Review
Fungal morphology and metabolite production in
submerged mycelial processes
Maria Papagianni *
Department of Hygiene and Technology of Food of Animal Origin,School of Veterinary Medicine,
Aristotle University of Thessaloniki,54006Thessaloniki,Greece
Accepted 29September 2003
Abstract
The use of fungi for the production of commercial products is ancient,but it has increased rapidly over the last 50years.Fungi are morphologically complex organisms,differing in structure at different times in their life cycle,differing in form between surface and submerged growth,differing also with the nature of the growth medium and physical environment.Many genes and physiological mechanisms are involved in the process of morphogenesis.In submerged culture,a large number of factors contribute to the development of any particular morphological form.Factors affecting morphology include the type and concentration of carbon substrate,levels of nitrogen and phosphate,trace minerals,dissolved oxygen and carbon dioxide,pH and temperature.Physical factors affecting morphology include fermenter geometry,agitation systems,rheology and the culture modes,whether batch,fed-batch or continuous.In many cases,particular morphological forms achieve maximum performance.It is a very difficult task to deduce unequivocal general relationships between process variables,product formation and fungal morphology since too many parameters influence these interrelationships and the role of many of them is still not fully understood.
The use of automatic image analysis systems during the last decade proved an invaluable tool for characterizing complex mycelial morphologies,physiological states and relationships between morphology and productivity.Quantified morphological information can be used to build morphologically structured models of predictive value.The mathematical modeling of the growth and process performance has led to improved design and operation of mycelial fermentations and has improved the ability of scientists to translate laboratory observations into commercial practice.However,it is still necessary to develop improved and new experimental techniques for understanding phenomena such as the mechanisms of mycelial fragmentation and non-destructive measurement of concentration profiles in mycelial aggregates.This would allow the establishment of
0734-9750/$-see front matter D 2003Elsevier Inc.All rights reserved.doi:10.1016/j.biotechadv.2003.09.005
*Fax:+30-2310-999829.
E-mail address:mp2000@vet.auth.gr (M.Papagianni).
https://www.doczj.com/doc/4317190152.html,/locate/biotechadv
Biotechnology Advances 22(2004)189
–259
a process control on a physiological basis.This review is focused on the factors influencing the fungal morphology and metabolite production in submerged culture.
D 2003Elsevier Inc.All rights reserved.
Keywords:Filamentous fungi;Morphology;Shear effects
1.Introduction
The use of fungi for the production of commercially important products has increased rapidly over the past half century.The exploitation of fungi by man is not a recent phenomenon.Numerous examples which indicate that man has been aware of the value of fungi since the dawn of civilization are known.The fermentation of alcoholic beverages,practiced in the days of the Pharaohs,is one of the earliest known examples of the exploitation of the biochemical activities of a fungus by humans.The use of yeast to leaven bread also dates back to biblical times.The higher fungi have long been used by man.Fruit bodies of basidiomyces and ascomyces have been collected and eaten by civilizations throughout the world (Hayes and Nair,1978).The production of alcoholic beverages,biomass and the manufacture of therapeutic compounds,together with the production of simple organic compounds,still remain the major fields in which fungi are used.
Apart from the rather unsophisticated techniques used for the production and mainte-nance of yeast in the brewing and baking industries,the deliberate growth of fungi for commercial purposes did not commence until well into the twentieth century.The development of the sulfite process for the production of glycerol by a yeast fermentation,which was widely used during World War I,probably marks the beginning of industrial mycology.However,it is since the advent of the submerged culture techniques used in the penicillin fermentation that the greatest expansion in the use of fungi in the industry has taken place.At present,increasing numbers of commercially important products are being produced from fungi.Tables 1–3present the major classes of filamentous fungal enzymes,antibiotics and organic acids of commercial importance and some of their sources.For extensive lists of industrial products and the preferred names of the filamentous fungi that produce them,the reader is referred to the ‘‘ATCC names of industrial fungi’’(Jong et al.,1994).
Filamentous fungi are morphologically complex microorganisms,exhibiting different structural forms throughout their life cycles.The basic vegetative structure of growth consists of a tubular filament known as hypha that originates from the germination of a single reproductive spore.As the hypha continues to grow,it frequently branches repeatedly to form a mass of hyphal filaments referred to as mycelium.When grown in submerged culture,these fungi exhibit different morphological forms,ranging from dispersed mycelial filaments to densely interwoven mycelial masses referred to as pellets.The particular form exhibited is determined not only by the genetic material of the fungal species but also the nature of the inoculum as well as the chemical (medium constituents)and physical (temperature,pH,mechanical forces)culturing conditions (Atkinson and Daoud,1976;Kossen,2000).With some fungal fermentations a particular morphological
M.Papagianni /Biotechnology Advances 22(2004)189–259
190
M.Papagianni/Biotechnology Advances22(2004)189–259191 Table1
Major classes of filamentous fungal enzymes of commercial importance and some of their sources
Enzyme Sources
a-Amylase As.niger,As.oryzae,As.awamori,Au.pullulans,Rhizopus oryzae,
Trichoderma viride
h-Amylase R.niveus
Amyloglucosidase As.niger,As.oryzae,As.awamori,A.phoenicis,Au.pullulans,R.niveus,
Rhizop.oryzae
Catalase As.niger,Eupenicillium javanicum,Penicillium vitale
Cellulase As.niger,A.soyae,As.terreus,P.citrinum,P.funiculosum,
T.longibrachiatum,T.reesei,Tr.viride
Dextranase A.carneus,Chaetomium gracile,P.funiculosum,P.lilacium,P.pinophilum
a-Galactosidase As.awamori,As.niger,As.oryzae,Mortierella vinaceae,P.dupontii
h-Galactosidase As.awamori,As.nidulans,As.niger,As.oryzae,Fusarium oxysporum,
N.crassa,P.funiculosum
h-Glucanase Acremonium persicinum,As.niger,As.oryzae,E.javanicum,G.candidum,
Tr.viride,T.reesei
Glucoamylase As.niger,As.awamori,As.oryzae,Au.pullulans,Rhizop.oryzae,R.niveus Glucose aerohydrogenase As.niger
Glucose oxidase As.niger,E.javanicum,P.amagasakiense,P.simplicissimum,P.vermiculatum a-Glucosidase As.awamori,Aspergillus flavus,A.fumigatus,As.niger,Mu.circinelloides
a-D-Glucosidase As.niger
h-Glucosidase As.niger,As.oryzae,T.reesei
Hemicellulase As.niger,As.oryzae,A.phoenicis,T.longibrachiatum,Tr.viride Hesperidinase As.niger
Invertase As.awamori,As.niger,As.oryzae,N.crassa
Lipase As.niger,As.oryzae,G.candidum,Humicola sp.,Rhizomucor miechei,
Rhizomucor sp.,R.arrhizus,R.niveus,P.roqueforti
Pectinase A.alliaceus,As.niger,Aspergillus sp.,Rhizop.oryzae,Rhizopus sp.,
Sclerotinia libertiana
Phytase As.niger,A.ficuum
Protease As.niger,A.melleus,As.oryzae,A.saitoi,P.dupontii,Penicillium sp.,
Rhizopus sp.
Rennets Cryphonectria parasitica,R.meihei,R.pusillus
Tanase As.niger,A.tamarii
Xylanase T.reesei
form may be preferred to achieve maximal performance.Filamentous growth of Asper-gillus niger is preferred for pectic enzyme production,whereas the pelleted form is preferred for citric acid production(Steel et al.,1954;Kristiansen and Bullock,1988). Table2
Major classes of filamentous fungal antibiotics of commercial importance and some of their sources Antibiotics Sources
Cephalosporins Acremonium chrysogenum,A.kiliense,Ac.persicinum,Fusarium solani,Nectria lucida Cyclosporins F.solani,Tolypocladium geodes,T.inflatum,Trichoderma polysporum Echinocandin B As.nidulans,A.rugulosus
Fusidic acid Calcarisporium arbuscula,F.coccophilum,Mortiella ramannianua
Griseofulvin Penicillium aurantiogriseum,P.griseofulvum,P.italicum
Penicillins Ac.chrysogenum,Ac.persicinum,As.flavus,A.giganteus,As.nidulans,As.niger,
As.oryzae,A.parasiticus,Aspergillus sp.,P.baculatum,Pe.chrysogenum,P.turbatum
Also,Konig et al.(1982)showed that the pelleted form of Penicillium chrysogenum was desired for production of penicillin in a tower bioreactor.
The change in morphology during growth affects nutrient consumption and oxygen uptake rate in submerged culture (Schu ¨gerl et al.,1983).Also,the morphological growth forms can have a significant effect on the rheology of the fermentation broth and thus the performance of the bioreactor.Filamentous growth results in highly viscous broths with non-Newtonian,pseudoplastic flow behavior (Kristiansen and Bullock,1988).The high viscosity has a negative impact on the mass transfer properties of the broth,specially the gas–liquid mass transfer rate.Pelleted growth exhibits low viscosities and approach Newtonian flow behavior (Chain et al.,1966).As expected,higher power inputs are required for filamentous versus pelleted growth in achieving adequate agitation and oxygen transfer.However,frequently,the central region of larger pellets undergoes autolysis as a result of nutrient limitation.This autolysis can have a significant effect on both cellular metabolism and product synthesis (Philips,1966;Elmayergi et al.,1973).Thus,small pellets as opposed to large ones would generally be considered desirable in developing filamentous fungal fermentations.
The successful production of a fungal metabolite requires a detailed knowledge of the growth characteristics and the physiology of the fungus in question.Not only does the production of different metabolites require different physiological conditions but also each fungus is unique in its anatomical,morphological and physiological development.Thus,for each fermentation,the precise physiological conditions and the correct stage of development must be established for maximal product formation.In other words,the control of the form of these microorganisms is a real issue that needs great attention in order to make optimal use of their potential production capacities.Many scientists have been studying this problem from an engineering point of view for more than five decades,while the contribution from physiologists is growing steadily as more and more details of the transport processes and the kinetics involved in the morphogenesis become known.The outcome is an impressive landscape of results about fungal morphology and metabolite overproduction and this extensive review is about this landscape.It is a survey
Table 3
Major classes of filamentous fungal organic acids of commercial importance and some of their sources Organic acid
Sources Citric acid
Aspergillus citricus ,A.clavatus ,As.niger ,A.phoenicis ,Penicillum decumbens ,P .isariiforme ,Tr.viride Fumaric acid
Rhizop.oryzae ,Rhizop.stolonifer Gluconic acid
A.carbonarius ,As.niger ,As.oryzae ,A.wentii ,E.javanicum ,Pe.chrysogenum ,P .luteum ,P .simplicissimum Itaconic acid
A.itaconicus ,As.terreus Kojic acid
A.candidus ,As.flavus ,As.oryzae ,A.parasiticus ,A.tamarii ,P .jensenii ,R.microsporus ,Rhizop.oryzae ,Rhizop.stolonifer D -Lactic
acid Rhizop.oryzae L -Malic acid A.atroviolaceus ,As.citricus ,As.flavus ,As.niger ,A.ochraceus ,As.oryzae ,
A.wentii ,Rhizop.oryzae
D -Araboascorbic
acid Pe.chrysogenum
Erythorbic acid
P .griseoroseum M.Papagianni /Biotechnology Advances 22(2004)189–259
192
M.Papagianni/Biotechnology Advances22(2004)189–259193 of all the main lines of development of a very interesting area of biotechnology research: growth mechanisms,dynamics of mycelial aggregation,transport phenomena,fragmen-tation,growth in submerged culture and process parameters,quantification of morphology and modeling of the growth and product formation.
