Current trends in biotechnological production of xylitol and future …abap.co.in/sites/default/files/2-Paper 8-36.pdf · 2010-03-25 · Current trends in biotechnological production

Current trends in biotechnological production of xylitol andfuture prospects

R S Prakasham*1, R Sreenivas Rao and Phil J. HobbsNorth-Wyke Research

Okehampton, Devon, EX20 2SB, UK1Permanent Address - Bioengineering and Environmental Centre

Indian Institute of Chemical Technology, Hyderabad – 500 607, India* For Correspondence: [email protected]

AbstractThis review describes recent research

developments on biological conversion ofhemicellulosic biomass towards production ofxylitol by taking advantage of power ofbiotechnology. Xylitol is a five-carbon sugaralcohol with established commercial uses indifferent healthcare sectors and especially as analternative sweetener for diabetic persons. Xylitolcan be synthesized either by chemicalhydrogenation of xylose or by fermentation. Theprecursor xylose is produced from biomass bychemical or enzymatic hydrolysis and can beconverted to xylitol primarily by yeast strainswhich offer the possibilities of economicproduction by reducing required energy whencompared to chemical production. Biomasshydrolysis under an acidic environment is the mostcommonly used practice and is influenced byvarious process parameters. Several microbialgrowth inhibitors are produced during chemicalhydrolysis that reduce xylitol production fromxylose, a detoxification step is therefore essential.Enzymatic hydrolysis has advantages overchemical conversion although more research isnecessary to reduce inhibition due to structuralvariation from different substrates or plantspecies. Enzymatic xylitol production is mostlyan integral process of microbial species belongingto the Candida genus. Extensive research hasbeen performed to screen for xylitol producingmicrobial strains as well as to understand

microbial metabolism, the xylitol metabolicpathway, cofactor requirements, development ofrobust recombinant strains, optimization ofbioconversion parameters and xylitol productionstrategies using free and immobilized cells. Theimperative role of hydrolysis of xylose containingbiomass and subsequent process parameters hasmajor impact on economis of bioconversion. Thereview identifies ways forward for improvedenzymatic xylitol production to compete withcurrent chemical processes.

Key words: Candida, Detoxification,Hemicellulosic material, Hydrolysis,Bioconversion, Xylitol, Xylose.

IntroductionXylitol is a polyol and a C

5 sugar, also

known as wood or birch sugar, obtained from thereduction of xylose. It is a rare sugar that existsin low amounts and is the constituent of manyfruits and vegetables, such as raspberries,strawberries, yellow plum, lettuce and cauliflower.Xylitol was first produced from birch trees in 19th

century in Finland. It has attracted global interestdue to its sweetening power similar to that ofsucrose; equivalent to 2.4 kcal.g-1 and laxativenature (145 J.g-1caloric content) (104, 22, 33).Xylitol has applications and potential for at leastthree types of industries namely food (for dietaryespecially in confectioneries and chewing gums),odontological (for its anticariogenicity, tooth

Current Trends in Biotechnology and PharmacyVol. 3 (1) 8-36, January 2009. ISSN 0973-8916

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rehardening and remineralization properties) andpharmaceutical (for its toothfriendly nature,capability of preventing otitis, ear and upperrespiratory infections and its possibility of beingused as a sweetener or excipient in syrups, tonicsand vitamin formulations). However the majoruse is for the prevention of dental caries as xylitolinhibits growth of microorganisms responsible fortooth decay (44, 69, 70, 157). In addition, xylitolis accepted for consumption for diabetics andhelps in treatment of hyperglycemia as itsmetabolism is independent of insulin (157). Thexylitol market is increasing and at present isestimated to be $340 million yr-1 and priced at$4–5 kg-1.

Currently, xylitol is manufactured at theindustrial level by a chemical hydrogenation ofthe five-carbon sugar D - xylose, in the presenceof nickel catalyst at elevated temperature andpressure. This chemical process is laborious, costand energy intensive. In addition, the processneeds expensive refining treatments necessaryfor xylose production. In order to produce thisxylitol in economically and eco-friendly manner,research was initiated for alternative strategies.One of the alternatives is bioconversion ofrenewable biomass sources which requireshydrolysis followed by bioconversion of xylosefrom crude hydrolysate to xylitol employingspecific microbial strains for fermentation (132,129, 130).

Photosynthetic biomass as raw material forxylitol production

In view of the disadvantages associatedwith the chemical production of xylitol processsuch as conversion efficiency, environmentimpact and energy input parameters research hasidentified alternative raw materials andproduction processes. One of the potentialalternative raw materials is xylo-oligosaccharides

(hemicellulosic materials) from plant biomass; asthe annual growth of plant-derived biomass isestimated to be 73.9 terra grams per year on adry matter basis (54) of which 20-35% is xylose.Biomass material is widespread, abundant,renewable, cost-effective and inexpensive sourceof polysaccharides which can be used forproduction of wide variety of biotechnologicalproducts including xylitol, these sources includeforests, agricultural and agro-industrial residues(Table 1).

Table 1: Lignocellulosic biomass producedannually in dry mass basis (54)

Crop Lignocellulosic

biomass (Tg)

Barley 058.45

Corn 203.62

Oats 010.62

Rice 731.34

Wheat 354.35

Sorghum 010.32

Sugarcane 180.73

Rye grass* 20.00$

*Source: Booth et al. (5) $ tons.hectar-1

According to estimates, hemicellulose isthe second most common polysaccharideavailable in nature (105) consisting ofheterogeneous polymers of hexoses (glucose,mannose and galactose) and pentoses like xyloseand arabinose (58). In order to use thesematerials they must be hydrolyzed into simplemonomeric sugars either by chemical orenzymatic methods for fermentation usingmicroorganisms. Several studies on hydrolysis ofxylose-rich hemicellulosic materials (Table 2)

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have been performed for utilization as substratesfor biotechnological xylitol production (78, 129).A variety of plant biomass materials wereevaluated as source of raw materials such as corncobs (129), sugar cane bagasse (14, 129),eucalyptus (146), brewery’s spent grain (12, 78),olive tree pruning (102), soyabean hull (114), palmoil empty fruit bunch fiber (96), and rice straw(65). Residue particle size reduction wasperformed by grinding for all the pretreatmentsof lignocellulose residue as it reduces cellulosecrystallinity, especially in case of photosyntheticbiomass as raw material (134). However,utilization of these resources mainly depends onthe degradation of these polymeric materials tosimple sugars, with hemicelluloses being importantin the overall conversion process (102, 129, 146).

Table 2: Xylan content in different materials

Feed stock material Xylan content(%) dry weight

Corn stover 22.4Corn fiber 16.8Wheat straw 21.2Switch grass 20.4Office paper 12.4

Hydrolysis methodologiesPhotosynthetic biomass mainly composed

of cellulose (34-50%), hemicellulose (19-34%),lignin (11-30%) and smaller amounts of pectin,protein, extractives and ash. Composition of thesecomponents differs with the source of plantspecies, age and growth conditions (4). Amongthese, cellulose (a homo-polysaccharide ofconsisting of polymerized D-glucose up to 10 000or more linked by â-1, 4-glucosidic bonds) formsa skeleton. Hemicellulose is a complexheterogeneous polysaccharide consisting of 200degree of polymerization composing of glucose,galactose, mannose, xylose, arabinose, glucuronic

acid with acetyl side chains. Cellulose isinterlinked by hemicellulose to build a structuralmatrix. This structure is further encrusted withlignin. Lignin, polymer of phenyl propane, is non-polysaccharidic in nature consisting of ñ-coumaryl-, coniferyl- and sinapyl alcohol unitsbonded by alkyl-, aryl, and combination of bothether bonds. In fact, cellulose, hemicellulose, andlignin are closely associated with covalent cross-linkages, hence, biomass can be regarded as acomposite material, in which the lignin serves asa protective layer. In addition, the composition oflignocellulosic materials varies with the biomassmaterial such as hard wood, soft wood andgrasses. Because of this, the plant biomassexhibits a remarkable stability against chemicaland biological attack and can rarely be convertedinto simple sugars under normal conditions.Therefore pretreatment is necessary in order toalter the structural integrity, remove the lignin andincrease the surface area to make this materialavailable as fermentable sugars (45).Performance of pretreatment depends onselected material harvesting nature, lignin andother components composition, storage type andtime, temperature and chemicals used. Ingeneral, processes used to produce xylo-oligosaccharides from xylan-rich materials areessentially hydrolytic in nature and can beperformed either by chemical means using basicor acidic media, or catalyzed by enzyme sources(78). Since, the scope of this review is limited toxylitol production, detailed information onpretreatment methodologies are delt very limited.

