Overview of the Oldest Existing Set of Substrate-optimizedAnaerobic Processes: Digestive Tracts

Jean-Jacques Godon & Laure Arcemisbéhère &

Renaud Escudié & Jérôme Harmand & Edouard Miambi &Jean-Philippe Steyer

Published online: 25 May 2013#

Abstract Over millions of years, living organisms have ex-plored and optimized the digestion of a wide variety of sub-strates. Engineers who develop anaerobic digestion processesfor waste treatment and energy production can learn muchfrom this accumulated ‘experience’. The aim of this work is asurvey based on the comparison of 190 digestive tracts(vertebrate and insect) considered as ‘reactors’ and their an-aerobic processes. Within a digestive tract, each organ ismodeled as a type of reactor (continuous stirred-tank, suchreactors in series, plug-flow or batch) associated with chemi-cal aspects such as pH or enzymes. Based on this analysis,each complete digestion process has been rebuilt and classi-fied in accordance with basic structures which take into ac-count the relative size of the different reactors. The resultsshow that all animal digestive structures can be groupedwithin four basic types. Size and/or position in the structureof the different reactors (pre/post treatment and anaerobicmicrobial digestion) are closely correlated to the degradabilityof the feed (substrate). Major common features are: (i) grind-ing, (ii) an extreme pH compartment, and (iii) correlationbetween the size of the microbial compartment and the de-gradability of the feed. Thus, shared answers found by animalsduring their evolution can be a source of inspiration forengineers in designing optimal anaerobic processes.

Keywords Animal mimicry . Anaerobic digestion .

Digestive tract . Degradability

Introduction

The degradation efficiency of animal digestive tracts is obvi-ous. As an example, some insects can process up to 400 kg ofcarbon organic demand (COD).m−3.d−1, whereas the loadingrate of industrial reactors treating organic solids is usually only4 kg of organic dry matter .m−3.d−1, and can reach up to 10 kgof organic dry matter .m−3.d−1 for high-solids technology [1].Over millions of years, animals have developed sophisticateddigestive tracts to recover useful molecules from organic mat-ter. More recently, in less than 100 years, (human) engineershave developed a large range of different digesters to recoverenergy from biomass. The line of enquiry pursued in thisarticle is how animals have proved better than engineers atdeveloping anaerobic digestion processes. This analysis shouldprovide guidelines for future improvements of anaerobic di-gestion processes based on a bio-mimetic approach [2, 3].

The forms of digestive systems are highly diversified, andhave become more efficient and complex through the evolu-tionary process. First to appear was the production of exteriorenzymes, and most fungi still rely on it. In this process,enzymes are secreted by the organism into the surroundingenvironment where they break down organic matter; some ofthe products of these enzymatic reactions then diffuse back tothe organism. Later, animals evolved, acquiring a vesicle or asac-like structure in which digestion took place. Thus, theabsorption of hydrolyzed products by the organisms wasimproved by the action of several specialized organs; thephysico-chemical environment in which the enzymatic reac-tion took place was more efficiently controlled. Further alongin the evolutionary process, in the sac-like structure, alloch-thonous anaerobic microorganisms partially replaced the en-zymes for the hydrolysis of organic matter (i.e., 100 trillionmicrobes in the human colon [4]). Thus anaerobic digestionwas born and, over time, each animal has selected its own‘domesticated’ microbial consortia and evolved in concert

J.<J. Godon (*) : L. Arcemisbéhère :R. Escudié : J. Harmand :J.<P. SteyerINRA, UR0050, Laboratoire de Biotechnologie de l’Environnement,Avenue des Etangs, Narbonne 11100, Francee-mail: godon@supagro.inra.fr

E. MiambiUMR211 – BIOEMCO, Equipe Interactions Biologiques dans lesSols, IBIOS, Université Paris Est Créteil (U-PEC),61 avenue du Général de Gaulle, 94010 Créteil, France

Bioenerg. Res. (2013) 6:1063–1081DOI 10.1007/s12155-013-9339-y

European Union 2013

with them [4]. Associated with such changes, during theanimals’ evolution, several other parameters have been se-lectively evolved to improve the degradation of biomass:cutting and grinding, chemical treatment, size and shape ofthe digestive organs, water content, retention time [5].