2.Growth mechanisms in filamentous fungi
Under appropriate conditions,the vegetative mycelium gives rise to a reproductive mycelium that supports the production of reproductive spores.The type of sporulation and the morphology of the spores and spore-bearing structures are key characteristics in fungus identification.The fungal spore,therefore,can be considered as the beginning and the end of the differentiation process.In mycelial fungi,hyphae extend by a highly polarized process of cell extension known as tip extension.As the tip extends,periodic branches are formed at or near the apex of the tip.These branches also extend in a polarized manner as new tips.The two processes of tip extension and branching permit the organism to colonize and efficiently utilize the substrate,and they are rarely found in organisms other than fungi,leading to their being termed hallmarks of the fungal kingdom(Heath,1995).
2.1.Hyphal tip extension
Spore germination results in the formation of a germ tube,whose early growth is supported by mobilization and utilization of storage compounds in the spore.As the germ tube develops,it contributes to biosynthesis and extension by uptake and metabolism of nutrients from the medium.Hyphal extension is an extreme example of polarized cell growth since cell extension is restricted to a narrow zone defined by the tapering hyphal apex.The rate of wall synthesis in the apical1A m of a hypha may be50times that50A m behind it(Gooday,1971;Gooday and Trinci,1980).Although this apical growth pattern has been known for more than a century(Reinhardt,1892),the underlying mechanisms that account for polarized hyphal growth are not yet understood.Extension rate accelerates as germ tube length increases and growth becomes autocatalytic.
Tip growth is a process that has many similarities in diverse walled cells such as hyphae,pollen tubes and hairs.Much research has focused on the mechanism of tip extension.Analysis of the literature on fungi,with selected comparison with other tip-growing plant cells,shows that the growth rate and morphology of hyphae are sensitive to factors,which influence intracellular Ca2+.These factors include variations in extracel-lular Ca2+concentrations,Ca2+ionophores,inhibitors of Ca2+transport and buffers introduced into the cytoplasm(Jackson and Heath,1993;Parton et al.,1997).The effects of these agents appear to be mediated by a tip-high gradient of cytoplasmic free Ca2+, which is obligatorily present and involved in active growth.Most recent observations agree that the gradient is very steep,declining rapidly within10–20A m of the tip(Jackson and Heath,1993).This gradient seems to be generated by the combined effects of an influx of Ca2+,via plasma membrane,possibly stretch-activated channels localized in the hyphal tip,and subapical expulsion or sequestration of these ions.It is suggested that the regulation of the Ca2+gradient,in turn,modulates the properties of the actin-based
component of the cytoskeleton,which then controls the extensibility,and,possibly the synthesis of the hyphal apex (Heath and Geitmann,2000).
Mitosis,septation and branching have been studied in undifferentiated mycelia and main hypha of Aspergillus nidulans ,which forms incomplete septa (Fiddy and Trinci,1976).Following spore germination,nuclei divide synchronously until germ tube hyphae contain either 8or 16nuclei.Mitosis occurs when the volume of cytoplasm per nucleus is about 60A m 3.Intercompartment development will not synchronize and mitosis in the mycelium as a whole eventually becomes asynchronous.During the stage of asynchronous compartment development,the nuclei,septa,branches and total length of undifferentiated mycelia are increased exponentially at approximately the same specific rate (Fiddy and Trinci,1976).The mean time required for the formation of a group of septa is reported to be about 9min.Each hypha forms up to nine septa in a group at a time.The mean interval between successive cycles of septation in a hypha is approximately the same as the doubling time of the organism.A very high correlation coefficient was reported between septation,branch initiation and most intercalary compartments initially formed a single branch (Fiddy and Trinci,1976).
Synchronous mitosis has also been observed during the early stages of growth of As.nidulans from spores (Rosenberger and Kessel,1967)and in apical compartments of main hypha of Alternaria solanis (King and Alexander,1969)and As.nidulans (Clutterbuck,1970).In both species,mitosis in main hyphae is followed after a small interval by septation.Such observations suggest the existence of a duplication cycle in apical compartments which is analogous to the cell cycle of uninucleate cells.The main events of this duplication cycle are the following:(1)reduction of the apical compartment to almost half of its length by septation;(2)the newly formed apical compartment continues to increase in length at a linear rate;(3)the volume of cytoplasm per nucleus increases up to a critical ratio when the nuclei are induced to divide more or less synchronously;and (4)mitosis followed by septation,which is completed when the apical compartment is about twice its original length (Fiddy and Trinci,1976).
Consequently,hyphal length increases exponentially at a constant specific rate which may be significantly greater than the maximum specific growth rate in the equivalent liquid medium because of contribution from endogenous spore reserves.Exponential growth cannot proceed indefinitely and extension rate eventually reaches a constant value,i.e.,extension is linear.This occurs when the tip can no longer incorporate the increasing amount of material being supplied or,more likely,when transport of material from regions distant from the tip is limited.The latter may result from a breakdown in apical polarity,such that transport occurs at a rate less than the hyphal extension rate (Prosser,1995).A more common explanation is the formation of septa which prevent transport of material to the tip.Extension rate will then be dependent on biosynthesis within the apical compartment only (Prosser,1995).
Both hyphal extension and the length of hypha supporting tip growth vary considerably within fungi.For example,linear growth occurs in Rhizopus stolonifer when the germ tube is only 40A m in length,whereas exponential growth of sporangiophores of Phycomyces blakesleeanus continues until they are 4mm in length (Trinci,1969).Hyphal extension rate will depend on the amount of material supplied to the tip and on the surface area of the extension zone,which will increase with hyphal diameter and with increased tapering of
M.Papagianni /Biotechnology Advances 22(2004)189–259
194
M.Papagianni/Biotechnology Advances22(2004)189–259195
the tip.Little work has been done on the relationship between tip shape and extension rate, but the extension rate is generally found to increase with hyphal diameter within a single organism.The relationship is not always well defined.Zhu and Gooday(1992)found a direct relationship between hyphal extension rate and the square of the diameter for hypha of Botrytis cinerea,suggesting extension rate to be dependent on the rate of supply of material to the tip.In Mucor rouxii,however,although extension rate increased with diameter,no precise quantitative relationship could be identified(Zhu and Gooday,1992).
2.2.Branching
2.2.1.Exponential growth
Microbial growth is normally associated with exponential increases in biomass when conditions are favorable for growth and when nutrients are in excess.Exponential growth requires that all,or a constant percentage of the mass of the microorganisms present, contribute to new growth.If all growth takes place in the apical segment of the hyphae and the individual hypha extend at a constant linear rate,then exponential growth will require that new branches are produced at a rate proportional to the rate of increase in cell mass. The first branch is usually formed from the germ tube towards the end of the period of exponential extension(Prosser,1995).This rarely affects the extension rate of the parent hypha,even though early growth of the branch is supported by the material provided by the parent hypha.Branch length increases at an accelerating rate before reaching a constant value,which,in young mycelia,is equal to that of the parent hypha.In terms of growth kinetics,branch formation may be considered equivalent to cell division in unicellular organisms.
It has been reported that the frequency of branching in As.nidulans was proportional to the specific growth rate when growth on different media was compared(Katz et al.,1972). Individual hyphae may also grow exponentially rather than linearly in certain circum-stances.Exponential growth has been reported during germ tube outgrowth in As.nidulans and also in the critical period of growth after branch formation.Fungal growth continues at a rate proportional to the length of the hypha until it reaches the maximum characteristic of the organism(Katz et al.,1972).The rate of apical growth can be considered to depend upon the biosynthetic capacity of the hypha.When it exceeds the capacity of the apical region to utilize the products of biosynthesis a new branch is initiated.Exponential growth therefore occurs through an exponential increase in the number of branches,each of which extends at the same constant rate.This was first demonstrated experimentally for Geo-trichum candidum,Neurospora crassa,Pe.chrysogenum and As.nidulans by Trinci (1974).This work also demonstrated that the specific rates of increase in total mycelial length and the total number of branches were equal to the specific growth rate of these organisms growing in equivalent media but in liquid culture,with biomass concentrations determined by the dry weight measurements.
A linear rate of extension imposes a restriction to overall growth,which in unicellular organisms proceeds at an exponential rate,and this is solved by branch formation:Smith (1924)observed an exponential increase in the combined lengths of parent and branch hyphae,though individual hyphae were extending linearly,and Plomley(1959)observed an exponential increase in total mycelial length in Chaetomium globosum.
Trinci (1974)studied early colony growth kinetics of As.nidulans ,N.crassa ,Mucor hiemalis and G.candidum on agar surface and found that all four species showed similar kinetics.Four features are exhibited:(1)total mycelial length increases exponentially for at least 11h;(2)the first branch is formed before the germ tube hypha is grown linearly,though possibly not before the deceleration phase;(3)branch production is initially discontinuous,but after 10branches have been formed the number increases exponentially with a specific rate equal to that for the total mycelial length;and (4)this specific rate is found to be equal to the specific growth rate during exponential growth in liquid medium,measured in terms of dry-weight increase.The results in that study (Trinci,1974)supported the hypothesis that mycelial growth involves the duplication of a ‘‘growth unit’’containing a tip and a certain mean length of hyphae.Plomley (1959)first suggested that filamentous fungi have a growth unit,which is duplicated at a constant rate (linear growth)while the whole mycelium grows exponentially.
Caldwell and Trinci (1973)studied the hyphal growth unit (HGU)of G.candidum grown in batch culture.The mycelium grew in the filamentous form while hyphal fragmentation occurred during growth.During the early part of the stationary phase,the hyphal fragments had a mean length of 300–400A m.The dry weight,total hyphal length,number of tips and the turbidity of the culture increased exponentially in a similar trend to the specific growth rate.The results suggested a functional unit of growth consisting of a hyphal tip associated with a constant mean length of hyphae.The hyphal growth unit,G ,was defined as the ratio of total hyphal length to the total number of branches and is therefore the average length of hypha associated with a growing tip.For G.candidum ,this unit was about 100A m and remained constant,while the specific growth rate varied by changes in temperature and the source of carbon (Steele and Trinci,1975).This unit therefore represents the mean length of the hypha required to support tip growth,while the peripheral growth zone represents the maximum length.Whereas the peripheral growth zone and the specific growth rate are related by the maximum rate of extension,the hyphal growth unit (G )is related to the specific growth rate by the mean extension rate (Steele and Trinci,1975).This is calculated as:
E ?2eH t àH 0T
B 0tB t e1T
where E is the mean extension rate,H 0and H t represent the total hyphal lengths 0and 1h later,and B 0and B t represent the respective numbers of hyphal tips.This is related to the specific growth rate by the equation:
E ?G l e2TValues of G increase during mycelial development and then oscillate until branches are formed continuously,when G reaches a constant value.Thus,whereas the specific growth rate provides an indication of the kinetics of branch formation,the hyphal growth unit provides information on the branching density,increasing as branching becomes sparser (Bull and Trinci,1977).The hyphal growth unit is a property of the mycelium and its mathematical properties and its relationship to other hyphal and mycelial growth parameters have been extensively analyzed by Kotov and Reshetnikov (1990).The
M.Papagianni /Biotechnology Advances 22(2004)189–259
196
M.Papagianni/Biotechnology Advances22(2004)189–259197 relative constancy of the hyphal growth unit length indicates the existence of a regulatory mechanism,in that a branch is formed somewhere in the mycelium when the value of G, characteristic of the organism and growth conditions,is exceeded.