Chemical hydrolysis is a simple and rapidmethod for hemicellulosic material howevertreatment conditions vary with agro-industrialmaterial and with respect to chemical agent typeand concentration, incubation temperature andtime (129, 134). When aged or fully grownagricultural residues or hardwoods are used asraw materials, xylose is the most abundant sugar

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in hydrolysates in addition to small fractions ofother sugars. For acid hydrolysis different mineralacids such as sulfuric (102, 134, 147), hydrochloric(40), nitric, hydrofluoric (25), acetic acid (17) andphosphoric (27) acids are used at high temperatureand pressure (commonly 160oC) and (10 atm).In general, acid hydrolysis performed underconcentrated (50–70%) or diluted (below 2%)conditions. Preferences are for diluted acidconditions and high temperatures due to highreaction rates with less microbial growth inhibitors,which is a low cost technology compared to otherchemical approaches (46, 101, 102).

The chemical hydrolysis reaction is acomplex process (27) that is a multi-step reactionthat occurs in following sequence (i) diffusion ofprotons through the wet lignocellulosic matrix; (ii)protonation of the oxygen of a heterocyclic etherbond between the sugar monomers; (iii) breakingof the ether bond; (iv) generation of a carbo-cationas intermediate; (v) solvation of the carbo-cationwith water; (vi) regeneration of the proton withcogeneration of the sugar monomer, oligomer orpolymer depending on the position of the etherbond; (vii) diffusion of the reaction products inthe liquid phase. All these process steps areinfluenced by pH of the hydrolysis medium, solid-liquid ratio, incubation temperature and time (65,129). Sun and Cheng (134) and Cara et al. (10)concluded that acid hydrolysis with the use ofconcentrated acids is toxic, corrosive andhazardous.

Auto-hydrolysis is an alternative methodfor the chemical depolymerization ofhemicelluloses with limited solubilization of lignin(29) and reduced quantities of sugar derivatives(furfurals and hydroxymethylfurfurals) (78). Inaddition auto-hydrolysis presents some technicaland environmental advantages too as no chemicals(acid or alkali) are used other than water. Auto-hydrolysis performed at mild temperatures yieldsa high mass of xylo-oligosaccharides without

modifying the cellulose and lignin structure sub-stantially, allowing improved recovery duringfurther processing (76, 117). The xylo-oligosaccharides produced are associated with asignificant fraction of acetyl and uronic acid groupswhich has the characteristic of very high watersolubility unlike that of chemical hydrolysis. Theauto-hydrolysis process efficiency and hydroly-sate chemical composition depends on incubationtemperature and time, solid to liquid ratio,structural integrity of raw material employed.Nabarlatz et al. (81) working with six agriculturalresidues namely corncobs, almond shells, olivestones, rice husks, wheat straw and barley strawas feedstocks for the production of xylo-oligosaccharides by auto-hydrolysis, reported thatthe yield of xylo-oligosaccharides depended onthe content of xylan and its accessibility, and wasproportional to the acetyl content of the rawmaterials. In fact, by regulation of auto-hydrolysisconditions, it is possible to influence characteristicsof the xylo-oligosaccharides (the acetyl contentand the molar mass distribution), but the natureof the raw material also has an influence (81).Hydrolysate analysis revealed that partiallyacetylated oligomeric and polymeric xylan frag-ments were attached with acetyl groups at 2 and3 positions and some monosaccharides andpartially O-acetylated 4-O-methylglucuronoxylanin addition to degradation products were present(80).

Biological or enzymatic hydrolysis hasbeen proven as an alternative hydrolysis methodoffers conceptual edges like low chemical andenergy use, but depends on enzyme accessibilityto the heterogeneous biomass structure. The rateand extent of enzymatic hydrolysis oflignocellulosic biomass is dependent on catalyticproperties of enzymes, their loadingsconcentrations, the hydrolysis period, reactionparameters employed, biomass type, pretreatmentmethod employed and compounds producedduring pretreatment process (159). Reduction of

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hemicellulosic crystallinity improves the enzymatichydrolysis rate and time in addition to the enzymeloading. Among all biomass components, lignin isidentified as a major deterrent to enzyme attackon cellulose indicating the importance of reducingthe structural integrity caused by lignin beforehydrolysis. Cellulase and xylanases are the majorenzymes employed in most of the pretreatmentstudies (95, 159). Biomass digestibility by enzymesis found to be regulated by the surface area ofthe material and an increase in surface area bypretreatment or decreasing particle size improvesbiomass hydrolysis (95).

Use of xylanase alone may not besufficient in view of the complex nature ofphotosynthetic biomass material. Xylanasescatalyzes the â-1, 4 bond in the xylan backboneyielding short xylooligomers. They are group ofenzymes work synergistically and differ withmicrobial origin. The selection of critical xylanaseblend consisting of xylosidase, Mannanases,arabinofuranosidaes, glucuronidases, esterases(ferulic and cumaric acid, acetyl-mannan, acetyl-xylan, etc.) and hemicellulolytic esterases is oneof the important factors for effective productionof xylose from hemicellulose fraction. Thisselection again related with the nature of xylanstructure which vary with type of biomass (soft,hard wood, grass, etc). Pre-hydrolysis either bymild chemical treatment at elevated temperaturesand/or by other specific enzyme treatment wouldoffer the better hydrolysis process for the efficientproduction of xylose. Use of non catalytic proteinssuch as expansins and swollenins decreases thecrystallinity structure thereby increases theaccessibility to enzymes may be novel approach.However, the applicability and feasibility is yet torequire further study. Our laboratory studiesindicated that xylanase from certain specificmicrobial strains could be used as an efficientxylose production from palm seed fibre (95).However, enzyme treatment parameters have to

be optimized for maximization of xylose produc-tion. Although enzymatic hydrolysis results in highyields in bioconversion of sugars from pretreatedphotosynthetic biomass, the cost of enzymes is akey aspect and needs to be costed. Use ofhemicellulosic hydrolytic enzyme blend is anotheralternative; however, one has to identify andoptimize process environment of the specificenzyme blend for each material. Wet oxidationpretreatment process proven to be efficient forlignocellulosic materials as crystallity decreasewas noticed along with lignin degradation to CO

2

and H2O and carboxylic acids. Recently use of

ionic liquids such as 1-butyl-3-methylimidazoliumcaution for biomass pretreatment revealedoptimistic results but indeapth studies are essentialfor its after effects like microbial/enzyme inhibitorproduction, process environment, etc. In nutshell,upstream to pretreatment, the choice of sourcematerial structure is an important in selection ofeffective pretreatment methodology.