Thus, over time, animals from different and independentlineages have carried out billons of ‘tests’ on different sub-strates, powerfully developed by natural selection. In compar-ison, engineers are only able to carry out relatively few tests,costly in time and money, by using empirical run-of-the-millmicrobial inocula. The main challenge to both systems is thehydrolysis of organic matter, and animals and engineers con-verge on several points: (i) anaerobic microbial consortia arethe least costly choice for degrading biomass, (ii) the type oforganic matter to be degraded (e.g., solid, liquid, easily-degradable or not) leads to different types of reactor, and(iii) the integration of physical and/or chemical treatmentbefore or after microbial degradation can improve the overallprocess (e.g., pH, temperature, grinding).

Assuming that animals have already ‘optimized’ the anaer-obic digestion of all existing substrates, the purpose of thissurvey is to analyze the features of animal digestive tracts. Theoriginality of this survey is that it considers substrates andanimal digestive tracts as digestive processes without takinginto account any other aspect (e.g., organ evolution, phylog-eny). This survey, based on available physiological studies,was organized as follows. First, we reduced animal digestivetracts to a sequence of basic reactor-like units. To this end, wetried to free our analysis of the classic nomenclature used inphysiological studies by considering the function of the organinstead of its position based on comparative evolution. Then,from the basic reactor-like units, the structure of the variousoverall digestion processes was rebuilt and classified into afew basic structures. Finally, these basic structures wereranked according to the degradability of the substratesingested by animals.

Deconstruction: from Digestive Tract to a Sequenceof Reactors

Shape Classification of different Organs

The first step in the bibliographic synthesis was to analyze alldigestive tracts composed of several organs as though theywere digestion processes including a sequence of basic reactorunits. The structure and the design of each such ‘element’ hadto be classified. For this, we assumed that all organs werecylindrical, and anatomical drawings were used to obtain thelength/width ratio. Based on this length/width ratio, we clas-sified vertebrates (size: 10 g to 10 tons) into three shapecategories: (i) a ratio ranging from one to five corresponds toa sac-like shape, (ii) a ratio with an order of magnitude of 10

corresponds to a large tubular shape, (iii) a ratio with an orderof magnitude of 100 corresponds to a narrow tubular shape.For insects (mostly termite size, less than 0.1 g), a ratioranging from one to five corresponds to a sac-like shape,and a ratio greater than five corresponds to a narrow tubularshape. Taking the human digestive tract as an example, thestomach was considered as a sac-like shape, the large intestineas a large tubular shape, and the small intestine as a narrowtubular shape (Fig. 1).

From Organ to Reactor: Modeling the Type of Reactor

The mixing mode and fluxes are important parameters to takeinto account when modeling and designing a digestive tract asa process. Flow-rate is ensured by peristalsis in the largetubular shape. In addition, in the sac-like shape the substrateis mixed homogeneously by contractions. Two types of sac-like shape exist: a sac-like shape with in- and outflow at thesame position or close by (e.g., caecum in rabbit), here called‘closed sac-like shape’, or a sac-like reactor with in- andoutflow at distal position [e.g. rumen of bovids (cattle) orthe human stomach], here called ‘open sac-like shape’(Fig. 1).

To convert a digestive tract into a process, organs previ-ously described on the basis of their shape, flux, and mixingmode must be considered as "gut reactors". As explained byCaton and Hume [6], gut reactors do not have exactly thesame characteristics as those defined in chemical engineering,notably because of the complex phenomena used by livingorganisms to maximize the efficiency of the transportation ofreactants through the intestinal gut, and because of the con-tinuous absorption of chemicals by the reactor walls (intestinalepithelium). Based on the classic chemical engineering ap-proach [7], two kinds of ideal continuous so-called reactorsare defined: the plug flow reactor (PFR) through which thefluid is assumed to progress as small parallel "slices" in theradial direction without exchanges between them; and thecontinuous stirred-tank reactor (CSTR) [8, 9], where the fluidis perfectly mixed.

In addition, whenever processes are not run continuously,operations can be differentiated as semi-continuous and batch.Some real systems can be modeled as combinations of suchideal reactors. For instance, a real tubular reactor exhibitsdiffusion which can be formalized on the basis of ainterconnected continuous stirred-tank reactor with recyclingfluxes to take into account back-mixing in the reactor.