The above discussion refers to hyphae in young developing mycelia.As a colony forms,the kinetics of hyphal growth and branching at the center of the colony will be influenced by reduction in concentrations of nutrients and oxygen,accumulation of inhibitory end products,production of secondary metabolites and changes in environ-mental factors such as pH(Prosser,1995).The relative importance of these factors on biomass formation in developing mycelia is unknown,but they will obviously lead to a decrease in growth rate,with associated effects on branch formation and the morphology of freely dispersed hyphal elements.
Trinci(1971)has related the rate at which fungal colonies grew on agar to their growth rate in submerged culture by using the concept of a peripheral annulus in which the mycelium grows exponentially.By measuring the width of the peripheral growth zone(w) and the linear colony radial growth rate(K r),Trinci used the equation:
d r
?l w?K re3T
d t
to calculate the specific growth rate(l)for nine species of fungi.He found that the computed value was equal to the specific growth rate measured in submerged culture.The colony radial growth rate would also be affected by the hyphal density or branching frequency,which may be a determinant of the width of growth zone.Bainbridge and Trinci (1971)noted that a mutant of As.nidulans was more branched than its parent organism under the same conditions.It had an almost identical specific growth rate in submerged culture but a substantially lower colony radial growth rate and a smaller peripheral growth zone when grown on agar.
Eq.(3),used by Trinci(1971)and Pirt(1967)to express the linear colony radial growth can be re-written as:
d r
?l bke4T
d t
where b is the hyphal density and k a constant.Hyphal density can be directly related to branching frequency if the internode length is used or the total hyphal length divided by the number of growing tips.Morisson and Righelato(1974)have used Eq.(4)to relate the measurements of the specific growth rate and hyphal branching in submerged culture to colony radial growth rate on agar.Their results suggest that the width of the peripheral growth zone of colonies growing on agar could change as the specific growth rate changes.
Recently,the use of image analysis in the study of Christiansen et al.(1999)permitted an on-line determination of the growth kinetics of the single hyphae of Aspergillus oryzae in a flow-through cell at different glucose concentrations.The tip extension rate of the individual hyphae were described with saturation type kinetics with respect to the length of the hyphae.It was observed that the maximum tip extension rate was constant for all hyphae measured at the same glucose concentration,whereas the saturation constant for the hyphae varied significantly between the hyphae even within the same hyphal element.
The tip extension rate decreased temporarily when apical branching occurred.The number of branches formed on a hypha was proportional to the length of the hypha that exceeds a certain minimum length required to support the growth of a new branch.
Attempts to understand tip growth and branching have employed various approaches.Cytological analysis has identified several key substances involved in the process,most notably actin and calcium (Heath,1995;Grinberg and Heath,1997;Hyde and Heath,1997;Jackson and Heath,1993;Kaminsky and Heath,1996).Ultrastructural studies have demonstrated the importance of tip-growth vesicles (Bartnicki-Garcia,1990;Bartnicki-Garcia et al.,1989;Prosser and Trinci,1979;Trinci,1969).Genetic analysis of induced and naturally occurring mutants has identified over 100loci that encode products that can affect tip growth and branching in N.crassa (Perkins et al.,1982;Scott,1976).Tip extension proceeds via the polarized exocytosis of tip-growth vesicles (Bartnicki-Garcia,1990;Bartnicki-Garcia et al.,1989;Prosser and Trinci,1979;Scott,1976).Vesicle deposition appears to be orchestrated by the Spitzenko ¨rper,a loose collection of vesicles near the hyphal apex (Grove and Bracker,1970;Howard,1981).
2.2.2.Modeling tip growth and branching
Any comprehensive model for tip growth and branching must incorporate the main phenomenology associated with these processes.Tip extension occurs via apical exocy-tosis of tip-growth vesicles manufactured subapically and transported to the tip.Thus,the tip concentration of vesicles and any other tip extension factors depends on the balance between the rates of supply (synthesis and transport)and consumption (either deposition or destruction).Branching,which is triggered by the rate of accumulation at the tip,is proportional to the excess of vesicle production over tip deposition.Branching has been shown to be at least partially controlled by factors at or proximal to the previous branch point (Watters et al.,2000a).
The vesicular basis of hyphal growth and branching was incorporated into a model by Trinci (1974).A key element of this model was the hyphal growth unit.The initiation of a new branch has been proposed to be controlled by changes in the cytoplasmic volume,so that branching occurs when a critical value of the mean hyphal growth unit is attained.In this way,the protoplasm considerably distant from the growing tip could have a contributing role in branch initiation.In a further elaboration of this model,Prosser and Trinci (1979)proposed that the concentrations of vesicles and nuclei regulate the increase in hyphal length and the occurrence of branches and septa.Prosser (1979)has also developed a model for hyphal growth and branching,which relates cytological events to growth kinetics.The model quantifies qualitative theories of hyphal growth that vesicles containing wall precursors and/or enzymes required for wall synthesis are generated at a constant rate throughout the mycelium and travel to the tips where they fuse with the plasma membrane.This is followed by the liberation of their contents into the wall and increasing of the surface area of the hyphae to give elongation.The hypothesis states that there is a duplication cycle in the hyphae,which is equivalent to the cell cycle observed in unicellular organisms.
Watters et al.(2000b)showed that in N.crassa the distribution of branch intervals is independent of tip extension rate,as controlled by temperature.Although rapid cooling disturbs this distribution,the normal default distribution of branch intervals was soon
M.Papagianni /Biotechnology Advances 22(2004)189–259
198
restored at the new temperature.Thus,the statistical distribution of branch-to-branch intervals along a hypha seems to constitute a homeostatic set point.The lack of dependence of branch distribution on temperature(or growth rate)was explained in the work of Watters and Griffiths(2001)who developed and tested a model in which the formation of a lateral branch in N.crassa was determined by the accumulation of tip-growth vesicles caused by the excess of the rate of supply over the rate deposition at the apex.The model explains how branching can be independent of tip extension rate under steady-state conditions while responding dramatically to changing conditions.
Apart from the above described vesicular models,many other different approaches have been adopted in modeling the early growth of filamentous fungi.Hyphal population models,in which the average properties of all hyphae within a mycelium are considered, have been described by Edelstein(1982),and Edelstein and Segal(1983).Stochastic models,which take account of natural variability within a system and the consequences of such variability,have been described by Hutchison et al.(1980).Yang et al.(1992) proposed a model for mycelial growth which combines the model of Prosser and Trinci (1979)with the stochastic approach adopted by Hutchison et al.(1980)to describe the direction of tip growth and branching,and the site of branch formation.
A link between mycelial population models and macroscopic growth was recently provided by the model of Viniegra-Gonzales et al.(1993),which is based on the mathematics of symmetric trees.The model considers the mycelium as a population of interbranch segments of average length L av,defined as:
L av?L t
N s
e5T
where L t is the total hyphal length and N s the number of segments and equals2(N tà1), where N t is the total number of tips.This model predicts that the specific growth unit G will be greater at higher levels of branching.This has been suggested by Caldwell and Trinci(1973),but model expressions provide quantitative relationships which show good agreement with the experimental data on growth of young mycelia of G.candidum.This model is developed further to allow prediction of the specific growth rate,determined in terms of biomass,incorporating a frequency distribution for the proportion of biomass which is inactive.This‘‘macroscopic’’model predicts the observed difference in specific growth rate between germ tubes and exponentially growing mycelia.It is also used to predict growth during batch culture of As.niger and provides a new approach to descriptions of mycelial growth and it is valuable in linking morphological properties to kinetics.Testing of the model will be facilitated with image analysis techniques now used for quantification of mycelial morphology.
Quantified morphological information obtained with on-line image analysis was employed in the model reported by Christiansen et al.(1999)for the early growth of As.oryzae.The kinetics observed in that study were used to simulate the outgrowth of a hyphal element from a single spore using a Monte Carlo simulation technique.The simulation showed that the observed kinetics for the individual hyphae resulted in an experimentally verified growth pattern with exponential growth in both total hyphal length and number of tips.
M.Papagianni/Biotechnology Advances22(2004)189–259199
3.Dynamics of mycelial aggregation
In submerged cultures,many filamentous microorganisms tend to aggregate and grow as pellets the compactness of which varies considerably.Pellets are spherical or ellipsoidal masses of hyphae with variable internal structure,ranging from loosely packed hyphae,forming ‘‘fluffy’’pellets,to tightly packed,compact,dense pellets (Yanagita and Kogane,1963).Wittler et al.(1986)proposed the existence of four regions.The outer region consists of viable hyphae and surrounds a layer of hyphae showing signs of autolysis.In hollow pellets,a third layer is found containing hyphae with irregular wall structure,while the center of the pellet contains no recognizable mycelia.The density of hyphae within pellets is of significance for diffusion of nutrients and oxygen to the mycelial biomass,with consequent effects on growth,particularly at the center of compact pellets.
Control of mycelial morphology in fermentation is often a prerequisite for industrial application.In some processes,free mycelia are required,as in the production of penicillin from Pe.chrysogenum .Whereas in other processes,pellets or immobilized cells are preferred for increased yields,as in the production of citric acid from As.niger (Solomons,1980;Metz,1976).The disadvantages of dispersed mycelial growth have been discussed and include a reduction in efficiency of mixing and oxygen supply as well as increased wall growth.These problems may be solved to some extent by growth in the form of pellets,which also improve harvesting through improved filtration characteristics of the broth.The methodology of pellet formation for several microorganisms has been reviewed (Steel et al.,1954;Whitaker and Long,1973;Brown and Zainudeen,1977;van Suijdam et al.,1980).
Application of mycelial aggregates to metabolite production depends upon obtaining uniform pellets of a desired size.This is not easily accomplished,since many factors influence pellet formation.Among the factors influencing cellular aggregation are inoculum size,type and age (Steel et al.,1954;Calam,1987;Gerlach et al.,1998;Papagianni et al.,1999d,2001;Papagianni and Moo-Young,2002),genetic factors and ability to produce bioflocculants (Prosser and Tough,1991;Braun and Vecht-Lifshitz,1991),medium composition (Charley,1981;Gerlach et al.,1998;Papagianni et al.,1999d;Papagianni,1999),biosynthesis or addition of polymers,surfactants and chelators (Pirt and Callow,1959;Jones et al.,1989;Papagianni,1999),shear forces (van Suijdam and Metz,1981;Braun and Vecht-Lifshitz,1991),temperature and pressure (Smith and Anderson,1973;Bull and Bushell,1976)and medium viscosity (Gerlach et al.,1998;Papagianni et al.,2001).The morphology of a filamentous fungus developing in any fermentation system could be considered as a final result of competing influences,an equilibrium between forces of cohesion and disintegration.Shear forces may be unambiguously assigned the role as disintegrating factors.At pH values above 5.5,cell walls of most microorganisms are negatively charged,tending to cause separation of aggregating cells by electrostatic repulsion.This may be suppressed by an increase in ionic strength,or bridging cells with Ca 2+ions (Braun and Vecht-Lifshitz,1991).Addition of polycations usually induces aggregation,whereas poly-anions suppress it (Elmayergi et al.,1973;Elmayergi,1975;Domingues et al.,2000).The chemico-thermodynamical basis for the effect of growth conditions on the pellet formation of As.niger was investigated by Ryoo and Choi (1999).The surface
M.Papagianni /Biotechnology Advances 22(2004)189–259
200
M.Papagianni/Biotechnology Advances22(2004)189–259201 thermodynamic balance between fungal cell and liquid media was found to be responsible for pellet formation,since the Gibbs free energy of pellet formation of the initial culture media(à73toà81ergs/cm2)were increased toà13toà46ergs/ cm2at48h.FTIR analysis showed that factors inducing pellet formation simultaneously increased the cell wall hydrophobicity of As.niger.