Components of biomass hydrolysateA range of products such as glucose

(mainly from cellulose and hemicellulose), xylose,mannose, galactose and acetic acid (fromhemicellulose) and phenolic compounds (fromlignin) are produced during the hydrolysis process.In addition, other compounds are also producedduring hydrolysis especially when chemicalhydrolysis is employed. Without exception, allsugar liquors obtained by chemical hydrolysiscontain furan derivatives, aliphatic acids andphenolic compounds. Furan derivatives commonlyknown as furfurals and hydroxymethylfurfural(HMF) are produced from the degradation ofpentoses and hexoses, respectively. Furtherdegradation of furfurals leads to the productionof formic acid. HMF is normally produced inless concentration compared to furfurals byhexoses degradation mainly due to the lowquantities of hexose in hemicellulose. This isbecause the conditions employed in the

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hemicellulosic material hydrolysis process do notdegrade hexoses in large quantities. Acetic acid,the major aliphatic acid present in chemicalhydrolysates, is mostly released from thehemicellulosic acetyl groups. During the acidhydrolysis, a minor part of lignin is also degradedto a wide range of aromatic compounds includinglow molecular mass phenolics (90). With the useof strong alkali solutions, depolymerized xylanmay be extracted from lignocellulosics, but theproduct obtained is completely deacetylated andhas very limited solubility in water hence is notthe preferred hydrolyzing reagent. In additionother compounds such as acidic resins, tannic,terpene, syringic, vanillic, caproic, caprylic,pelargonic, and palmitic acids are reported to beproduced during chemical hydrolysis (6, 78).

Microbial fermentative inhibitors of biomasshydrolysates

The major disadvantage of chemicalhydrolysis is the reduction of availablemonosaccharides and production of theirderivatives (furans, hydroxymethylfurfurals andother phenolic toxic compounds which aremicrobial growth inhibitors and hinder furtherbiotransformations (78). However, the type andconcentration of microbial fermentative inhibitorycompounds mainly depend on raw material as wellas the operational parameters. Microbial toxicityis also associated with fermentation variables likemicrobial physiological growth conditions,dissolved oxygen concentration and pH of themedium. In general, biomass hydrolysateinhibitors can be categorized as sugar or lignindegradation products, derived from lignocellulosicstructure and heavy metal ions (78).

Furfurals derived from pentose are themajor microbial growth inhibitor compoundspresent in chemical hydrolysates for xylitolbioconversion. They inhibit the growth of microberanging from 25 – 99% relative to the furfuralconcentration (0.5 – 2.0 g/l) and cell mass yield

per ATP by interfering with the respiration process(90, 94). Delgenes et al. (20) and Martinez etal. (71) reported that Pitchia stipitis and Sac-charomyces cerevisiae growth was reduced by100% when the HMF in the concentration of 1.5and 1.0 g/l was supplemented in the growthmedium indicating the inhibitory effect varies withthe type of microbial strain. Presence of lowconcentrations of these compounds in thefermentation medium showed better microbialgrowth (94) indicating the microbial strainproperties role during bioconversion ofhydrolysates. In addition, the antagonistic effectof furfural and HMF along with acetate, formicand levulinic acid on microbial growth was alsoreported with P. tannophilus and P.stipitis duringxylose fermentation (148).

A variety of lignin degradation productsthat included aromatic, polyaromatic, phenolic andaldehydic compounds present in hydrolysate alsocause inhibitory effects on microbial growth byintegrating into biological membranes andaffecting the membrane permeability. Villa et al.(145) reported that phenolic compounds at morethan 0.1 g/l concentration affect the xyloseconsumption, cell growth and xylitol productionin C. gluilliermondii. Acetic acid toxic effectis mainly associated with its pKa property as atthis value acetic acid is liposoluble, diffuses acrossthe plasma membrane and discharges protonsresulting in cell death due to dropping the internalpH. However, presence of acetic acid at lowconcentrations (1.0 g/l) in the fermentationmedium reported to improve the xylose-to-xylitolbioconversion (24) probably due to more diffusionof internally pooled xylitol during xylosemetabolism because of limited acetic acid effectat cell membrane. Heavy metals (iron, chromium,nickel and copper) produced during hydrolysismainly originate from corrosion of hydrolysisequipment causes cell toxicity by inhibitingmetabolic pathway enzymes (93).

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Detoxification methodologiesIn order to remove the microbial growth

inhibitors and increase the hydrolysatefermentability, several detoxification treatments,including chemical, physical and biologicalmethods have been developed. However, theneeds for detoxification must be evaluated in eachcase since it depends on the chemical compositionof the hydrolysate and is strain specific. Theeffectiveness of a detoxification methodologydepends on raw material, type of hydrolysisprocess and microorganism employed (129).Taherzadeh et al. (136) reported four differentapproaches for minimizing the inhibitory effectof hemicellulosic hydrolysates; (1) use ofbioconversion friendly hydrolysis methods; (2)detoxify the hydrolyzate before fermentation; (3)use of inhibitor resistant microorganisms; (4)convert toxic compounds into non-toxic. Sincedetoxification increases the cost of the process,it is important either to overcome detoxificationsteps or to develop cheap and efficient methods.Development of a new metabolically engineeredmicrobial species which tolerate inhibitors couldbe the better option which can eliminatedetoxification.

Vacuum evaporation is the best physicaldetoxification method with limited scope and helpsto reduce volatile toxic compounds that includeacetic acid, furfural, hydroxymethylfurfural andvanillin. Mussantto and Roberto, (78) reportedthat more than 90% these compounds areremoved from wood, rice straw and sugarcanebagasse hemicellulosic hydrolysates by employinga vacuum evaporation method. However, thisprocess enhances the concentration of non-volatile toxic compounds and reduces volumes ofthe hydrolysate (61). Neutralization, over-liming,sulfite treatment, extraction with organic solvents,treating with ion-exchange resins and adsorptioninto activated charcoal or diatomaceous earthreduce the ionization properties of inhibitory

compounds by precipitation of toxic compounds.pH adjustment is effective and the most cost-effective chemical detoxification method amongavailable treatments. Calcium hydroxide andsulfuric acid are commonly used for treatment ofhemicellulosic hydrolysates for removal ofphenolic compounds, ketones, furfurals andhydroxymethylfurfurals (84, 129). Activatedcharcoal is the other process attracting muchattention because of its low cost and a highcapacity to absorb pigments, free fatty acids, n-hexane and other oxidation products (98, 129).The effectiveness of activated charcoal treatmentdepends on different process variables such aspH, temperature, contact time and solid-liquid ratio.Acidic pH favours removal of the neutral or non-ionized phenolic molecules while alkaline pH fororganic bases during activated charcoal treatment.Increase of contact time is reported to influencethe clarification process. The absorption processincreases at elevated temperatures duringcharcoal treatment basically due to a faster rateof diffusion of absorbate molecules from thesolution to the absorbent and temperature inducedorientation of charcoal surface (77). Comparativeevaluation of different chemical detoxificationmethodologies indicated that anion exchange resinsremove high percentages of toxic compoundssuch as acetic acid (96%), phenolic compounds(91%), furfural (73%), HMF (70%) in addition tosubstantial removal of aldehydes and aliphaticacids from hydrolysates compared to cation-exchange resins (78, 129). Grzenia et al. (35)reported use of hollow fibre based liquid extractionsystem for removal of acetic acid from corn stoverhydrolysate using two different LiquiCelMembranes.