In this work, in order to convert digestive systems intoreactors, we have only considered four types of idealizedreactor or their combinations.

& In a first approximation, depending on the characteris-tics of input and output flows, sac-like organs wereassimilated to either batch-reactor completely-mixed

1064 Bioenerg. Res. (2013) 6:1063–1081

systems or to CSTR. In practice, differentiating betweenthese two options was largely based on the location ofthe input with respect to the output: if they were distinct,a sac-like organ was considered to be a CSTR (e.g.,simple stomachs or ruminant forestomachs) while abatch reactor was retained used if the input and outputwere near close to each other, or the same (e.g., caecum).

& For long organs, a representation based on a PFR orCSTRs in series was adopted. The choice depended onthe ratio between their length and diameter, on thecontinuous/discontinuous mode, and on the uni/bi-direc-tional character of the fluxes, On the one hand, the smallintestine was modeled by a perfect PFR; on the other,more complex organs in which gentle mixing takesplace (e.g., large intestine) were approximated as a com-bination of a limited number of batch reactors or con-tinuous stirred-tank reactors. A PFR is equivalent to aseries of an infinite number of CSTRs. For simulationpurposes, ten to 30 reactors of the same volume areconsidered sufficient, and only two or three are neededif their volume is well-chosen. Thus, in practice thenumber of CSTRs used depends on their application.For the sake of simplicity, the representation of suchorgans has thus been reduced to an undefined finiteseries of CSTRs.

Other Specialized Compartments

Some animals also possess digestive organs that serve toseparate. These compartments can separate liquids from

solids or sort solids according to their size. For example,the bovids recycle the larger food particles from the rumento the mouth to facilitate the reduction in particle size. Inrabbits, particles are separated by a different mechanism: anorgan (fusus coli) located between the proximal and thedistal colon sends small particles back to the caecum,whereas larger indigestible particles continue on to the distalcolon.

Storage organs exist in some animals at different placesalong the digestive tract: hamsters have capacious cheekpouches, birds have a crop [10], the hematophagous bathas a modified expendable stomach, the emperor penguinand rabbit use the stomach, sloths and bees store their fecesin a specialized organ. A crop is also present in some in-sects, and in ants it has the role of a reservoir for ‘socialized’food. In the case of honeypot ants (genus Myrmecocystus),the reservoir is capable of great distension [11].

Finally, almost all animals have some form of press alongthe digestive tract to compress the undigested particles,reabsorb water, and form excrement.

Treatment for Hydrolyzing Organic Matter in a Reactor

Mechanical (Cutting, Grinding)

In every reactor, the substrate undergoes some form ofmechanical treatment as it passes through the digestive tractto facilitate access to further chemical and enzymatic treat-ment. Cutting and grinding can be carried out in specialized

Fig. 1 Correspondencebetween organ shape, organname, modelized reactor, andreactor scheme

Bioenerg. Res. (2013) 6:1063–1081 1065

organs upstream where cutters and/or grinders may be teeth(e.g., mammals) or mandibles (e.g., arthropods) inside themouth. Grinding can be carried out in an open sac-like reactorfunctioning as a grinder, as in the gizzard of birds for example,where grinding is facilitated by sand or pebbles. Keratinizedepitheliums may also serve as a rasp and help in grinding in,for example, a tubular reactor (e.g., the foregut of termites).

Chemical

Various compartments of the digestive tract are associated withchemical treatment, including exposure to acidic or alkalinepH conditions and digestive enzymes under a controlled tem-perature. These two types of chemical treatment are intimatelylinked.

pH

The pH of the different digestive compartments is crucial in thedigestion of some compounds. Different pHs, ranging from 1to 12, occur depending on the degradation function. In almostall animals, one compartment only along the digestive tract isdedicated to extreme pH (e.g., the stomach or similar organ invertebrates and the first segment of the termite hindgut). Mostvertebrates use an acid pH (pH 1 to 3), whereas some arthro-pods use a basic pH (pH 10 to 12.5) [12–15] (e.g., for ligno-cellulosic materials, both alkaline and acid pretreatment seemto have a similar effect on the cleavage of ester bonds [16]).

Enzymes

Enzymes are the main agent in degrading organic matter. Theyare present in different organs and are produced in two differ-ent ways: either by animal cells or by symbiotic microbe cells.