Genetic factors influence the cell wall composition and surface properties and determine the formation and composition of a slime layer.Genetic and environmental factors are responsible for the production of surface-active agents and of lectins.Both of these affect forces of cohesion and/or repulsion between cells.According to the classification of Takahashi et al.(1958)and Takahashi and Yamada(1959),two types of pellet formation may be distinguished.Pellet formation in As.niger belongs to the coagulative type(Yanagita and Kogane,1963),where spores coagulate while germinating and give rise to a net of intertwining hyphae.Pellets of Pe.chrysogenum belong to the non-coagulative type(Metz,1976),where one pellet is produced from one spore.Spore coagulation may play a role in pellet formation,provided that trap-nets develop,although pellets frequently form in the absence of any spore agglutination.Even in As.niger,the number of pellets equals the number of initial spore clumps only at low power input;with increased power input,the spore to pellet ratio tends towards unity in As.niger(Vecht-Lifshitz et al.,1989),while in other pellet-forming organisms,this ratio may reach several orders of magnitude below unity(Elmayergi et al.,1973).
Assessing the factors that influence pellet formation in filamentous fungi,it is often difficult to define a mechanism for pellet formation from reported results,as often more than one parameter is adjusted by changing only one variable.Even for the most studied, industrially important Aspergillus and Penicillium species reports are contradictory (Solomons,1980;Whitaker and Long,1973;Metz et al.,1979;Gomez et al.,1988). Attempts to treat pellet formation as a general phenomenon are frequently met by industrial microbiologists with mistrust.Remarkably,the effect of various fermentation parameters on pellet formation seems to be quite similar in filamentous systems as genetically remote as fungi and actinomycetes.For example,in Pe.chrysogenum(Metz, 1976),As.niger(van Suijdam et al.,1980),Streptomyces tendae(Vecht-Lifshitz et al., 1989)and S.griseous(Braun and Vecht-Lifshitz,1991),pellets are formed at inoculum levels below1011spores mà3,while at higher inocula,filamentous growth predominates. Similarly,factors favoring increased growth rates,such as media rich in easily assimilable nutrients,reduce pellet formation in fungi(Hemmersdorfer et al.,1987)and actinomycetes (Vecht-Lifshitz et al.,1989).Such observations led to a limitation hypothesis which suggested that the lack of any nutrient,including oxygen,induces pellet formation (Hemmersdorfer et al.,1987).Indeed,increased mycelial aggregation was noted as a consequence of nitrogen limitation in many cases(Vecht-Lifshitz et al.,1989;Hemmers-dorfer et al.,1987).There are,however,a few reports contradicting the limitation hypothesis with respect to oxygen.Hockenhull(1980)stressed that pelleted morphologies predominate in the early life of a culture when oxygen supply is sufficient,while older cultures tend to be filamentous.
Formation of mycelial pellets is considered,in some instances,a prerequisite for successful production of certain metabolites,such as the itaconic and citric acids(Metz et al.,1979;Gomez et al.,1988),and some fungal enzymes such as glucose oxidase(Zetelaki
and Vas,1968),polygalacturonidase (Hemmersdorfer et al.,1987),phytase (Papagianni et al.,1999d),and glucoamylase (Papagianni and Moo-Young,2002).Differentiation of mycelia during pellet formation results in striking effects on enzyme production.Poly-galacturonidase synthesis is well associated with the fungal morphology of As.niger .The more compact the pellet,the greater the polygalactorunidase synthesis.Regardless of the medium used,an increase of two orders of magnitude in enzyme concentration and rate of production between the free filamentous mycelium and the pelleted type was observed (Hemmersdorfer et al.,1987).Similar increases were observed in glucoamylase production rates by pellets of As.niger (Papagianni and Moo-Young,2002).Such phenomena may be related to diffusional limitations in pellets,which either reduce the extent of catabolic repression in pellets or limit the oxygen supply,preventing this way an oxidative inactivation of a specific set of enzymes.
The magnitude of the difference between enzyme production in pellets and in free filamentous mycelia at different concentrations of catabolites (Hemmersdorfer et al.,1987)seems to indicate the existence of additional factors,such as gradients of metabolic products in pellets which serve as biological signals (modulators).In fact,the high level of cell-to-cell interaction and signaling resulting from short diffusional distances in mycelial aggregates leads to a state of differentiation qualitatively different from that of free filamentous mycelia.In a review on the factors affecting mycelial aggregation,Braun and Vecht-Lifshitz (1991)have stated that mycelial aggregates may be viewed not merely as mechanical conglomerates,but rather as complex differentiated tissues phenotypically characterized by specific metabolic activities.According to them,the closest analogy to this concept would be the obvious distinction between the unicellular and multicellular forms of a slime mould.
4.Growth of fungal pellets
Pelleted cultures are traditionally assumed to follow cube-root kinetics,following the early observations of Emerson (1950),on growth of N.crassa ,and Marshall and Alexander (1960)who investigated a number of fungi.These kinetics are described by the equation:
M 1=3?M 1=30tkt e6Twhere M represents the biomass concentration and k is a constant.Pirt (1966)explained these kinetics by considering the heterogeneity within pellets in similar manner to that within colonies growing on solid substrate:A pellet is considered as a spherical mass of non-growing mycelia surrounded by an outer shell of active hyphae.Thus,a pellet is assumed to increase in radius at a constant rate through exponential growth of mycelia within the outer shell of active hyphae.The width of the outer shell,w ,is determined by the diffusion properties of material through the mycelium and depends on the pellet structure.Therefore,it is not directly equivalent to the peripheral growth zone.Thus,steady-state concentrations of biomass and substrate and the critical dilution rate depend not only on diffusivity but also on the pellet size and fragmen-
M.Papagianni /Biotechnology Advances 22(2004)189–259
202
tation and the growth conditions reflecting on them.Changes in pellet radius are described by the equation:
r?r0tw l te7Twhere,r and r0represent pellet radii at times t and0.The outer active shell has a w width and l specific growth rate.If the pellet is assumed to be spherical and to have a constant biomass density q,the above equation can be written in terms of biomass,as:
M?M0t
4
3
pq n
1=3
w l te8T
where M and M0are the biomass of pellets at times t and0,respectively,and n is the number of pellets.This is equivalent to Eq.(6),with
k?
4
3
pq n
1=3
l te9T
The model predicts exponential growth in batch culture until restrictions to diffusion of nutrients through the pellet mass reduce growth rates in the center of the pellets. Subsequent growth will then follow cube-root kinetics.Cube-root kinetics have been observed experimentally(Trinci,1970),but practical difficulties in accurately measuring biomass concentrations make distinction of different types of growth kinetics difficult. Although cube-root kinetics are predicted for pellet growth,it is difficult to distinguish them from exponential kinetics.Koch(1975)constructed a model that provides a unified approach for growth in liquid and solid substrate and predicts the majority of growth kinetics observed experimentally,such as linear colony expansion,exponential,square and cube-root kinetics for biomass and explains these in terms of the capacity of mycelia to colonize unoccupied regions of substrate.
Koch(1975)fitted Trinci’s data to the logistic equation,modified to account for mycelial growth.The logistic equation was originally constructed to describe growth of individuals within a population and it is based on the assumption that the specific growth rate decreases as a negative linear function of population size,having the following form:
d N d t ?rNà
rN2
k
e10T
where N is the number of individuals,r the intrinsic rate of increase and k the yield or the carrying capacity of the environment.The above equation gives:
N?
kN0exp rt
00
e11T
where N0is the initial cell number.The equation predicts a sigmoidal growth curve.To describe mycelial growth,Koch replaced the number of individuals by the mycelial biomass W,occupying unit volume of space,d V.N0and k were replaced by the initial(S) and maximum(K)mycelial biomass per unit volume and t was replaced by(Tàt).T is the M.Papagianni/Biotechnology Advances22(2004)189–259203
time since growth of the colony started,while t is the time at which growth first occurred within the volume element considered.The first equation can then be rewritten for (T àt )>0,as
d W d t ?SK exp r eT àt T
K àS àS exp r eT àt Td V e12T
Restriction of growth to two dimensions to form a colony of height h ,which expands at a constant radial growth rate a ,and considering the lag and exponential growth periods as negligible,leads to the expression:
W ?Z
T 0SK exp r eT àt TK àS àS exp r eT àt T2p h 2t d t e13T
while an equivalent expression was derived for three dimensional growth,describing growth in liquid media in the form of pellets.The model was solved using numerical approximation techniques.The growth kinetics were fount to depend on the ratio of maximum to initial biomass density,K /S .For K /S values in the range 102–104,growth is predicted to be exponential and the curve linear.Experimental data for As.nidulans pellets were best characterized by a K /S value of about 1and predicted exponential growth followed by cube-root kinetics for total biomass and linear radial expansion.Pellet growth is characterized by rapid and simultaneous colonization of the medium surrounding the pellet by densely packed hyphae covering the pellet surface.This effectively leaves no unoccupied space for subsequent growth,giving the low observed K /S ratio.
5.Effects of diffusional limitations inside pellets
Although the above approach provides information on changes in pellet density,there is a little indication of the heterogeneity characteristic of pellets and a missing link between the microscopic description of mycelial and pellet growth,and the overall process.The major cause of heterogeneity within pellets is diffusional limitation of nutrients and oxygen which arises from dense hyphal packing.The extent of this limitation depends on the density of the structure,thus in compact pellets,biomass production in the center of the pellet will cease,and,eventually,cell autolysis will occur.This pellet morphology has been observed frequently,e.g.,in submerged citric acid fermentation (Clark,1961).Dense As.niger pellets at the end of fermentation (140h)consisted of a shell of mycelium occupying less than 50%of the pellet volume (Clark,1961).The proportion of the metabolically active biomass in this case is restricted to the outer zone of less dense mycelium.In less dense pellets,the actively growing outer zone is wider,substrates diffuse freely inside the pellet and mass transfer may occur via turbulent diffusion and convective flow according to Wittler et al.(1986)and Gerlach et al.(1998).
The importance of nutrient and oxygen limitation in pellet growth and pelleted biomass physiological heterogeneity has been taken into account in modeling the pellet growth.Pirt’s (1966)model of pellet growth was based on growth of an active peripheral zone
M.Papagianni /Biotechnology Advances 22(2004)189–259
204
surrounding a spherical pellet and was used to derive an expression for the pellet radius, R c,at which diffusion of nutrients to the core is limited by increased biomass density.The nutrient which is likely to limit growth first is oxygen and use of the diffusion coefficient for oxygen and growth kinetic parameters for Pe.chrysogenum predicted a critical radius which responded well with the experimentally determined radius.The critical radius,R c, was given by the expression
R c?m
6D V Ys m
ql
1=2
e14T
where D V is the diffusion coefficient,Ys m is the yield of biomass,q its density and l the specific growth rate.
More detailed models for oxygen diffusion and utilization inside pellets were presented and elucidated the importance of oxygen limitation in pellet growth.In the model described by Aiba and Kobayashi(1971),both respiration and inward diffusion were considered in calculating the oxygen balance within a pellet.Diffusion equations were applied and the relative rates of respiration within the pellet and in the surrounding
mediumeq V O
2=q O
2
Twere determined using the expression:
D d2C
d r
t
2d C
r d r
?2q q O
2
C
K mtC
e15T
where r is the pellet radius and q the biomass density inside the pellet.
