Serine proteases produced by Bacillus strains. Proteases from several bacteria have been purified and characterized. Genus Pseudomonas a gram-negative bacterium that predominantly produces alkaline proteolytic enzymes and the proteases has been purified. Fungal alkaline proteases are  advantageous  because  of  the ease  of downstream  processing  to  prepare  a microbe-free enzyme at low cost production.The protease and more specifically alkaline protease have shown numerous complication with the silk during the process of degumming. First of all the silk is highly sensitive to the alkali  and  alkaline  protease  which  hampers  it  physical  appearance.  The Mechanical properties  also  get  affected  under  the  exposure  of  alkaline  protease. Another complication  with  the  conventional  protease  is  their  range  of  activity  and stability in various pH and Temperature. Another significant complication especially in protease  based silk  degumming  incorporation  of  chemicals  in  certain  instants  which also inactivates the conventional enzymes.

Thermo-stable Protease

The optimal activity of conventional protease ranges 30-40`C which is not appropriate for compete degumming process. Even some time process has to run for more than 24 hour in at lower temperature which again causes  deactivation  of  conventional  enzyme.  Often the degumming process runs at higher temperature which leads to denaturation of conventional proteases which are not thermo-stable. In some cases during the process a lots of chemical are required to complete the process and many circumstances presence of chemical also leads to the deactivation of enzyme and process subsequently.The proteases are generally classified into two broad categories (exopeptidases, that cleave off amino acids from the ends of the protein chain and endopeptidases, which cleave peptide bonds within the protein) are becoming major industrial enzymes, and constitute more than 65%  of the  world  market. These protease enzymes have been extensively used in the food, pharmaceutical, leather and textile industries.

The Bacilli provides 70% of protease hence the diverse sources has made these organisms the focus of attention in biotechnology. In a chemically defined medium, thermophilic and alkaliphilic Bacillus sp.  JB-99 was also reported to produce thermostable alkaline proteases. Dominant producers  of  proteases  in  fact,  are  the  microorganisms  of  the genera  Pyrococcus, Thermococcus and Staphylothermus. Extremely thermostable serine proteases are produced by the hyperthermophilicarc  haeum  Desulfurococcus  strain, and thermostable metalloroteases are reported from a gram-negative thermophilic bacterium.In  the  last  decades  numbers  of  commercial  uses  have  been  established  for thermostable amylases and roteases. In the brewing industry, starchy materials used as adjuncts must first be liquefied by the addition of thermostable amylases. Thermostable amylases are used in desizing textiles to remove unwanted starch. They also may be used to aid in the clarification of fruit juices in the manufacture of jelly and chocolate syrups. Proteases are used in the leather industry, particularly in baiting hides to remove unwanted inter fibrillar material.Protease based silk degumming has been shown higher efficiency, expressed over removal of  sericin  and  energy  save,  achieved  through  lower  process  temperature.  Milder treatment conditions  under  which  the  fibers  were  processed  during  enzymatic degumming  prevented fibrillation and dusting i.e. fiber damage. Damage to the soap degummed fibers was enhanced in  subsequent  dyeing  process,  which  was  the  reason of  inability  to  spectrophotometrically measure dyestuff concentration in dye-bath. When dyeing silk fibers, method of degumming should be considered. After the degumming fibers still had lustrous, soft and smooth surface. Result of staining test with direct dyes was appeared pink, indicating that there was a small amount of sericin remaining. This process can be used instead of conventional degumming method with low cost and do not harm the environment.

References:

1.  Ole Kirk*, Torben Vedel Borchert and Claus Crone Fuglsang: Industrial enzyme applications. Current Opinion in Biotechnology 2002, 13:345–351

2.  http://biotech.uplb.edu.ph/index.php/en/research3/food-feeds-and-specialty-products-biotechnology?catid=35:static&id=52:enzyme-research-lab

3. Silkroad Foundation. 2000. http://www.silk-road.com/artl/silkhistory.shtml . Accessed September 23, 2014.

4. Republic of the Philippines, Department of Agriculture. Fiber Industry Development Authority. http://fida.da.gov.ph/Templates/silk_history.htm . Accessed September 23, 2014.

5. OISCA. 2014. http://www.oisca-international.org/programs/sustainable-community-development-program/philippines/development-of-sustainable-communities-in-negros-province-through-silk-production-/ . Accessed September 23, 2014.

6. Rajasekhar, A., Ravi, V., Reddy, M. N., & Rao, K. R. S. S. (2011). Thermostable Bacterial Protease - A New Way for Quality Silk Production, 3(4), 43–58.

7. P. H. NIELSEN, H. KUILDERD, W. ZHOU and X. LU, Novozymes A/S, Denmark8.