Enzymes produced and secreted by an organism’s ownglands are used to catalyze the digestion of dietary constit-uents such as amino acids, proteins, lipids, saccharides, andpolysaccharides. Some organisms, including spiders andflies, simply secrete digestive enzymes into the extracellularenvironment prior to ingestion of the resultant ’mixture‘. Inothers, catalytic reactions begin in the mouth with salivaryenzymes such as α-amylases, followed by other reactions inthe stomach where gastric enzymes such as pepsins aresynthesized. Digestion is further helped by the action ofintestinal secretions combined with pancreatic juice. Suchjuices contain different enzymes, including lipases, malt-ases, saccharases, lactases, pancreatic amylases, peptidases(trypsine and chymotrypsine), phosphatases, nucleosidases,and nucleases. Host enzymes are secreted into organs thatdo not contain microbes. In this way, animals optimize thecost of enzyme production and avoid competition withmicrobes for hydrolysed substrates. In addition, the optimalefficiency of enzymatic reactions is related to the pH of the

organ and to the body temperature of the animal (ectothermicor endothermic organisms).

Microbial reactors such as the gastrointestinal tract arecolonized by microbes just after birth [4]. These anaerobicsymbiotic micro-organisms produce both endogenous andexogenous enzymes. Exogenous enzymes permit the break-down of large molecules such as proteins and polysaccha-rides (i.e., cellulose, chitin, etc.) into shorter molecules.These molecules can then be taken up either by the epithelialcells of the host or by microbes. Microbial cells hydrolyzeshorter molecules with endogenous enzymes and producebiomass and by-products such as short-chain fatty acids(acetate, propionate, and butyrate) but also lactate, CO2,etc. These molecules can be taken up either by the epithelialcells of the host or by other microbes within the trophic foodweb. The contribution of microbes to the production andconservation of nutrients has been reviewed by Stevens andHume [17].

Temperature

Depending on their environment, the temperature of somepoikilothermic animals with active digestion can vary from−2 °C (e.g., Antarctic icefish [18] ) to 50 °C (e.g., the ant inthe Sahara desert [19]). Even so, during evolution, the regu-lation of body temperature appeared in at least two lineages(birds and mammals). In both these lineages, temperaturesremain within a mesophilic range, from 32 °C to 42 °C:between 32 °C–36 °C for primitive mammals (e.g., mono-tremes and marsupials), 37 °C–39 °C for most other mam-mals, and 39 °C–42 °C for birds. When regulated at amesophilic temperature, the yield of the digestive processincreases, but being warm-blooded is costly in energy.Futhermore, for a similar weight mammals and birds havemuch greater energy requirements than reptiles. In manycases, enzymes (from the host or from bacteria) are adaptedto body temperature [20]. Moreover, the mass-specific re-quirement of homeothermic animals is related to body mass[21].

Combination of Reactor Types and Treatment Selectedby Animals

To hydrolyze organic matter within the four reactor types previ-ously defined, three different pH conditions (acid, neutral, basic)and two different origins of enzymes (from the host, frommicrobes) are possible. The results give 24 combinations.Among these 24 possible reactor units, only eight have actuallybeen selected during animal evolution (Fig. 2); three others existas exceptions (see section “Other original features”). Withinthese eight, only three basic reactors are widely colonized bymicrobes (bacteria and archaea). Their growth is possible under

1066 Bioenerg. Res. (2013) 6:1063–1081

neutral pH and longer retention times. These microbes aresometimes associated with indigenous protozoa and fungi, asin the ruminant and lower termite groups [22, 23].Within reactorunits, host enzymes and microbial enzymes are never presenttogether; suchmutual exclusiveness avoids competition betweenthe host and microbes for the use of the hydrolyzed compoundsproduced by the enzymatic reactions. In fact, microbial popula-tion growth is ‘controlled’ through the type and condition of thebasic reactor. This control can be ensured by either extreme acidor basic pH in CSTR or batch reactors. Rapid transit (retentiontime) also limits the growth of microorganisms. Acid CSTRs arepresent in almost all vertebrates, but exclusively within thevertebrate group. Basic CSTRs are present in some arthropodgroups, including the larvae and adults of butterflies, scarabbeetles, higher termites, and nematoceran diptera [13], but onlywithin the arthropod group [15].