In another model,Kobayashi et al.(1973)introduced an effectiveness factor to describe the reduction in respiration with increasing distance from the pellet surface.They investigated several situations,with constant respiration within the pellet,respiration varying with age and also respiration dependent on adaptation of mycelium to oxygen limitation.Experimental data derived from pelleted cultures of As.niger indicated the last of these situations to be the most likely.
Michel et al.(1992)compared experimental data from pelleted cultures Phanerochaete chrysosporium with model predictions for V max and K m for oxygen inside the pellets. These values were then used to predict oxygen limitation as a function of pellet size and dissolved oxygen concentration.Predictions of CO2evolution from populations of pellets in batch culture were also obtained and agreed well with experimental data.
6.Cell aging and autolysis
An important aspect of pellet growth is fragmentation,or breakup of pellets.It has been observed that the initial increase in pellet concentration in fungal cultures is followed by a rapid decrease which coincides with a decrease in the specific growth rate(Nielsen et al., 1995).This breakup is caused by cell lysis within pellets,whereby the stability of the pellet is lost,and it becomes more susceptible to damage by mechanical forces.Besides pellet breakup,hyphal elements are torn off at the pellet surface,weakened by the natural aging process of vacuolation.Due to the fragmentation of pellets and the loss of hyphal M.Papagianni/Biotechnology Advances22(2004)189–259205
elements from the pellet surface,the macroscopic morphology of a fungal culture grown in liquid substrate changes drastically,e.g.,from the pelleted to the dispersed form (Ju ¨sten et al.,1996;Paul et al.,1999),thus changing the rheological properties of the broth and its mass transfer capabilities.
In spite of the differences brought about by the cultivation conditions,hyphae retain some characteristics,which can be indicated as symptoms of aging.A growing septate hypha can be separated into at least three zones:(1)the apical zone (see Section 1);(2)the subapical zone,rich in plasma components;and (3)the vacuolation zone in which the size of vacuoles increases with distance from the apex,i.e.,with the age of the compartments.The cell wall also undergoes an aging process.Structural differences between the wall of the apex and that of the lower part of the hypha have been indicated by Marchant and Smith (1968)and Strunk (1963).Autoradiography studies of wall synthesis showed a progressive thickening of the walls in parts more distant from the apex.Similarly,the autolysis of the walls is relative to their age.Observations on the compositional and structural inequality of the hyphal walls caused by aging have been made by many researchers (Katz and Rosenberg,1971;Gooday,1971;Gull and Trinci,1974;Chang and Trevithich,1974),and the relationship between the morphological and physiological functions of hyphal parts of different age is apparent.However,it is often difficult to distinguish whether the observed biochemical changes in hyphae during the course of aging are evoked by aging or are only due to cultivation conditions.Autoradiography studies on As.niger during different phases of culture development showed that during the stationary phase some parts of the hypha irreversibly lose their ability to synthesize RNA and protein and they begin to autolyse.In other parts,the rate of synthesis was decreased by 15–20%as compared to hyphae from the exponential phase (Fencl,1978).This was explained by the assumption of protoplasmic streaming and transport of nutrients from the older to the younger parts of the hyphae,resulting in an exposure to starvation of the older parts.The apical parts are not exposed to starvation and they retain a higher regeneration ability.However,this hypothesis is not generally valid since in some older parts of hyphae,there is the potential possibility of passing to a physiologically younger state if a compartment forms a new branch.Then this part will increase its rates of RNA and protein synthesis to the levels of the apical part (Machek and Fencl,1973).
In contrast to a culture growing in the free filamentous form,in pellets,there is a more substantial differentiation in the filaments caused by the transport of nutrients from the outer zone to the inside,and by the excretion of the metabolic products to the outside of the pellets.This differentiation may be influenced by self-toxicity induced by excreted metabolites,as observed with a N.crassa mutant which secretes a mucopolysaccharide that inhibits growth (Reining and Glasgow,1971).Only a thin layer in the peripheral zone of pellets is biosynthetically active,as shown in studies with Aspergillus and Penicillium .
Autolysis is considered as the last stage of culture development even when it is observed in parts of hyphae during the stationary phase (Fencl,1978).The main cause of lysis is the material imbalance in hyphae caused either by internal or external factors.Of the internal factors,it could be a disturbance of the organelles or an accumulation of toxic metabolites.The external could be physical and chemical factors,the lack of nutrients,as well as enzyme influences which disturb the cell wall structures (Fencl,1978;Nombela et al.,1993).Exhaustion of nutrients from the medium represents one of the most common
M.Papagianni /Biotechnology Advances 22(2004)189–259
206
M.Papagianni/Biotechnology Advances22(2004)189–259207 reasons for a culture gradually reaching autolysis.The transition to autolysis is gradual and lack of nutrients is reflected in the hyphae during the pre-autolytic phase by increased differentiation.Righelato et al.(1968)observed that glucose supply at the maintenance rate in continuous cultures of Pe.chrysogenum caused increased vacuolation and predisposed the culture to mechanical damage and increased fragmentation.Righelato et al.(1968)concluded that the age of the mycelium does not appear to control the aging process but rather that aging and lysis are determined by the amount of available energy. Transition to the pre-autolytic phase in Pe.chrysogenum occurs at the moment when the amount of glucose supplied to the culture approaches the maintenance energy which was calculated to be0.022g glucose/g mycelium dry weight/h.Below this level,autolysis sets in.The rate of degeneration of the individual biomass components depends on the cultivation conditions under which the culture has been grown.
Nutrient limitation resulted in heavily vacuolated hyphae and subsequent fragmentation in fed-batch cultures of Pe.chrysogenum in the work of Paul et al.(1994a).During the rapid growth phase there was little vacuolation and the mean main and total hyphal lengths and the mean number of tips per mycelium rose or remained steady.Following glucose limitation,the values of the parameters declined sharply,indicating a fragmentation process which was more severe when significant hyphal vacuolation was established, i.e.,during the production phase.The authors based on quantitative information on vacuolation and morphology obtained by image analysis,concluded that,in addition to shear,physiological effects can enhance fragmentation,and this supports the idea that shear might only be effective when the hyphae have been significantly weakened by internal decay processes.The process of autolysis in batch cultures of Pe.chrysogenum under a range of stirrer speeds was also investigated by Harvey et al.(1998)who reported degradation of penicillin V as a result of culture autolysis.The relationship between vacuolation,fragmentation and citric acid production by As.niger was investigated in batch and fed-batch culture by Papagianni et al.(1999a,b,c,d).Quantitative information on morphology and vacuolation obtained by image analysis,together with specific growth and production rates,were used to establish a link between these under various agitation conditions and glucose levels.Increased vacuolation and low specific production rates were observed at low glucose levels,while vacuolation weakened the hyphae and made the mycelium more susceptible to shear forces at increased agitation levels.
When the fungus is grown in excess nitrogen and growth is limited by the carbon source,the protein component of the cell is degraded most rapidly(Lahoz et al.,1970; Lahoz and Miralles,1970).Conversely,if excess carbon is present in the media,autolysis raises the amount of reducing substances.Furthermore,while polysaccharides are normally lysed slowly,lipids are preferentially degraded(Lahoz et al.,1967).Mono-saccharides,thus,can always be detected in the lysing mycelium(Lahoz et al.,1970). Growth of Pe.chrysogenum in excess of the penicillin precursor phenylacetic acid,has been associated with increased cellular autolysis,reduced biomass and penicillin produc-tion levels,while precursor concentration controlled within the optimal range for penicillin production has little impact on differentiation or degradation within an industrial culture of Penicillium(White et al.,1999).
Autolysis does not proceed synchronously in the entire filament but only in its individual compartments(Trinci and Righelato,1970).In a lysed compartment,the
resistance of individual organelles is not the same.Mitochondria are much more stable than ribosomes,and the decomposition of organelles of the same type is synchronous,i.e.,it is catalyzed by cell-free enzymes (Trinci and Righelato,1970).In contrast with other organisms,lysosomes and autophagy do not play a substantial role in the autolysis of cytoplasm in fungi (Fencl,1978).Hyphal protoplasm undergoes lysis because of either a decreased amount or defects in composition of organelles,or to a lack of provided maintenance energy.Vacuolation and disruption of organelles,including an increased hydrolase activity may be observed in the older parts of hyphae,however,the physio-logical age and the age of the compartment need not coincide (Fencl,1970).For this reason,there is no regularity in the lysis of hyphae from the base toward the tip and the lysed compartments are localized irregularly in different parts of the hyphae.Excretion of lytic enzymes,such as h -N -acetyl-glucosamidase,h -1-3glucanase,chitinase,invertase and acid phosphatase was found to be consistent with the degree of autolysis (Lahoz et al.,1967),however,hyphae contain their own protective substances through which they withstand lysis by their own enzymes and it has been demonstrated that only after removal of their protective barrier will intracellular glucanases and chitinases attack the walls (Wessels and Koltin,1972).Although the autolytic enzymes are localized directly in hyphal walls,because of its protection the cell wall is only slowly autolysed (Trinci,1974).Both Lahoz et al.(1986)and Trinci and Righelato (1970)noted the persistence of intact fungal cell walls after many days of autolysis,which involved extensive proteolysis.In contrast to the inside of hyphae,where autolysis affects compartments irregularly,the lysis of walls proceeds regularly from the tip to the older sections,thus reflecting the chemical composition of the walls.
Despite its importance,fungal autolysis has received much less attention compared to the lysis of bacteria and commercially important yeasts.The methods used to assess the extent of autolysis in fungal cultures involved the mean decline of biomass,cellular breakdown products,e.g.,NH 4+release enzyme activity assays,and direct measurement of the autolysing regions by image analysis techniques.The latter,relatively recent improve-ments in our ability to follow the processes of growth and differentiation in submerged fungal cultures,allowed extraction of detailed quantitative information on the micro-morphology of filamentous fungi grown in dispersed form in submerged fermentations.The studies of Packer and Thomas (1990),Makagiansar et al.(1993),Nielsen et al.(1995),Paul et al.(1994a,b),Papagianni et al.(1999b)and McIntyre et al.(2001)describe image analysis studies which measure growth and differentiation in submerged culture and investigate the relationship between hyphal degeneration and vacuolation,and productiv-ity.Image analysis is,therefore,a more direct method than the ‘‘filtration probe’’of Nestaas and Wang (1983)for implementing control strategies for antibiotic fermentations based on simple differentiation.