Biological detoxification can be doneeither by using specific enzymes ormicroorganisms. Laccases and peroxidases aregenerally employed for detoxification (78). Theprobable enzymatic detoxification mechanisminvolved is oxidative polymerization of low



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molecular weight phenolic compounds (49)whereas, microbial detoxification of hydrolysateinvolves utilization of toxic compounds formicrobial growth or adaptation of specific microbefor hemicellulosic hydrolysate (129). Schneider(116) reported that acetic acid in the hydrolysatecan be removed more than 90% by S. cerevisiaemutant from wood hydrolysate. Silva andRoberto (118) and Sreenivas Rao et al. (129)successfully demonstrated that adaptation of C.tropicalis as an effective and inexpensiveapproach to alleviate the inhibitory effect of toxiccompounds on xylose utilization for xylitolproduction from rice straw and corn cobhydrolysates.

Sreenivas Rao et al. (129) working onxylitol production from sugarcane bagasse andcorncob hydrolysate reported that variouschemical and biological detoxificationmethodologies i.e., more than one method, wereeffective compared to single treatment processes.The authors reported pH adjustment followed byactivated charcoal and resin treatment only helpedup to certain level and adaptation of microbialstrain would be the better option for effectiveand efficient use of sugar compounds fromhemicellulosic hydrolysates. In summary, eachdetoxification method is specific to certain typesof compounds. Choosing detoxification methods(more than one) and their sequence was importantfor improved yields, however identification ofinhibitory compounds and their concentrations inthe hydrolysate was necessary.

Xylitol producing microbial strains

In the last few decades, several paperspublished on xylitol production using bacteria(157, 158), fungi (19), and yeasts (55, 62, 119,126, 132). Among the microorganisms, yeasts areconsidered as the best xylitol producers (Table3). Candida strains have been extensively studied

for the production of xylitol as they have an ad-vantage over the metabolically engineered S.cerevisiae for being natural D-xylose consum-ers and maintaining the reduction– oxidation bal-ance during xylitol accumulation.

Table 3: Some of the best xylitol producing yeasts

Yeast References

Candida boidinii 106

Candida guilliermondii FTI-20037118, 119Candida intermedia 28Candida maltosa 36Candida mogii 122Candida parapsilosis 55C. tropicalis HXP 2 32C. tropicalis 129Debaromyces hansenii 107Hansenula polymorpha 135Pachysolen tannophilus 110Pichia caribica 126Pichia miso 88

Screening programmes for xylitol productionfrom D-xylose

Xylitol is an intermediate metaboliccompound produced in all microbial strains whosexylose metabolism occurs in a sequential catalyticactivity of xylose reductase and xylitoldehydrogenase enzymes. Keeping this in view,several scientific researchers have been involvedin microbial screening programs to isolate effi-cient microbial strains for xylitol production.Hiroshi and Toshiyuki (41) tested 58 strains andP. miso emerged as the best xylitol producingstrain with an yield of 3.77g of xylitol from 8.50gof D-xylose was consumed. Ojamo (88) screened30 yeast strains for a xylitol metabolizing pathwayand reported that C. gluilliermondii and C.

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tropicalis were the highest yielding strains. While,Sirisansaneeyakul et al. (122) reported xylitol yieldof 0.62g.g-1 of xylose using C. mogii from 11strains they tested for xylose utilization. Suryadiet al. (135) tested four methanol-utilizing yeastsfor xylitol production from D-xylose. H.polymorpha was found to be the better strainout of 4 strains tested with 43.2 g.l-1 xylitolproduction from 100 g.l-1 D-xylose after 4 daysof cultivation. Whereas, Yablochkova et al (155)tested 13 strains and noticed only 6 strainsemerged from Candida genus as the best xylitolproducers in the range of 0.50 to 0.65 g.g-1 xylitolproduction. After screening 274 yeasts for xylitolproduction Guo et al. (36) selected 5 strains forfurther production and observed that C.gluilliermondii and C. maltosa were the bestxylitol produces. Recently Sreenivas Rao et al.(126) tested a total of 35 yeasts isolated from thegut of beetles collected from Hyderabad city,India. Twenty of these yeasts utilized xylose as asole carbon source but only 12 of these strainsconverted xylose to xylitol. The authors alsoreported that the ability to convert xylose to xylitolvaried among the isolates and ranged from 0.12to 0.58 g.g-1 xylose. Out of these strains Pichiasp. was the best xylitol producer (0.58 g xylitol.g-

1 from xylose). In another study, Sampaio et al.(107) tested 270 yeast isolates for xylitolproduction using xylose as the sole carbon source.The authors reported that D. hansenii UFV-170was the best isolate with production capacity of5.84 g.l-1 xylitol from 10 g.l-1 xylose after 24 hoursincubation. A report with xylose transport capacityas a screening parameter was reported byGardonyi et al (2003) to isolate xylose-utilisingyeasts.

Molecular characterization of xylitolproducing yeasts

The approach to yeast identification hassignificantly changed in just a few decades dueto the rapid increase in basic biological knowledge,

increased interest in the practical applications andbiodiversity of this important microbiologicalgroup, and technological advances. Thedevelopment of molecular techniques hassignificantly widened the tools available forunderstanding and documenting speciesdesignations and phylogenetic relationships.Analyzing ribosomal DNA (rDNA) is nowstandard in molecular techniques and have madeit possible to construct phylogenetic trees of allknown species, with the capacity to betterunderstand interspecific and intergenericrelationships. As a result, it is a common practiceto deposit the sequences of key molecular regions,such as the 600-nucleotide variable region D1/D2 of LSU (large subunit) (26S) rDNA and theITS1 and ITS2 (internal transcribed sequences)of 18S rRNA, with database servers such asGenebank. From D1/D2 sequence analysis,greater than a 100 species have been assigned tothe genus Pichia and Candida which aredistributed across the Saccharomycetales (59)and there is a specific distinct clade that containsxylose utilizing species (The xylose-fermentingclade). Molecular characterization studies helpto understand the relationship between xyloseutilizing yeasts that fall in this specific clade.Sreenivas Rao et al. (126), reported thatphylogentic analysis helped to characterize thexylitol producing yeasts (Table 4).

The best xylitol producer YS54 based ontheir D1/D2 domain sequence, showed similaritywith Pichia caribbica and this strain is identifiedwithin the xylose utilizing clad in the phylogenetictree. Suh et al. (133) isolated several xyloseutilizing strains and demonstrated that the LSUrDNA sequence data helped to identify the xylosefermenting yeasts and noticed that they also in tospecific xylose fermenting clade. Similar trendwas also observed by Nguyen et al. (83) whoisolated two yeasts which ferment xylose, and



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based on molecular characterization the authorsreported that these strains belong to novel speciesand named as Spathaspora passalidarum gen.sp. nov. and Candida jeffriesii sp. nov.

Construction of recombinant yeasts forxylitol production

Screening of different xylitol producingmicrobial strains confirmed that xylitol productionmetabolic process is mostly associated with yeastin general and particularly with the Candidagenus. Among different species in this genus, C.tropicalis is the best strain for xylitol productiondue to its high xylose uptake rate and xylitolproduction capacity (33, 132) and has application

potential at industry level. In addition, this genushas an advantage, due to the lack of sexual stage(33), for further development of recombinantstrains with high xylitol production potential. Infact, the major genetical differences of Candidaand Saccharomyces genera are that the latterspecies is more tolerant in terms of their xylosefermentation, toxicity and growth tolerance in thepresence of inhibitors of hemicellulosichydrolysates. This has created new horizons todevelop recombinant strains of Saccharomycessp. with Candida sp. XYL1 gene for improvedbiological production of xylitol (82, 151). In orderto make S. cerevisiae an efficient xylose-utilizerfor the production of xylitol, an efficient enzyme

Table 4: Tentative identification of xylitol producing yeasts from insect guts based on D1/D2domain sequence of the 26S rRNA gene (126)

Yeast Accession no. ofisolate D1/D2 domain Identification Isolated from Xylitol yield XR activity

(g-1 of xylose) (U/mg protein)