The volume of the different organs is also a key parameterin hydrolytic reactions, because the time needed for the deg-radation of the substrate in an organ’s specific operatingconditions is related to such volume. To determine the relativescale of each reactor unit within the process, the organs of 190animals were measured by analyzing anatomical drawings ofdigestive tracts. It was assumed that the organs were cylindri-cal in shape. Some of the most relevant results are summarizedin Table 1. Taking into account the variation within an animalspecies of the volume of each organ, and the low accuracy ofthe estimates made, volumes indicated in this table must beconsidered as a rough indication only.

From Reactor to Process: Definition of Basic Structures

The next challenge was to determine how the eight types ofreactor unit identified are arranged together to form a digestive

tract. We have called these eight types: acidic CSTR, acidicbatch, alkaline CSTR, alkaline batch, microbial CSTR, mi-crobial CSTR in series, microbial batch, and enzymatic PFR.The relative sizes of the different reactor units within thedifferent overall structures were also considered. Finally, tocorrelate overall structure and the size of a reactor unit, animalphylogeny and animal diet were taken into account.

This analysis of animal digestive tracts has permitted theirgrouping into four basic structures incorporated in the differ-ent reactor units (Fig. 3). Almost all the 190 digestive tractsconsidered belong to the following schematized structures:

& A ‘human-like structure’ composed of, first, an acidicCSTR, followed by an enzymatic PFR and a microbialCSTR in series.

& A ‘bovid-like structure’ starting with a microbial reactor,then followed by the same structure as in the ’human-likestructure’. The configuration of this pre-fermenter can be abatch reactor, a CSTR, CSTRs in series, or a combinationof all these types.

& An ‘ant-like structure’ made up of an enzymatic PFR,followed by a series of microbial CSTRs.

& A ‘termite-like structure’ starting with an enzymatic PFR,then followed by an alkaline CSTR and a microbial CSTR.

The first two structures are characteristic of the vertebratasubphylum, while the others are typical of the hexapodasubphylum. These structures are analyzed in the next section.

From Diet to the Biodegradability of Substrates

Traditionally, animals are roughly classified into three maingroups according to their diet: carnivorous, herbivorous, and

Fig. 2 Combinations betweenprevailing pH, enzymaticactivity and type of reactorevolved by animals. Squares,horizontal rectangles and theseries of vertical rectanglescorrespond to, respectively,CSTR, PFR, and CSTRs inseries. Black, grey and whitecorrespond to, respectively,basic, neutral and acid pHconditions. Dark grey spotsindicate the presence of largemicrobial communities. Arrowscorrespond to the location ofinput and output within CSTRand batch reactor. Xcorresponds to no occurrence inanimal gut. O corresponds torare occurrence in animal gut