7.Fragmentation of hyphal elements and pellets
Fragmentation of hyphal elements and pellets during submerged fermentations often results in growth renewal since fragments may act as centers for new growth,enabling reseeding of the pellet population.Unfortunately,despite their importance,there have been M.Papagianni /Biotechnology Advances 22(2004)189–259
208
《语言学纲要》名词解释 导言 4.交际工具：人类交际活动所使用的工具。语言是人类最重要的交际工具。此外，身势等伴随动作是非语 言的交际工具；旗语之类是建立在语言、文字基础上的辅助性交际工具；文字是建立在语言基础之上的一种最重要的辅助交际工具； 5.思维：是认识现实世界时的一种动脑筋的过程，也指动脑筋时进行比较、分析、综合以认识现实的能力。 是人脑能动地反映客观现实的机能和过程。根据思维活动的不同形态可分为三种类型：直观动作思
维、形象思维、抽象思维。 6.社会：指生活在一个共同的地域中、说同一种语言、有共同的风俗习惯和文化习惯的人类社会的共同体， 即一般所说的部落、部族和民族。与此相关联的现象就是社会现象。 7.社会现象：指那些与人类共同体的一切活动——产生、存在和发展密切联系的现象。 取渐变，不能爆发突变。 8.语言发展的不平衡性：指语言结构体系发展变化是不平衡的，即词汇、语义、语音、语法的发展速度是 不一样的。与社会联系最直接的词汇、语义变化最快，语音次之，语法最慢。 9.表层结构和深层结构：表层结构和深层结构相对，表层结构赋予句子以一定的语音形式，即通过语音形
式所表达出来的那种结构，表层结构是由深层结构转换而显现的；深层结构是赋予句子以一定的语义解释的那种结构。 10.组合关系：符号和符号组合起来的关系。符号和符号的组合形成语言的结构。 11.聚合关系：在链条的某一环节上能够相互替换的符号具有某种相同的作用，它们自然聚集成群。它们彼 此的关系称为聚合关系。 23.音高：声音的高低，是由发音体形状及振动频率快慢决定的。 24.音重：声音的强弱，它取决于声波振幅的大小，而振幅的大小与发音时用力大小有关。 25.音质：也叫音色，指声音的品质或个性。
中国传媒大学语言学及应用语言学专业方 向介绍 （感谢凯程罗老师对本文的有益指导.) 语言学及应用语言学专业（050102） 1．应用语言学方向 应用语言学方向是适应我国应用语言学发展需要设立的研究方向，它以“人机交际”和人际交际的语言理论和技能、技术为主要研究对象。作为跨学科的语言应用学科，它同人文科学、自然科学（数学、物理学、信息科学、计算机科学等）都有密切关系。 本方向有社会语言学和计算语言学两个研究领域。社会语言学，重在研究语言在社会生活中的应用。它结合中国社会的文化背景，对语言应用进行多角度、全方位的研究，总结社会生活不同领域（如旅游、交通、商贸、公关、司法、教育等）人际交往中语言应用的特殊规律，对语言实践提供理论指导，并为我国语言文字规范化工作服务。计算语言学是利用计算机对人类自然语言处理和加工的科学，它是适应信息时代的要求而产生和发展起来的一门边缘交叉性学科。机器翻译、自然语言理解、语音识别与合成等都是本方向研究的重要课题，这些课题的研究对人工智能计算机的研制，起着举足轻重的作用。 本方向将发挥中国传媒大学文、理、工多科院校、以及与教育部语言应用研究所合作的优势，为二十一世纪的应用语言学培养高层次专门人才。 2．对外汉语教学方向 本研究方向以对外汉语教学研究为重点。汉语教学法历来都是汉语言基础理论体系中的重要组成部分，汉语教学法研究，也是汉语言基础理论研究中不可缺少的部分。 自现代汉语诞生之日起，其教学过程即随之开始；应用语言学的产生和发展，更进一步推动了汉语教学研究的理论升华。特别是近几十年来，汉语教学研究，无论是对内的，还是对外的，都越来越受到重视。从建立完整的理论体系的角度说，我校有国内著名语言学家为学科带头人的现代汉语语法、修辞研究的深厚基础，最近又增添了应用语言学研究的雄厚实力，如果再加上汉语教学研究理论的参与，必将使汉语语言理论研究体系更趋完善。我校作为全国广播电视人才培养基地，多年来一直担负着为广播电视事业培养专门人才的重要任务。如何在汉语教学中把结构、意义和交际功能三者有机地结合起来，更好地培养学生实际运用语言的能力，也是多年来面临的理论与实际的大问题。随着时代要求的不断提高，矛盾日益突出。汉语教学如何借鉴心理学、教育学等相关学科的研究成果，结合语言应用实际，力求走出一条新路子。 对外汉语教学专业人才的需求，近年来不断加大，国内各汉语教学机构为了在来华留学生的市场竞争中占有更多的份额，都纷纷把对外汉语教学高学历师资的竞争放到了首位。此外，全球性“汉语热”方兴未艾，特别是在进入新世纪之后，国内外汉语教学师资的需求量呈上升趋势。本方向将竭力培养对外汉语教学方面的高层次专门人才。 3. 语言信息处理方向 语言信息处理是语言学与计算机科学交叉形成的一门新型学科，是应用语言学的重要组成部分。 我国“十五”发展规划的重要方针之一，是大力推进信息化进程，以信息化带动工业化。
Chapter 3 Morphology形态学 Nothing is more important to language than words. Words can carry meaning. Words are the fundamental building blocks of a language. So, is word the most basic or the minimal unit of meaning? If not, then what is? How words are formed?---morphology 3.1 what is morphology? Morphology refers to the study of the internal structure of words and the rules by which words are formed. 3.2 Open class and closed class (开放词类和封闭词类) Open class words----content words of a language to which we can regularly add new words, such as nouns, adjectives, verbs and adverbs. Closed class words----grammatical or functional words, such as conjunction, articles, preposition and pronouns. New words can be added to open class words regularly with the development of human civilization. However, the number of closed class words is small and stable since few new words are added. 3.3Morphemes--the minimal units of meaning（词素，最小的意义单位） Word is the smallest free from found in language. Word can be further divided into smaller meaningful units---morphemes. So, morpheme is--- the smallest unit of language that carries information about meaning or function. Words are composed of morphemes. Words may consist of one morpheme or more morphemes, e.g. 1-morpheme boy, desire 2-morpheme boyish, desirable 3-morpheme boyishness, desirability 4-morpheme gentlemanliness, undesir(e)abl(e)ity 5-morpheme ungentlemanliness 6-morpheme anti+dis+establish+ment+ari+an+ism Free morpheme & bound morpheme Free morpheme----is one that may constitute a word (free form) by itself, such as bed, tree, sing, dance, etc. Bound morpheme----is one that may appear with at least one other morpheme. They can not stand by themselves, such as ―-s‖ in ―dogs‖, ―al‖ in ―national‖, ―dis-‖ in ―disclose‖, ―ed‖ in ―recorded‖, Allomorph
绪论、第一章、第二章 一、名词解释 1．语言学 2．语言 5．文言文 6．符号 7．符号形式 8．符号的任意性 9．语言符号 10．组合关系 11．聚合关系 二、填空 1．__________、________ 、________________ 具有悠久的历史文化传统，是语言学的三大发源地。 2．__________ 是我国古代的书面语，用它写成的文章称为__________。 3．__________ 、__________ 、__________ 是我国传统的语文学。 4．研究语言的结构，主要是研究 __________、____________ 、__________ 三个部分。 5．运用语言传递信息的过程，可以分为________、________ 、_________ 、__________ 、__________ 五个阶段。 6．语言是人类社会的______________，而且也是思维的______________。 7．在一定条件下，身体姿势等伴随动作还可以离开语言独立完成交际任务。例如汉民族点头表示____________，摇头表示____________，送别时挥手表示 ______________，____________表示欢迎，咬牙切齿表示_____________，手舞足蹈表示____________。 8．人的大脑分左右两半球，大脑的半球控制语言活动，右半球掌管不需要语言的感性____________。 9．汉语的“哥哥”、“弟弟”，英语用________________表示，汉语的“舅舅、姨父、姑父、叔叔、伯伯”，英语用______________表示。 10．英语可以直接用数词修饰名词，汉语数词修饰名词一般要加上一个 _____。 11．儿童最早的智力活动就是学习_________。 12．任何符号，都是由和两个方面构成的。 13．一个符号，如果没有_______，就失去了存在的必要，如果没有_______，我们就无法感知，符号也就失去了存在的物质基础。 14．语言符号是_________和_________的统一体，声音是语言符号的______。
语言学纲要名词解释 导言 1.语言学：以语言作为专门研究对象的一门独立学科，其主要任务是研究语言的性质、结构和功能，通过考察语言及其应用的现象，来解释语言存在和发展的规律。 2.语文学：指19世纪历史比较语言学之前的语言研究，这时的语言研究尚未独立，语言学作为其他学科的附庸而存在，语言研究的主要目的是为了阅读古籍和语言教学，从而为统治者治理国家或为其他学科的研究服务。 3.小学：中国传统的语文学，由于汉语书面语使用的文字——汉字的特点，中国传统语言研究抓住汉字，分析它的形体，探求它的古代读音和意义，形成了统称“小学”的文字、音韵、训诂之学，也就是中国传统的语文学。 4.理论语言学：也称普通语言学，是关于语言的一般规律的理论研究。理论语言学的水平在很大程度上决定于具体语言学的研究成果。 5.共时语言学：以同时的、静态的分析方法，研究语言相对静止的状态，描写分析语言在某一时期、某一阶段的状况，是从横的方面研究语言。 6.历时语言学：研究语言发展的历史，观察一种语言的各个结构要素在不同发展阶段的历史演变，是从纵向的方面研究语言的历史。涉及到一种语言的叫做历时语言学，如历史语音学，历史词汇学，历史语法学等；涉及到多种语言和方言的叫做历史比较语言学。 7.索绪尔：瑞士语言学家，现代语言学的奠基人，结构主义语言学的开创者，著有《普通语言学教程》，被人们誉为“现代语言学之父”。他提出语言是符号体系；符号由能指所指两部分构成，这两部分的关系是任意的，一旦形成以后又是约定性的；符号系统内部存在“组合关系”和“聚合关系”；区分了“语言”和“言语”；“内部语言学”和“外部语言学”；“历时语言学”和“共时语言学”。他的学说标志着现代语言学的开始，在不同的程度上影响着20世纪的各个语言学派。 8.布龙菲尔德：美国描写语言学派的代表人物，它的主要贡献是将语言学从哲学理念建设成为一门科学。早期的著作是出版于1914年的《语言研究导论》，立足于心理学来阐释语言、刺激—反应论来解释语言的产生和理解的过程。1933年他的最有影响的著作《语言论》出版。这时，他已经从构造心理学转到行为心理学。他在该书中提出了美国结构语言学派研究语言的基本原则和描写语言结构的总框架。 9.乔姆斯基：转换生成语法的创始人，著有《句法结构》。最出他用结构主义的方法研究希伯来语，后来发现这种方法有很大的局限性，转而探索新的方法，逐步建立了转换生成语法，1957年出版的《句法结构》就是这一新方法的标志。这种分析方法风靡全世界，冲垮了结构语言学的支配地位，因而被人们成为“乔姆斯基革命”，对语言学的发展方向产成了巨大影响。 10.韩礼德：英国语言学家，功能主义语言学派的代表人物，继承了以弗斯为首的伦敦学派的基本理论，并吸收布拉格学派和哥本哈根学派的某些观点的基础上发展起来和创立了系统功能语法，著有《普通语言学教程》《语法功能论》等著作。他从人类学和社会学的角度出发研究语言与社会的关系，把语言看做“社会符号”，其包括功能系统、层次和语境等概念，在语法中认为系统中存在连锁系统和选择系统，在功能语法中他用功能的配置来解释语法结构，提出了概念功能、人际功能、语篇功能。20世纪70年代后，他将注意力转移到语言与社会学、符号学的关系上，对社会语言学进行研究。 11.赵元任：字宣仲，江苏常州人，生于天津。赵元任是“中国语言学之父”。他将科学运用于语言学研究的结果：用自然科学中的基本概念说明语言问题；用自然科学的先进成果记录和分析语音；把自然科学中的研究方法引入语言学；引入科学的描述事物的方式以及解决问题的程序等等。中国的传统语言学在他和同时代的一批学者的努力下，逐步走向现代化。 12.历史比较语言学：出现于18世纪末19世纪初，中心在德国。该学派采用历史比较的方法，通过语音和词形的比较研究语言的发展和演变，发现了语言之间的亲缘关系并建立了语言的谱系分类。历史比较语言学的兴起，是语言学成为一门独立的学科。经过一个多世纪的探索研究，弄清了世界上很多语言的同源关系，建立了世界语言的谱系分类，为语言学的发展做了很大贡献。历史比较语言学也有局限：他强调了语言的历史比较，忽视了语言的共时研究；孤立地研究语言单位，缺乏对语言系统性的研究。 13.结构主义语言学：1916年索绪尔的《普通语言学教程》标志着结构主义语言学的诞生。索绪尔语言理论的核心是语言是符号系统，突破了历史比较语言学的局限，开拓了语言研究的新领域，给语言学带来革命性的变化。