YS 5 AM159103 Issatchenkia sp. Euetheola sp. 0.14 1.5

YS 6 DQ358865 Candida sp. Nicrophorus sp. 0.30 4.4

YS 21 AM159101 Candida sp. Strategus sp. 0.54 8.0

YS 24 AM159108 Candida sp. Diplotaxis sp. 0.40 6.2

YS 43 AM159105 Candida sp. Calligrapha sp. 0.26 2.6

YS 44 DQ358867 Candida sp. Blepharida sp. 0.48 6.5

YS 47 DQ358868 Candida sp. Copris sp. 0.52 8.0

YS 54 AM159106 Pichia sp. Megalodacne sp. 0.58 9.1

YS 60 AM159102 Clavispora sp. Epicauta sp. 0.12 1.4

YS 27 AM420304 Candida sp. Anoplophora sp. 0.36 5.4

YS 34 AM420306 Candida sp. Pseudomorpha sp. 0.26 2.4

YS 19 AM420305 Candida sp. Calosoma sp. 0.16 1.6

YS 5 AM159103 Issatchenkia sp. Euetheola sp. 0.14 1.5

YS 6 DQ358865 Candida sp. Nicrophorus sp. 0.30 4.4

YS 21 AM159101 Candida sp. Strategus sp. 0.54 8.0

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system for the conversion of xylose to xylitolshould be introduced into the S. cerevisiae.Cloning of XYL1 gene from C. tropicalis to S.cerevisiae improves the latter yeast for utilizationof xylose from hemicellulosic material andconversion of xylose to xylitol (82, 130).

Several investigators have cloned thenecessary genes responsible for xylosemetabolism in S. cereviceae and constructed therecombinant strains for production of xylitol (16,38, 39, 73, 138). In the construction of a xylosemetabolizing S. cerevisiae the gene encoding XRwas cloned from the xylose metabolizing yeastsand transferred to S. cerevisiae. The authors ofthis review observed that these transformantscould not produce xylitol for prolonged periodsdue to an imbalance of the redox potential in thecell (130).

One of the main possible limitations ofutilization the XYL1 gene recombinant strainsduring continuous production of xylitol was thelack of reducing cofactors for the xylose to xylitolcatalyzing enzyme, NADPH. The redox balanceon substrate uptake in the yeast xylosemetabolism has therefore been studied (38, 48).Different co substrates were evaluated, asgenerators of reduced cofactors for xylitolproduction by recombinant S. cerevisiaeexpressing the XYL1 gene, encoding xylosereductase. Glucose, mannose, and fructose, whichare transported with high affinity by the sametransport system as xylose inhibit xyloseconversion rates by 99, 77 and 78 respectively.Competitive inhibition of xylose transport wasindicated and xylitol yields varied widely withdifferent co-substrates (48). Galactose as co-factor generator gave the highest xylitol yield, 5.6times higher than that for glucose. This may beattributed to the observed difference in redoxmetabolism of glucose and galactose andsubsequent enhanced availability of reduced

cofactors for xylose reduction with galactose(130). Granstrom et al. (33) evaluated formateas a co-substrate to increase the intracellularconcentration of NADH and based on the resultsthe authors have hypothesized that excess NADHwould result in higher oxygen and xyloseconsumption and correspondingly increase xylitolproduction by inhibiting xylitol dehydrogenaseenzyme.

In this context, addition of cofactor in thegrowth medium may be a possible solution.Experimental evidence of 25% enhanced XRactivity in galactose supplemented xylose mediafurther supported that cofactor limitation is animportant drawback for enhanced production ofxylitol in recombinant strain studies (130). Similarobservations are also noticed by Granstrom et al.(33) where the authors reported the metabolism(Metabolism Flux Analysis (MFA)) of xylose byC. tropicalis in oxygen-limited chemostatconditions. Furthermore, in vitro enzyme assayindicated that glycolytic and gluconeogeneticenzymes are expressed simultaneously, facilitatingcofactor recycling. Moreover, enhancing the redoximbalance by co feeding of formate increasedxylose and oxygen consumption rates and ethanol,xylitol, glycerol and CO

2 production rates at a

steady state. MFA indicated that fructose 6-phosphate is replenished from the pentosephosphate pathway in sufficient amounts withoutcontribution of the gluconeogenetic pathway (33).Overall, the observed enhanced XR activity ingalactose supplemented xylose medium bytransformant S. cerevisie suggested the cofactoravailability importance for xylose metabolism inrecombinant strain and improved xylitolproduction.

Metabolic pathways for xylose utilization

In 1960, Chiang and Knight found thatthe filamentous fungus Penicillium chrysogenum



19

converted D-Xylose to D-xylulose through atwo-step reduction and oxidation and noticedxylose utilizing enzyme in the bacteria wasdifferent. This finding, as well as some furtherinvestigations (15) led to the conclusion that thetwo-step conversion of D-xylose to D-Xyluloseis specific for yeasts and fungi, whereas inbacteria the same conversion is catalyzed byxylose isomerase in a single step. The detectionof xylose isomerase in the yeasts Rhodotorula(42) and C. boidinii no. 2201 (149) is one of thefew exceptions to this generalization.

In xylitol producing yeasts, xylose is re-duced to xylitol either by NADH- orNADPH-dependent xylose reductase (aldosereductase EC 1.1.1.21). The produced xylitol iseither secreted from the cell or oxidized to xylu-lose by NAD- or NADP-dependent xylitol dehy-drogenase (EC 1.1.1.9). These two reactions areconsidered to be limiting for D-Xylose fermenta-tion and xylitol production. The ratio of xylosereductase and xylitol dehydrogenase in additionto cofactor regenerating system is the major meta-bolic regulator for xylitol production. However,certain strains of yeast are known to utilize xy-lose as a carbon source via the phosphorylationof xylulose to xylulose-5-phosphate which is cata-lyzed by xylulokinase (EC 2.7.1.17) (60, 124). Adetailed study of biochemistry and physiology ofthe yeasts metabolizing xylose was published byHahn-Hagerdal et al. (37). In fact, the conver-sion of D-xylose to xylitol in yeasts cannot beseparated from the conversion of D-Xylose toother metabolic products such as carbon dioxide,ethanol, acetic acid and polysaccharides.

Coenzyme specificityThe first two enzymes, D-xylose

reductase (XR) and xylitol dehydrogenase (XDH),of xylose utilization in xylitol producing microbialstrain, are regulated by the ratio of cellular pools

of NAD(P)H/NAD(P). These two enzymes re-quire pyridine nucleotide cofactors and their speci-ficity which differ with different yeast strains. Itwas reported that XR from, e.g. Candida utiliscan utilise only NADPH (8), the XR fromPachysolen tannophilus CBS4044 and Pichiastipitis can use either NADH or NADPH as acofactor (143, 144). The dual cofactor depen-dence of XR on NADH and NADPH may pre-vent a complete regeneration of NAD+ which isneeded for the XDH reaction (47, 57), and hencexylitol is secreted into the medium. Xylitol mayalso be formed due to the action of unspecificreductases, like GRE3 (139).

Under anaerobic or oxygen-limited con-ditions, the difference in the cofactor requirementsof these enzymes causes a redox imbalance whichinfluences xylitol production in yeasts. In gen-eral xylitol formation is favored underoxygen-limited conditions because of the NADHaccumulation and subsequent inhibition ofNAD-linked xylitol dehydrogenase. Cell growthdepends on some of the above metabolic prod-ucts and it is also necessary that the cofactors beregenerated through different steps in the meta-bolic pathway. Therefore, for obtaining good yieldsof xylitol, the amount of xylose being convertedto xylitol and the amount of xylitol which is avail-able for further metabolism have to be well bal-anced (130).