Bioenerg. Res. (2013) 6:1063–1081 1067

Tab

le1

Volum

eof

thedifferentcompartmentsof

thedigestivetract.Animalsareclassified

basedon

thestructureof

thedigestivetract

Class

Order

Species/Latin

name

Com

mon

name

Weight/

Kg

Degradability

Feed

Volum

ereactor/totaldigestivetractvolume%

Enzym

atic

PRF*

Microbial

reactor

CSTR**

Enzym

atic

PRF*

Microbial

reactor

CSTR**

Microbial

total

Hum

anstructure/molesub-structure

Fish

Perciform

esScarus

rubroviolaceus

Ember

parrotfish

5Easily

Benthic

algae

100

0

Fish

Mugiliform

esMugilcephalus

Mullet

8Easily

Benthic

algae

59a

410

Fish

Perciform

esAcanthurus

nigrofuscus

Brown

surgeonfish

0.3

Easily

Benthic

algae

2575

0

Mam

mal

Chiroptera

Myotis

lucifugus

Little

brow

nbat

0.01

Moderate

Insect

7624

0

Mam

mal

Insectivora

Talpaeuropaea

Com

mon

Mole

0.1

Easily

Earthworms

8416

0

Mam

mal

Marsupialia

Dasyurus

maculatus

Tiger

quoll

2.5

Easily

Insects,birds,

small

anim

als

7426

0

Hum

anstructure/ostrichsub-structure

Mam

mal

Carnivora

Phoca

vitulin

aCom

mon

seal

130

Easily

Fish

8019

22

Bird

Accipitriformes

Buteo

jamaicensis

Red-tailed

Haw

k1

Easily

Small

mam

mals

b79c

174

4

Bird

Psittaciform

esMelopsitta

cus

undulatus

Budgerigar

0.03

Moderate

Seed

b66c

304

4

Bird

Casuariiformes

Dromaius

novaehollandiae

Emu

38Moderate

Seed

52c

432

46

Mam

mal

Carnivora

Ursus

americanus

Black

bear

100

Moderate

Omnivorous

6527

77

Reptile

Squam

ata

Crotalus

adam

anteus

Rattlesnake

2.3

Easily

Small

mam

mals

8111

88

Reptile

Crocodilia

Caiman

crocodilu

sCaiman

40Moderate

Insects,

crustaceans

7215

1313

Mam

mal

Carnivora

Canisfamiliaris

Dog

30Easily

Small

mam

mals

3848

h13

13

Mam

mal

Monotremata

Tachyglossus

aculeatus

Echidna

4Moderate

Antsand

term

ites

6422

1414

Amphibien

Anura

Bufoam

ericanus

Toad

0.02

Moderate

Insect

6419

1717

Monotremata

Monotremata

Ornith

orhynchus

anatinus

Platypus

1.5

Easily

Wormsand

insectlarvae

2457

1818

Mam

mal

Carnivora

Procyon

lotor

Raccoon

5Moderate

Omnivorous

3052

1818

Mam

mal

Cetacea

Physetercatodon

Sperm

whale

50000

Slowly

Squid

and

fish

3247

2020

Mam

mal

Carnivora

felis

sylvestris

Cat

4Easily

Birds

and

small

anim

als

5716

h25

26

Reptile

Squam

ata

Amblyrhynchus

cristatus

Marineiguana

1.5

Moderate

Seaweedand

algae

52c

1631

31

Mam

mal

Primates

Hom

osapiens

Omnivore

50Moderate

Omnivorous

4420

h35

36

1068 Bioenerg. Res. (2013) 6:1063–1081

Tab

le1

(con

tinued)