中国传媒大学2010年攻读博士学位研究生入学考 试 语言学理论试题 一.论述题。 1.必答题(40分) 就语言规划或语言规范化问题尽你所知进行论述。 2.选答题(30分,任选其一回答) A.就话语(也叫篇章、语篇)研究进行论述。 B.就形式主义语言学和功能主义语言学进行论述。 二.分析题(30分,每小题10分) 1.指出下面三个四字格之间的联系,分析形成的原因,并说明各自的表达效果。喜鹊登枝 喜上眉梢 喜上眉梢 2.下面两组句子,一组可逆,一组不可逆,请从认知角度解释。 我后悔死这件事了——这件事后悔死我了 我后悔这件事了——★这件事后悔我了 3.运用预设理论解释下面句子。
北京又要打击非法一日游了 中国传媒大学2010年攻读博士学位研究生入学考试 语言学理论试题 二.论述题。 1.必答题(40分) 就语言规划或语言规范化问题尽你所知进行论述。 (1)语言文字的合理发展,对一个国家或民族的发展具有不可估量的作用。国家或社会为了管理社会语言生活,对语言的现状及发展施加有计划有目的的影响以促进语言文字健康发展使其更好的尾社会服务而进行的各种工作就是语言规划。 (2)语言规划涉及的内容很多,主要包括民族共同语的确立和推广,民族共同语的规范和完善,语言选择,语言协调,语言调查,科技术语的统一和标准化,文字的创制和改革等。 (3)①语言文字立法是语言规划的一种体现。《中华人民共和国国家通用语言文字法》是语言政策的法律体现,对于加强语言文字工作有重要意义。②政府对语言的管理和影响表现在官方语言的选择上,外语教育也有语言选择问题,除了语言选择以外还有标准化问题,包括科技术语的标准化和语言规范化。 (4)语言规划的原则:语言规划必须尊重并且符合语言发展的客观规律,符合社会发展的需要,符合广大人民群众的意愿。避免两种倾向:一种是人在语言文字面前无能为力,无所作为;一种是过于夸大人的作用。
《网店美工视觉设计实战教程（全彩微课版）》 教学大纲 一、课程信息 课程名称：网店美工：店铺装修+图片美化+页面设计+运营推广（全彩微课版） 课程类别：素质选修课/专业基础课 课程性质：选修/必修 计划学时：21 计划学分：2 先修课程：无 选用教材：《网店美工视觉设计实战教程（全彩微课版）》，何晓琴编著，2018年；人民邮电出版社出版教材； 适用专业：本书可作为有志于或者正在从事淘宝美工相关职业的人员学习和参考，也可作为高等院校电子商务相关课程的教材。 课程负责人： 二、课程简介 随着网店的迅速普及和全民化，衍生了“淘宝美工”这个针对网店页面视觉设计的新兴行业。本书从淘宝美工的角度出发，为淘宝卖家提供全面、实用、快速的店铺视觉设计与装修指导。主要包括网店美工基础、图片调色、图片修饰、店铺首页核心模块设计、详情页视觉设计、页面装修、视觉营销推广图制作等，最后针对无线端进行首页、详情页视觉的设计与装修。本书内容层层深入，并通过丰富的实例为读者全方面介绍淘宝美工在日常工作中所需的知识和技能，有效地引导读者进行淘宝店铺装修的学习。 本课程主要对淘宝美工的设计基础和方法进行详细介绍，通过学习该课程，使学生了解网店美工的基本要求，以及掌握网店的设计与制作。 三、课程教学要求
体描述。“关联程度”栏中字母表示二者关联程度。关联程度按高关联、中关联、低关联三档分别表示为“H”“M”或“L”。“课程教学要求”及“关联程度”中的空白栏表示该课程与所对应的专业毕业要求条目不相关。 四、课程教学内容
五、考核要求及成绩评定 注：此表中内容为该课程的全部考核方式及其相关信息。 六、学生学习建议 （一）学习方法建议 1. 理论配合实战训练进行学习，提高学生的实战动手能力； 2. 在条件允许的情况下，可以申请一个网店，进行深入学习； 3. 提高学生的是设计感和审美能力; （二）学生课外阅读参考资料 《网店美工：店铺装修+图片美化+页面设计+运营推广（全彩微课版）》，何晓琴编著，2018年，人民邮电出版社合作出版教材
导言 一,名词解释(分,每小题分) ,研究语言的本质,语言的结构和发展规律. .指我国传统的语文学,包括文字学,音韵学,训诂学三方面的内容. ,个别语言学,以一种(或几种有联系的)语言为研究对象,研究某一种语言的结构. ,静态分析的方法,研究语言相对静止的状态,描写分析语言在某一个时期,某一个阶段的状况,是从横向的方面研究语言. ,动态的角度研究语言发展的历史,观察一种语言的各个结构要素在不同发展阶段的历史演变,是从纵向的方面研究语言的历史. ,探讨人类语言的共同规律,是在具体语言学基础上建立起来的,下面又分普通语音学,普通语法学,普通词汇学等分支学科. 二,填空题(分,每空分) .中国印度古希腊罗马 .文字学音韵学训诂学 .语音词汇和语义语法 .编码发送传递接收 .历时共时历史描写 .历史比较 .《语言论》 .索绪尔 三,问答题(分,每小题分) ①研究对象不同:古代的语言学主要以书面语为主要研究材料,不重视口头语言的研究,而今天的语言学则十分重视口语研究,如制定语言规范,确立共同语的各方面标准等,都要依据口语的研究成果; ②研究目的不同:古代语言学研究语言,主要是给政治,哲学,宗教,历史,文学方面的经典著作作注解,比如我国古代的语文学主要就是围绕阅读先秦经典著作的需要来研究文言的,而现代语言学的研究目的主要是分析语言的结构,以此探讨语言发展的共同规律. 正因为有这些差别,所以古代的语言学还不是独立的学科,处于附庸地位,而现代语言学已经发展成为一门独立的学科,随着现代科学的发展又产生了许多边沿性学科. 人类语言交际的过程,实际上就是信息的传递与接收问题,可分为编码,发送,传递,接收,解码五个阶段.编码就是发话人利用词语组织语句;发送就是把思维成果变成话语,通过发音器官表达出来;传递就是通过空气振动形成声波,把话语传达给受话人;接收是受话人利用听觉器官感知对方所说的话;解码则经过大脑的思维把声波还原成语言,理解对方话语的含义,从而完成信息传递接收.如果受话人收到语言信息有所反馈,那么上述五个阶段则又重复一遍,只是发话人与受话人调换了. (举例说明略,可结合分析任举一个句子作说明.) 语言是伴随着人类一起出现的,是人类社会生活必不可少的,所以人类很早就注意到了语言的重要性,很早就注意研究语言,所以语言学是古老的,但语言学直到世纪下半叶,产生了历史比较语言学,后来又建立了语言学的各个部门,语言研究才发展成为一门独立的学科,同其它学科相比,语言学的确是十分年轻的.语言是社会现象,与社会的政治,经济,文化,历史等密切相关,而语言的发生又与物理,生理,心理等学科密切相关,而现代社会语言与语言的信息处理(如机器翻译,语码转换等)又涉及到数学,计算机科学,所以语言学既与社会科学有密切的联系,又与自然科学有密切的联系.正因为如此,随着语言学与别的学科的交融,又产生了许多新的语言学分支学科,如社会语言学,心理语言学,统计语言学,话语语言学,数理语言学,宇宙语言学等等,这些也体现了语言学是一门年轻的学科. 语言学的作用主要表现在以下几个方面:①学习语言文字是掌握科学技术,提高文化水平的基础,而要学好用好语言,就必须利用语言学的研究成果;②制定语言文字的有关政策,制定语言规范,都要在对语言进行充分研究的基础上进行,而语言学的研究成果正好可以作指导;③新兴技术的出现扩大了语言学的应用范围.至于个人,同样可以利用语言学的成果,比如学习一种语言或方言,最好的办法是找到所学语言同自己母语的各方面的对应规律,这样可以收到事半功倍的效果,这就需要利用语言学的研究成果. (结合实际部分可以根据自己的学习,工作或生活经历举例) 语言学首先可以分为理论语言学和应用语言学,一般所说的语言学,主要是指理论语言学.根据研究对象的不同,理论语言学又分普通语言学和专语语言学(具体语言学).普通语言学以人类所有的语言为研究对象,探讨人类语言的共同规律,是在具体语言学基础上建立起来的,下面又分普通语音学,普通语法学,普通词汇学等分支学科.专语语言学以个别的,具体的语言为研究对象.探讨研究某一种语言的规律.从是静态研究还是研究语言的历史看,又分历时语言学和共时语言学:历时语言学研究具体语言的发展历史,是纵向研究,比如汉语史研究,共时语言学研究具体语言在某一时代的状态(相对静止的状态),规律,对之进行客观的描述,是横向研究.如描写语言学,又分描写语音学,描写语汇学,描写语法学等分支学科,现代汉语就是共时语言学. .在历史语言学产生以前,语言学
中国传媒大学2006年应用语言学考研试题 语言学理论 解释下列概念 互补分布 2、语义特征 3、屈折语 4、自源文字 5、隐喻 二、单项选择题 1、历史比较语言学出现在--------- A、19世纪中叶 B、20世纪初 C、19世纪初 D、20世纪中叶 3、从音质角度划分出来的最小语音单位是----------- A、音渡 B、音素 C、音位 D、音节 4、[p、t’、ts、k]这一组音的共同的区别特征是----------- A、不送气 B、清音 C、塞音 D、擦音 5、下列词语中的“子”是实语素的是---------- A、鸽子 B、傻子 C、莲子 D、日子 6、下列说法中正确的是 A、“老”可以同“新、旧、少、嫩”等构成反义词 B、“大”和“小”是绝对对立的反义词 C、“红”与“黑”这对反义词具有非此即彼的关系 D、“冷”和“热”是相对反义词 7、英语代词“我”，作主语时写作I，作宾语时写作me。这种变化时语法范畴中---------的表现。 A、性 B、树 C、格 D、人称 8、关于社会方言的形成，下列说法不正确的一项是--------- A、社会方言大多是在语言的相互接触中形成的 B、社会方言是随着社会的社群分化而产生的 C、一种语言的内部有可能形成社会方言 D、一种方言的内部有可能形成社会方言 9、克里奥尔语是指----------- A、用于特殊交际场合的皮钦语 B、功能单一，只能用于口头交际的一种混合语 C、结构简单，不可能得到充分发展的一种混合语 D、母语分化的皮钦语 10、下列各组内三个合成词构词类型不一致的是----------- A、天地宇宙阴阳 B、鱼子花儿锄头 C、改善揭露纠正 D、爸爸哥哥姐姐 三、分析题 1、请证明下列各组音素在汉语普通话中属于不同音位，还是同一音位的不同变体。[ t ]—[ t’ ] [ b ]—[ p ] [ i ]—[ u ] [ a ]—[ α ] 2用层次分析法分析下列词组（要求分析到词） 词汇是语言中反映社会发展变化最敏感的要素。 像流水一般晶莹剔透的月光。 3指出下列句子中划线词语的语义指向 小王把酒喝光了。 新买的球鞋也踢坏了。 4设置不同的语境，使“我等你半天了”这个语句形式表达不同的意思，呈现不同的交际效果（至少三个）。 5判断正误并说明理由。 语言是语言，言语是言语，二者完全不同，没有任何关系。 任何一种符号，都是由形式和内容两方面组成的。
导言 *中国、印度、希腊、罗马具有悠久的历史文化传统，是语言学的三大发源地之。 *形成了统称“小学”的文字、音韵、训诂的我国传统语文学。 *语言本身的构造很复杂，对它的研究大致可以分为语言、词汇和语义、语法三个部分。 *运用语言交际的过程是瞬间的事情，但却包含着一系列复杂的问题。如果借用信息论的术语来说，这一过程大体上可以分为“编码—发送—传递—接收—解码”五个阶段。 第一章 *语言研究的任务就是要把说话中反复使用的材料和规则找出来，把那些隐藏在无数话语中的坚守不见尾的语言找出来，使它的整体和每一片鳞甲都清清楚楚地展现在人们的面前。这当然是复杂细致的任务。 *除了语言之外，人们还可以使用其他的交际工具：文字，旗语，红绿灯，电报代码，数字符号，化学公式等。人们在使用语言进行交际的时候，不但要动嘴，而且要面部的表情、手的动作、乃至整个躯体的姿态等非语言的东西也都会参加进来。（非语言：手、眼神） *思维能力是全人类共同的，语言的各组民族不同的。 *为什么语言是人类最重要的交际工具？ 1、语言是最灵活的，最方便的，信息量最大的，所以是最重要的。语言是组成社会不可少的因素，人与人之间的联系要靠语言来维持。 2、语言是为满足人类交际的组要而产生的，其他动物是没有的。 3、其他的交际工具是建立在一定的语言基础上的语言或语言成分的代用品，而且都有特殊的服务领域。 第二章 *符号：是一个社会全体成员共同约定用来表示某种意义的记号标记。符号应该具备的条件：约定俗成、重复使用。语言符号的特点是：任意性和线条性。 *语言是一种分层装置，这种装置靠组合和替换来运转。语言必须是一种经济有效而又富于弹性和灵活性的装置。这装置的重要特点就是分层。 *语言的底层是一套音位，一种语言的音位的数目虽然只有几十个却能构成数目众多的组合，这些组合为语言符号准备了形式的部分。语言的上层是音义结合的符号和符号的序列，这一层又氛围若干级。第一级是语素，意义在这里被装进形式的口袋，成了音义结合的最小的符号。第二级是由语素构成的词，第三级是由词构成的句子。词和句子都是符号的序列。 *组合关系和聚合关系是语言两种最根本关系。 第三章 *音素：是从音节中划分出来的最小的语音单位。（从音质角度划分出来） *音标：记录音素的标写符号。国际音标：是国际语言协会于1888年制定的并开始使用的一套记录音素的标写符号。 *语音的发音---传递---感知三个环节，分别对应于语音的生理---物理---心理三个方面的属性，由发音学---音响学---听觉语音学三门学科加以研究，标志着人们研究语言的的进展过程。三个环节，三个方面，三门学科的研究都是自然科学和语言学的交界领域，都要利用大量的实验手段。生理、物理、心理的研究可以说是语音的自然属性的研究。 *元音和辅音的区别是什么？ 1、气流是否受阻，发元音气流不受阻，辅音受阻。 2、发音器官紧张状况。发元音时各器官的各部分都保持均衡紧张，发辅音时只有形成阻碍的那一部分特别紧张。 3、气流强弱不同，发元音气流弱，发辅音气流强。 *音位：是具体语言中有区别的语音形式的作用的最小语音单位。音位的分析对拼音文字的