Process regulatory factors on xylitolproduction

Bioconversion of xylose to xylitol usingmicrobial strains is generally influenced by,nutritional composition (substrate, nitrogen sourceand micro nutrients and their concentrations),culture and process conditions (temperature, pH,aeration, inoculum concentration, immobilizationand reactor conditions) as well as genetic natureof the microorganisms (native isolates, mutantsand recombinant strains).

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Impact of nutritional compositionAmong all nutritional parameters xylose

concentration (51, 107, 108, 125, 132) yeast extract,urea, corn steep liquor, casamino acids,hydrolysate composition play a vital role on cellularmetabolism and subsequent xylitol production. Ingeneral, in the presence of glucose, xyloseutilization was strongly repressed and glucosefollowed by xylose sugar utilization was observed.

Kim and Oh (56) demonstrated achemically defined medium with urea (5 gl-1) asa nitrogen source and various vitaminssupplementation as a substitute for a complexmedium containing yeast extract (10 g l-1) in theproduction of xylitol by C. tropicalis. C.gluilliermondii VTT-C-71006 growth on rarepentoses and their implications for production ofpure xylitol was studied by Granstrom et al. (33)and observed that this yeast strain grew on allthe tested pentoses like L-arabinose, L-ribulose,D-ribose and D-xylose and gave the fastestgrowth. Suryadi et al. (135) working withmethanol-utilizing yeasts reported that H.polymorpha produces 43.2g/l xylitol from 100g/l D-xylose after 4 days of cultivation with 1% (v/v) methanol supplementation and furtheradditions of urea, (NH

4)

2SO

4, and NH

4NO

3

proved to be effective for an increase of xylitolyield this yeast. The effect of different nitrogensources on xylitol production from D-xylose byCandida sp.L-102 was also reported by Lu etal. (67) and maximum xylitol production of 87 %was obtained with urea as the nitrogen source.Yeast extract at a maximum concentration of 10g/l was found to be optimum for xylitol productionby C. tropicalis DSM 7524 and concentrationshigher than 15 g/l blocked the conversion ofD-xylose to xylitol (120).

Increase in concentration of yeast extractfrom 5 and 10g/l increased the biomass productionbut a sharp decrease in xylitol productivity wasidentified for C. gluilliermondii FTI 20037 (121).

Similarly the addition of yeast extract and peptoneto the chemically defined medium enhanced cellgrowth of C. mogii ATCC 18364 but had littleimpact on the yield and specific productivity ofxylitol (122). However, in some yeasts, specialnutrient supplementation improved xylitolproduction. Lee et al. (63) reported that high-biotincontaining medium favored ethanol productionover that of xylitol with P. tannophilus NRRLY-2460, while in C. gluilliermondii FTI 20037,xylitol formation was favored under similarconditions.

Role of temperature and pH on xylitolproduction

In general, the most suitable temperaturefor xylitol production in yeasts is 30°C. However,the xylitol yield was temperature-independentwhen the yeast was cultured in a temperaturerange between 30°C and 37°C but above 37°Cthe xylitol yield decreases sharply (120).Exceptions to this were observed by SreenivasRao et al. (132) where a variation of 3oCinfluenced (27%) on xylitol production in C.tropicalis. No variation in xylitol formation in C.guilliermondii FTI 20037 was noticed intemperature range of 30 and 35°C but decreasedwhen the temperature increased to 40°C (Barbosaet al., 1988). The conversion of D-xylose to xylitolby Candida sp. B-22 was relatively constant overthe temperature range of 35-40°C and furtherincrease in temperatures to 45°C and higher, theconversion was sharply reduced (Cao et al., 1994).This was probably due to loss of the activities ofboth NADPH and NADH-dependent xylosereductase associated with the temperatureincrease (123). Sampaio et al. (108) reported asignificant observation, that xylitol production withD. hansenii UFV-170 was hardly affected eitherat lower (10–20 °C) or higher (40–45 °C)temperatures. Wilkins et al., (2008) reportedhigher xylitol production at above 45°C withthermotolerant yeasts.



The yeasts are generally cultivated at pHvalues between 4 and 6. However, variation alsoreported in literature. For example, C.parapsilosis ATCC 28474 (86) and C.guilliermondii NRC 5578 (75, 86) revealed themaximum growth at pH 6.0 while, Candidamogii ATCC (122) and P. stipitis NRRL Y-7124show optimum at pH 5 and 5.5, respectivelywhereas, pH 4 was optimum for C. tropicalisIFO 0618 (43). In general, the optimum initialpH value for the best xylitol yield in C. boidiniiwas 7.0 (142, 150), whereas under controlledconditions, a pH of 5.5 (142). Batch culture of C.parapsilosis ATCC 28474 (64) showed higherperformance in xylitol production at pH 6 whilefor continuous culture a pH of 4.5 was found tobe effective (26). Variation of pH from 4.5 to 5.5did not show any influence on xylitol productionby isolated C. tropicalis (130, 132). In contrastSilva and Afschar (120) reported that C.tropicalis DSM 7524 was not very sensitive topH and attained a maximum xylitol yield at pH2.5. Increasing the pH from 2.5 to 4.0 led to anincrease in xylitol productivity but a decrease inxylitol yield. Sampaio et al (108) noticed that thepercentage of xylose consumed for xylitolproduction progressively increased with pH anddecreased reaching nearly constant values at pH4.0. This process is associated with both biomassgrowth and catabolic reaction through the TCAcycle.

InoculumConflicting reports are available in the

literature on xylitol production versus innoculumloading. On studying the effect of initial cellconcentration of Candida sp. B-22 on xylitolproduction from D-xylose, Cao et al. (9) reportedthat the rate of xylitol production was linear andthe fermentation time was dramatically reducedover an initial biomass concentration range of 3.8to 26 g.l-1. The authors noticed 210g.l-1 of xylitolwith an initial yeast cell concentration of 26 g.l-1

and using 260 g.l-1 D-xylose indicating a high initial

cell mass concentration is beneficial for xylitolproduction by C. boidinii NRRL Y17213. Inanother study, Vandeska et al., (142) reported adoubled xylitol yield and specific productivity withthe increase of inoculum level from 1.3 to 5.1 g.l-

1 using initial D-xylose concentration of 50 g.l-1.Use of very high inoculum observed to improvethe xylitol formation under nitrogen limitationenvironments. In addition, xylitol formation wassimultaneously influenced by the physiologicalstate of the culture and the concentration ofbiomass (109). However limited variation in xylitolproduction was observed by C. tropicalis withthe use of inoculum concentration in the range of6%-10% (132).

The effect of inoculum size on themicrobial production of xylitol from hemicellulosehydrolysates was also investigated. A high initialcell density did not show any positive effect whenC. guilliermondii FTI 20037 when grown on ricestraw hemicellulose hydrolysate since increasingthe initial cell density from 0.67 g.l-1 to 2.41 g.l-1

decreased biomass formation, xylose utilizationand xylitol accumulation (99). On the contraryD. hansenii NRRL Y-7426 grown on woodhydrolysate produced more xylitol at higher initialcell densities (91). Overall, the relationshipbetween biomass and xylitol production wasobserved to be dependent the microbial strainphysiological growth and metabolic properties.

AerationWith respect to aeration, the oxygen sup-

ply rate is a key parameter for D-xylose metabo-lism in xylitol producing yeasts and determineswhether D-xylose will be fermented or respired.It is very important, therefore, for an effectiveprocess to determine the oxygen flux that willenable balanced utilization of carbon both forgrowth and xylitol production.