Class

Order

Species/Latin

name

Com

mon

name

Weight/

Kg

Degradability

Feed

Volum

ereactor/totaldigestivetractvolume%

Enzym

atic

PRF*

Microbial

reactor

CSTR**

Enzym

atic

PRF*

Microbial

reactor

CSTR**

Microbial

total

Mam

mal

Primates

Otolemur

crassicaudatus

Galago

1Moderate

Omnivorous

4510

539

44

Reptile

Testudines

Geochelone

carbonaria

Red-footed

tortoise

28Moderate

Omnivorous

4114

540

45

Reptile

Squam

ata

Iguana

iguana

Green

iguana

9Moderate

Leaves,

flow

ers,

fruit

3714

544

49

Mam

mal

Artiodactyla

Susscrofa

Pig

100

Moderate

Omnivorous

3016

747

54

Mam

mal

Primates

Pongo

pygm

aeus

Orangutan

75Moderate

Fruit

2714

h57

58

Mam

mal

Primates

Pan

troglodytes

Chimp

40Slowly

Omnivorous

2613

853

61

Mam

mal

Perissodactyla

Diceros

bicornis

Black

rhinoceros

1000

Slowly

Herbivorous

354

952

61

Bird

Struthioniformes

Struthio

camelus

Ostrich

100

Moderate

Seeds,

shrubs,

grass,fruit

16c

178

5967

Mam

mal

Proboscidae

Loxodonta

africana

African

bush

elephant

5000

Slowly

Herbivorous

266

1058

68

Mam

mal

Marsupialia

Vombatusursinus

Wom

bat

30Slowly

Herbivorous

179

7474

Hum

anstructure/horsesub-structure

Bird

Galliformes

Gallusdomesticus

Chicken

1Moderate

Omnivorous

b57c

2811

415

Mam

mal

Sirenia

Dugongdugon

Dugong

500

Slowly

Sea-grass

813

97

16

Mam

mal

Marsupialia

Isoodonmacrourus

Bandicoot

1.2

Moderate

Omnivorous

1935

1333

46

Mam

mal

Rodentia

Cavia

porcellus

Guineapig

0.7

Slowly

Herbivorous

336

583

61

Mam

mal

Lagom

orpha

Oryctolagus

cuniculus

Rabbit

1Slowly

Herbivorous

345

5012

62

Bird

Rheiformes

Pterocnem

iapennata

Rhea

20Slowly

Omnivorous

11c

1469

675

Mam

mal

Marsupialia

Phascolarctos

cinereus

Koala

10Slowly

Herbivorous

154

4041

81

Mam

mal

Perissodactyla

Equus

caballu

sHorse

500

Slowly

Herbivorous

4h

3163

94

Mam

mal

Rodentia

Hydrochoerus

hydrochaeris

Capybara

60Slowly

Herbivorous

42

922

94

Hum

anstructure/sea-chubssub-structure

Fish

Perciform

esKyphosus

sydneyanus

Silv

erdrum

mer

10Slowly

Benthic

algae

1826

56d

56

Reptile

Squam

ata

Saurom

alus

obesus

Chuckwalla

9Slowly

Herbivorous

318

54d

762

Mam

mal

Hyracoidea

Heterohyrax

brucei

Rockhyrax

4Slowly

Herbivorous

228

64d

569

Cow

structure

Mam

mal

Primates

Colobus

abyssinicus

Colobus

monkey

9Slowly

Herbivorous

64e

815

h12

77

Bioenerg. Res. (2013) 6:1063–1081 1069

Tab

le1

(con

tinued)