CH 5 Morphology 的上課筆記 1. What is morphology? 2. How to use it? Who is it for? 3. When to use it? 4. Are there rules in it? 5. Is there an infix in English? 6. The use/ significance of the morphology. Morphology (構詞學) is (1) the study of morphemes and their different forms (allomorphs), and the way they combine in word formation, i.e., the English word unfriendly is formed from friend , the adjective-forming suffix -ly and the negative prefix un-. (2) a morphemic system(詞素系統). Morphology helps people recognize possible words. 有沒有新字的出現？ 例子1： Bug → a possible word Bog → a possible word Sog → not a possible word 例子2： 木神 → Morphology ( it depends on people)
(1) 昨晚我的ㄅㄧㄤˋ痛得睡不著。 →從這句話大概知道句子要表達意思：某人遭受疼痛之苦， 但不是能清楚知道每個字(individual words)的意思。 在訓練聽力時，是訓練學生get the main concept (idea) in the context/ dialogue. (2) 我的手臂上長了一個ㄅㄧㄤˋ，很癢。 →ㄅㄧㄤˋ可能是瘤或青春痘。 (3) 我的腳上長了一個ㄅㄧㄤˋ，不舒服。 →ㄅㄧㄤˋ可能是皮膚病。 (1)→(2)→(3)：從不同的context，慢慢會懂“ㄅㄧㄤˋ”的意義，但還是有點模糊。 直到你看到實物的那一天，才能真正了解其意義。 因為文字本身就是抽象的，所以如果用文字去表達一個抽象的概念，會很難懂；但若是用具體的實物來講解，會比較容易明白。 教單字→可以把圖片運用在單字教學上。 Morphology可以幫助學習新單字。 例子4： A: Have you seen ―wuggles‖ before? B: What are ―wuggles‖ ? A: They are four-legged animals. B: What do they look like? Dogs, cats, rabbits, or horses? A: No, they look like spiders. B: They are six-legged insects. 因為―wuggles‖(a possible word)符合造字原則，所以B會問What are ―wuggles‖ ?
第三章语音和音系 一、名词解释 语音四要素——音高、音重（强）、音长、音质。 音素——是从音质的角度划分出来的最小的线性的语音单位 音位——具体语言中有区别词的语音形式的作用的最小的语音单位。也是按语音的辨义作用归纳出来的音类，从语言的社会属性划分出来的语言单位 音位变体——处在互补关系中的相似的音素彼此不对立，即不起区别词的语音形式的作用，我们可以把它们归并为一个音位。如果它们被归并为一个音位，则处于互补关系中的各个音素就被看成为同一音位在不同位置上的代表，是同一个音位的不同的变异形式，所以我们把它们叫做音位变体。 非音质音位——非音质音位指具有区别词的语音形式的作用的音高、音重、音长等。例如汉语普通话声调中的阴平、阳平、上声、去声，是由音高的变化形成的而不是音质变化形成的，就是非音质音位。 区别特征——具体语言中有区别音位的作用的发音特征，叫做该语言的区别特征。每一个音位都可以分解为几个不同的区别特征。运用区别特征比较容易说清楚音位在具体语言中的特点和具体语言语音系统的组织方式。 音节——由音位组成的语音中最小的结构单位，也是从听觉上感受到的最自然的单位。 语流音变——音位和音位组合的时候，由于受说话时快慢，高低、强弱的不同和邻音的影响，可能发生不同的临时性的变化。这种变化，我们叫做语流音变。 音步——语言的一种节奏中，语流是大致每隔两个音节就有一次小的轻重、高低、长短或松紧的交替，形成语流中大致等距离出现的两音节的节奏单元。这种节奏单元叫做音步。 二、填空或简答 1、画一张元音舌位图，用国际音标标出八个基本元音。 八个基本元音[i][e][ ][a][u][o][?][ɑ]
中国传媒大学硕士研究生入学考试 “语言学理论”考试大纲 一、考试的总体要求 本考试大纲适用于报考中国传媒大学语言学与应用语言学专业硕士研究生的入学考试。语言学理论的主要内容包括：语言和语言学、语言的本质、语言的功能、语言结构、语言的运用、语言的历史、应用语言学、文字以及语言研究简史，等等。要求考生熟练掌握语言学理论的基本概念、基本原理，并具备综合运用所学知识以分析具体语言现象、解决实际问题的能力。 二、考试的内容 （一）语言和语言学 1．语言学的研究对象 （1）语言研究对象的演变及语言的概念（2）言语活动、语言、言语 2．语言学的学科体系 （1）规定语言学、描写语言学、解释语言学（2）历史语言学、历史比较语言学，历时语言学与共时语言学（3）理论语言学、应用语言学（4）普通语言学、语种语言学 （5）语言学的内部分支：语音学与音系学，词汇学与语义学，语法学；语用学、语篇分析（5）语言学的交叉学科和边缘学科 3．语言研究简史现代汉语》（修订本）,胡裕树,上海教育出版社 ②《现代汉语》（增订三版）,黄伯荣、廖序东,高教出版社 ③《古代汉语》王力,中华书局 ④《语音学教程》林焘、王理嘉,北大出版社 （1）传统语言学（语文学）（2）历史比较语言学（3）现代语言学：四大流派 （4）当代语言学：形式主义、功能主义、语序类型学、认知语言学 （二）语言的本质 1．语言作为符号系统 （1）符号与语言符号的概念（2）语言符号的特点（3）语言作为一个层级体系 （4）语言符号的组合关系和聚合关系 2．语言作为人类特有的现象 （1）语言能力（2）动物的语言和人类的语言的区别 （三）语言的功能 1．语言作为交际工具 （1）语言是人类社会的交际工具（2）语言是人类最重要的交际工具 2．语言作为思维工具 （1）语言与思维的关系（2）思维离不开语言的实验根据
I. Decide whether each of the following statements is True or False: 1.Morphology studies the internal structure of words and the rules by which words are formed. 2.Words are the smallest meaningful units of language. 3.Just as a phoneme is the basic unit in the study of phonology, so is a morpheme the basic unit in the study of morphology. 4.The smallest meaningful units that can be used freely all by themselves are free morphemes. 5. Bound morphemes include two types: roots and affixes. 6.Inflectional morphemes manifest various grammatical relations or grammatical categories such as number, tense, degree, and case. 7.The existing form to which a derivational affix can be added is called a stem, which can be a bound root, a free morpheme, or a derived form itself. 8. Prefixes usually modify the part of speech of the original word, not the meaning of it. 9.There are rules that govern which affix can be added to what type of stem to form a new word. Therefore, words formed according to the morphological rules are acceptable words. 10.Phonetically, the stress of a compound always falls on the first element, while the second element receives secondary stress. II.. There are four choices following each statement. Mark the choice that can best complete the statement: 1. The morpheme “vision” in the common word “television” is a(n) ______. A. bound morpheme B. bound form C. inflectional morpheme D. free morpheme 2. 2.The compound word “bookstore” is the place where books are sold. This indicates tha t the meaning of a compound __________. A. is the sum total of the meaning of its components B. can always be worked out by looking at the meanings of morphemes C. is the same as the meaning of a free phrase. D. None of the above. 3.The part of speech of the compounds is generally determined by the part of speech of __________. A. the first element B. the second element C. either the first or the second element D. both the first and the second elements. 4._______ are those that cannot be used independently but have to be combined with other morphemes, either free or bound, to form a word. A. Free morphemes B. Bound morphemes C. Bound words D. Words 5._________ is a branch of grammar which studies the internal structure of words and the rules by which words are formed. A. Syntax B.Grammar C. Morphology D. Morpheme 6. The meaning carried by the inflectional morpheme is _______. A. lexical B. morphemic C. grammatical D. semantic 7. Bound morphemes are those that ___________. A. have to be used independently B. can not be combined with other morphemes C. can either be free or bound D. have to be combined with other morphemes. 8. ____ modify the meaning of the stem, but usually do not change the part of speech of the original word. A. Prefixes B. Suffixes C. Roots D. Affixes