Xylitol production by yeasts is alwaysassociated with micro aerobic conditions. Severalauthors reported aeration and agitation effects

21

Prakasham et al


22

on yeast growth and xylitol production (7, 14, 132,152). In general, under strict aerobic andanaerobic conditions, xylitol is not producedextracellularly (115). Kastner et al. (50) reportedthat the growth of the xylitol producing organism,Candida shehatae, is drastically affected whenthe culture was incubated under anaerobicconditions and a step change from aerobic toanaerobic improved product formation. Waltheret al. (152) reported that oxygen limitation andinitial xylose concentration had considerableinfluences on xylitol production by C. tropicalisATCC 96745. Under semi-aerobic conditions, themaximum xylitol yield was 0.62 g.g-1 substrate,while under aerobic conditions, the maximumvolumetric productivity was 0.90 g.l-1.h-1.Granstrom et al. (33) studied the metabolism ofxylose by C. tropicalis in oxygen-limitedchemostat and reported glycolytic andgluconeogenetic enzymes are expressedsimultaneously facilitating substrate cycling basedon an in vitro enzyme assay. The authors wereable to enhance the redox imbalance by co-feeding of formate which increased xylose andoxygen consumption. Santos et al. (112) workingwith immobilized cells of C. guilliermondi onporous spheres reported xylitol production influidized bed reactor using sugarcane bagassehemicellulose hydrolysate and reported amaximum xylitol (17.0 g.l-1) yield with an aerationrate of 75ml/min.

To determine the specific oxygen uptakerate at which C. boidinii NRRL Y-17213 beginsto produce xylitol, Winkelbausen et al. (154)cultivated yeast continuously under oxygen-limitedconditions and noticed that xylitol secretion wastriggered at 0.91 mm.g-1.h-1. No xylitol productionwas observed at specific oxygen uptake ratesabove this value. Upon a shift to lower specificoxygen uptake rates, as expected, xylitol produc-tion rates and yield increased more rapidly thanthose of ethanol. Branco et al. (7) studied the

influence of the aeration on ca alginateimmobilized C. guilliermondii cell concentrationand reported the highest conversion efficiency(41%) using 1.33 vvm aeration rate and 40%immobilized system. Whereas, Roseiro et al.,(103) reported a combinatorial influence ofsubstrate concentration and aeration rate on xylitolformation in yeasts. The authors noticed amaximum xylitol productivity of 2.67 g.l-1 whenthe initial k

La, D-xylose and yeast extract

concentrations were 172, 21 g.l-1 and 452 l.h-1,respectively.

Reports are also noticed in the literatureon relationship between co-factor generation andaeration. The general characteristic of mostxylose-fermenting yeasts is that their xylitoldehydrogenase uses predominantly NAD andvery rarely the NADP cofactor (30, 31, 60, 64).The varying ratio of NADH- to NADPH-linkedD-xylose reductase activity with aerationconditions was first found in P. tannophilus andsimilar variations were observed in the yeasts C.parapsilosis ATCC 28474 (86) and C. boidiniiNRRL Y17213 (141). It has been noticed thatoxygen may lower the ratio of NADH linkedD-Xylose reductase and NAD-linked xylitoldehydrogenase activities and consequentlyminimize xylitol accumulation in D-xylose-fermenting yeasts (123). This was also observedin C. boidinii NRRL Y-17213 (141). The NADH/NAD ratio decreased 2-fold with increasingoxygen availability from 10 - 30 mmol/h.

It is very difficult to compare data fromdifferent studies because oxygenation is mea-sured and reported differently. Yet it is evidentthat yeasts producing xylitol require smallamounts of oxygen that is specific for each yeaststrain. It is observed that D. hansenii has thehighest demand for oxygen compared to otheryeasts (107).



23

Optimization studiesNutritional, physiological, operational,

genetical and metabolic parameters are importantfor the economic xylitol production by microbialstrains at industrial scale. The scientificcommunity has performed elaborate optimizationstudies using several statistical approaches (127).When optimizing the xylitol production rate of C.tropicalis ISO 0618 by employing the Box-Wilsonmethod, Horitsu et al. (43) found that theinteraction between D-xylose concentration andaeration rate is related to cell biomassconcentration. Rodrigues et al. (100) usedresponse-surface methodology for xylitolproduction optimization from sugarcane bagassehydrolysate in a fed-batch process and reportedthe best experimental parameter for achieving amaximum of 0.78g of xylitol per g of xylose byusing C. guilliermondii. Whereas, Carla et al.(11) used a fractional factorial design for selectionof important variables on xylitol biosynthesis fromrice straw hydrolyaste by C. guilliermondii. Theauthors noticed that all four selected factors suchas xylose concentration, inoculum level, agitationspeed and nutrient supplementation have playeda critical role in the xylitol fermentation and themost important factor is initial xyloseconcentration. Genetic algorithms coupling neuralnetwork was used for optimization of six mediumcomponents for xylitol production by C. mogii byBaishan et al. (2) and noticed 0.65g xylitolproduction per g of xylose utilized. In anotherstudy, Sreenivas Rao et al. (132) optimizedincubation temperature, pH, agitation, inoculumsize, corn steep liquor, xylose, yeast extract andKH

2PO

4 requirements for maximum xylitol

production using Taguchi methodology andachieved 78.9% conversion at optimizedenvironment with isolated C. tropicalis.Optimization studies are also reported for fed-batch fermentation based xylitol production by C.tropicalis ATCC 13803 by Kim et al. (53) andnoticed 0.75% xylitol conversion rate per gramof xylose utilization.

Xylitol production by immobilized yeastsAnother way to improve the process

parameters is the use of immobilized cells since itallows obtaining high cell concentration in thereactor, with the increase in the efficiency andproductivity of the process. In addition, the useof immobilized cell systems make possible therecovery of cells for later use in repeated batchoperations. A good performance of animmobilization system depends on immobilizationmatrix properties, procedures employed, reactorconfiguration and bioconversion conditions (93,97). Reports on use of different matrices havebeen evaluated for immobilization of cells and forxylitol production. Carvalho et al. (13) andBranco et al. (7) working with alginateimmobilized C. guilliermondii cells reportedrepeated use of these cells for bioconversion instirred tank reactor with average productivityvalue of 0.43 g/l and 0.21 g/l/h, respectively. Inanother study, Santos et al. (111) observed morethan 70% bioconversion of xylose to xylitol withC. guilliermondii cells immobilized on naturalsugarcane bagasse fibers. The maximum yieldwas 0.73 g of xylitol per gram of xylose consumedwas noticed by Liaw et al. (65) with Candidasubtropicalis immobilized in polyacrylic hydrogelthin films whereas, Cunha et al., (18) reportedincreased productivity with increase in recyclingof polyvinyl alcohol immobilized C.guilliermondii. Silva and Afschar (120)immobilized the cells of C. tropicalis DSM 7524on a porous glass and used them in a fluidizedbed reactor. The authors intended to reuse theimmobilized cells several times by repeating thebatch fermentation with substrate shift. Howeverthe yeast was degenerated after completion ofthe first cultivation and addition of fresh medium.Under continuous conditions, the immobilized cellsof C. guillermondii converted D-xylose intoxylitol with a high productivity of 1.35 g.l-1.h-1.

Co-immobilization of different microbialstrains and their use in xylitol bioconversion

Prakasham et al


24

revealed improved productivity values. Thehighest conversion rate was observed whenbenzene-treated cells were co-immobilized in thephoto-crosslinkable resin prepolymers ENT 2000and 4000 (85). Almost 100% of the D-xylose (4.5g.l-1) was converted into xylitol after 33 h ofincubation when the volume ratio of immobilizedmethanogen to immobilized Candida pelliculosawas 1:2. In the co- immobilized cell system, thedegree of conversion and the conversion rate ofD-xylose were higher than those in the separatelyimmobilized cell system. Co-immobilized cellswere stable for about 2 weeks with approximately35% conversion. Lohmeier-Vogel et al. (66)studied the glucose and D-xylose metabolism inagarose-immobilized C. tropicalis ATCC 32113by nuclear magnetic resonance. NMR studiesshowed that neither glucose nor xylose metabo-lism was enhanced by use of an immobilizationprocess. Attempts to improve the rate of D-xylosemetabolism by increasing the oxygen delivery tothe entrapped cells were not successful.

Bioreactor process strategiesMost of the xylitol bioconversions by

employing the microbial strains are associated withbatch culture methods either at flasks or lab batchstirred tank reactors with the use of free orimmobilized cell systems and pure xylose or xylosecontaining hydrolysates (3, 31, 75, 79, 86, 87, 103,126, 129, 132, 142, 150, 153) with productivityvalues ranging from 0.55 to 0.78 gram substrateper gram xylitol. Application potential of thesebatch processes at industrial scale is timeconsuming as batch processes are associated withpreparatory activities such as regular inoculumdevelopment, sterilization of the reactor, etcinvolving considerable input of labour, energy andtime leading to decreased productivity. Effortshave been made to improve the productvolumetric productivity values using differentreactor configurations and varying the processparameters. In this context, continuous culture

techniques often provide better productivities andyields. Santos et al. (111) working on thedevelopment of a bioprocess for the continuousproduction of xylitol from hemicellulosichydrolysate using C. guilliermondii immobilizedcells reported 70% xylose to xylitol bioconversion.Similar xylitol productivity values with C.guilliermondii FTI20037 under continuousfermentation using sugarcane bagasse hydrolysatehave been reported by Martinez et al. (72)however, the authors noticed little impact of k

La

on volumetric productivity which is interestingphenomenon in xylitol production process. Fariaet al. (23) evaluated the role of membranebioreactor in a view to achieve the simultaneousseparation of xylitol during continuousbioconversion process and noticed the bestperformance (86% conversion) with 0.2 µm porediameter containing membrane at a dilution rateof 0.03 per hour. An improvement of 30% onxylitol production/conversion under continuouscultivation of D. hansenii was observed with thesupplementation of small amounts of glucose andat lower aeration environments (137). In factproduction of xylitol from hemicellulosichydrolysate may be more effective with the useof mixed culture as in continuous process andprocess efficiency depends on removal of othermonosaccharides from the hydrolysate by the co-microbial culture (21). In most of the continuousreactor configurations, a substantial improvementin productivity values can be achieved only byusing low dilution rates of xylose with highresidence time, which is very difficult to achievein practice for bulk production.

Research has therefore focused on xyli-tol production by fed-batch mode where substrateconcentration can be maintained at a suitable levelthroughout the course of fermentation, i.e., a levelsufficient to induce xylitol formation but not toinhibit microbial growth. In addition, these pro-cesses generally operate with high initial cell den-sity which normally leads to an increase in volu-



25

metric productivity. The yeast C. boidinii NRRLY17213 gave better results when cultivated in afed-batch fermentor compared to other ways ofcultivation. The highest xylitol yield was 75% ofthe theoretical yield, compared to 53% in the batchculture. The productivity of 0.46 g.l-1. h-1 wastwice as high as the highest obtained under batchconditions (140). Olofsson et al. (89) reportedxylitol production yield of 0.67% under fed-batchcondition using recombinant S. cerevisiae strainwith wheat straw hydrolysate. Whereas, Oh etal. (87) working with glucose-limited fed-batchcultivation of recombinant yeast strain observedan 1.9 fold increase in specific xylitol productiv-ity over a control strain containing only xylosereductase enzyme. In order to improve the volu-metric productivity and to overcome loss of xyli-tol producing biocatalysts in repeated fed-batchreactors, cell recycle attachment with hollow fi-ber membrane was employed and 3.8-fold in-creases were observed compared with the cor-responding values of batch-type xylitol produc-tion parameters (1). Xylitol productions by otherreactor configurations are also reported in the lit-erature. Branco et al. (7) reported only 41%conversion of xylose to xylitol in bubble columnbioreactor using immobilized C. guilliermondiiand sugarcane bagasse hydrolysate. More than70% xylitol yield was reported with the use ofsemi-continuous process in stirred tank reactorby alginate immobilized yeast cells (13).

Future prospects and conclusionsXylitol is gaining the commercial

importance due to its application potential in healthand pharmaceutical sectors. Xylose is the rawsubstrate used for xylitol production either bychemical hydrogenation or by bioconversion withcertain microbial species. Chemical productionof xylitol is cost-intensive, energy consumingprocess and production economics depend onpurity of the xylose and the main source of xyloseis xylan from hemicellulosic biomass.

Hemicellulosic xylan can be converted to xyloseeither by chemical or enzymatic hydrolysis whichis depend on the parameters related to biomass,hydrolysis and enzyme. Chemical hydrolysis ofbiomass produces microbial growth inhibitors andneeds detoxification. Detoxification ofhydrolysate can be performed by physical,chemical and biological methods. However, themajor challenge is for economic pretreatmenttechnology with energy efficiency, in addition tooptimum convertibility associated with reducedformation of degradation products. Developmentof species specific hydrolyzing enzymes wouldoffer selective hydrolysis of xylan from renewablebiomass as well as eliminate or reduce theinhibitory effects of some hydrolysates and xyloseutilization in presence of other monomeric sugars.However, combination of all these detoxificationmethods is most suitable and cost effectiveapproach but adaptation is necessary accordingto the microbial metabolic pathways. Manyscientific groups have screened for xyloseutilizers and noticed that Candida genus is thebest for xylitol production. Molecularcharacterization of xylose utilizing yeast strainsrevealed the presence of a xylose utilizing cladein the phylogenetic tree. Xylitol production byany microbial strains is related to the balance ofxylose reductase and xylitol dehydrogenase.Xylitol production depends on the nutritional,fermentation and physiological growth factorsassociated with micro-aerophillic conditions.Several studies have investigated the optimizationof xylitol production using free or immobilizedcells in batch or in continuous fermentationconditions using different reactor configurations.Considering the limitation of microbial conversionof xylose to xylitol, especially with the use of thenecessary high dilution rates and residence time,it is important to focus on the development ofxylose reductase dependent enzymatic biocon-version of xylose from hemicellulosic hydrolysate.The development of an independent microbial

Prakasham et al


26

metabolic cofactor regeneration system needsspecial attention. One of the other alternatives isto develop robust microbial systems by cloningthe xylose reductase gene by recombination alongwith reduced cofactor generation system,however this has not been successful due to lackof continuous cofactor regeneration system. Useof co-substrates such as galactose for cofactorregeneration increased xylitol productionindicating the need for further understanding andexploitation of this approach at the genetic levelfor successfull development of recombinantstarins. Screening and development of robust andnovel microbial strains with hydrolysate inhibitortolerance play a pivotal role in xylitol productionat the industrial scale. A focus should bemaintained on a common platform ofunderstanding of the hydrolysate material,hydrolysis procedure, microbial performance, bio-conversion environment and downstreamprocessing is one of the most essential aspectsfor development of integrated technologicalsolution for production of second generationbiorefinary products like xylitol via biotechnologi-cal process at an economic industrial scale.

Acknowledgements:Authors of this article acknowledge their

gratitude to Biotechnology and BiologicalSciences Research Council (UK) for BBSRC-India Partnering Award. One of the authors, DrR S Prakasham, is thankful to Department ofBiotechnology and Department of Science andTechnology, Government of India, New Delhi forfinancial support in the form research projectsand DBT Overseas Award.

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