Class

Order

Species/Latin

name

Com

mon

name

Weight/

Kg

Degradability

Feed

Volum

ereactor/totaldigestivetractvolume%

Enzym

atic

PRF*

Microbial

reactor

CSTR**

Enzym

atic

PRF*

Microbial

reactor

CSTR**

Microbial

total

Mam

mal

Marsupialia

Macropusgiganteus

Eastern

grey

kangaroo

66Slowly

Herbivorous

81e

133

h2

84

Mam

mal

Artiodactyla

Hippopotamus

amphibius

Hippo

1500

Slowly

Herbivorous

83f

66

487

Mam

mal

Artiodactyla

Bos

taurus

Cow

800

Slowly

Herbivorous

87d

32

24

93

Mam

mal

Edentata

Bradypustridactylus

3-toed

sloth

3Slowly

Herbivorous

89d

15

594

Mam

mal

Artiodactyla

Llamaglam

aLam

a130

Slowly

Herbivorous

92d

21

496

Bird

Opisthocomiformes

Opisthocomus

hoazin

Hoatzin

1Slowly

Herbivorous

91d

3c2

22

95

Termite

structure

Termite

Apicoterm

itinae

Labidotermes

celisii

>0.0001

Slowly

Soil-feeder

335

6262

Termite

Apicoterm

itinae

Trichoterm

esvillifrons

>0.0001

Slowly

Soil-feeder

122

8686

Termite

Nasutiterm

itinae

Nasutiterm

escostalis

>0.0001

Slowly

Wood-feeding

1823

5959

Termite

Termitinae

Noditerm

eslamaniasus

>0.0001

Slowly

Soil-feeder

337

655

61

Termite

Termitinae

Probosciterm

estubuliferus

>0.0001

Slowly

Soil-feeder

645

148

49

Termite

Termitinae

Microceroterm

esstrunckii

>0.0001

Slowly

Woodfeeding

514

8181

Termite

Termitinae

Foram

initerm

estubifrons

>0.0001

Slowly

Soil-feeder

548

4747

Ant

structure

Termite

Hodotermitidae

Hodotermes

mossambicus

>0.0001

Slowly

Grass-feeder

1090

90

Termite

Rhinoterm

itidae

Coptoterm

esgestroi

>0.0001

Slowly

Woodfeeding

694

94

Termite

Macroterm

itinae

Macroterm

eslilljeborgi

>0.0001

Slowly

Grass-feeder

35g

6565

*,PFRcorrespo

ndsto

PlugFlow

Reactor;**

,CSTRcorrespo

ndsto

continuo

usstirred-tank

reactor.a,correspo

ndto

noacid

compartment;b,

crop

considered

justas

astoragecompartmentisno

tinclud

ed;c,thevalueinclud

edtwoacid

compartments(gizzard

andstom

ach);d,

continuo

usstirred-tank

reactor(CSTR);e,CSTRin

series;f,batchreactor;g,

fung

alextern

reactorin

additio

n;h,

reactorpresentsbu

tthevo

lumeisless

than

1%

oftotaldigestivetractvo

lume

1070 Bioenerg. Res. (2013) 6:1063–1081

omnivorous. This classification does not take into account thedegradability of substrates. A carnivorous diet can includemeat, an easily-degradable substrate rich in proteins andlipids, and crustaceans, rich in slowly-degradable chitin.Similarly, an herbivorous diet can include plant fibers, richin slowly-degradable cellulose, and green algae or fruits,which are rich in easily-degradable cellular content. In thisreview, because the approach has been to consider a digestivetract as an anaerobic digestion process, a classification bysubstrate biodegradability has been adopted to establish threegroups: (i) easily-degradable substrate, such as meat, readily-accessible polysaccharides (fruit, roots, nectar, some algae),(ii) slowly-degradable substrates, mostly plant or animalstructural polysaccharides (grass, leaves, wood, crustaceans),and (iii) moderately-degradable substrates, mainly reservepolysaccharides (seeds) and a mix of slowly- and easily-degradable substrates in various quantities, formerly groupedas omnivorous (some examples are given in Table 1).Figure 4a presents the relative size of the reactor-like organscorresponding to the three types of hydrolysis (microbe, pH,and enzyme) according to the degradability of the substrate.

Structure and Biodegradability of Substrates

Human-like Structure

In this structure, the basic sequence is acidic CSTR – enzy-matic PFR – microbial CSTRs associated in series. The mi-crobial reactor is never of the batch type, and the size of themicrobial CSTRs associated in series varies from completeabsence to very large. The human-like structure is reportedthroughout all classes of vertebrates, whatever the diet. Themajor feature of this structure is the strong correlation between

the relative volume of each reactor and the diet of the animal(Table 1). Animals consuming easily-degradable nutrientshave no (or a very small) microbial CSTR and usually avoluminous acidic CSTR. In contrast, animals consumingslowly-degradable food have a very large microbial reactorand a small acidic CSTR. Animals with an intermediate diet(moderately-degradable) have compartments of a similar size.Four sub-structures from this basic digestive structure can bedefined (Fig. 5), and they differ in the type of the mainmicrobial reactor: none, CSTR in series, batch reactor orCSTR:

– The ‘mole sub-structure’: acidic CSTR – enzymatic PFR(Fig. 5), is characteristic of animals that consume easily-degradable substrates, such as fish that eat green algaeand some mammals (insectivorous bat, mole, spotted-tailed quoll, phascogale) [12]. Digestion occurs mainlythrough chemical reactions. The main feature of this sub-structure is the absence of a microbial reactor. The chem-ical compartment is very large, and represents from 60 %to 84% of the total volume of the digestive tract (Fig. 4b).Moreover, for some fish species, the CSTR reactor isabsent, and when present has a neutral pH (Table 1) [17].

– The ‘sea-chub sub-structure’: acid CSTR – enzymaticPFR – microbial CSTR, is found in herbivorous fishbelonging to the Kyphosidae family that eat red andbrown algae, as well as in at least one herbivorous mam-mal (rock hyrax) [12]. The microbial CSTR representsabout 60 % of the digestive tract (Table 1 and Fig. 4b).

– The ‘ostrich sub-structure’: acidic CSTR – enzymaticPFR – microbial CSTR in series (Fig. 5), is found in alladult amphibians (toad, salamander) that eat easily-,moderately- or slowly-degradable substrates, some rep-tiles (green iguana), some birds (ostrich), and somemammals (human, dog, rhinoceros, elephant). A micro-bial batch reactor is also often found, but of a small size,like a vestigial organ (e.g., the appendix in humans; seesection “Other original features” for the potential role ofthis organ). The size of the acidic CSTR ranges from16 % (ostrich) to 80 % (seal), the size of enzymatic PFRfrom 4% (rhinoceros) to 57 % (platypus), and the sizes ofthe microbial reactor (CSTR in series and a batch reactor)from 2% (seal) to 74% (wombat). In fact, the relative sizeof the three compartments is related to the food: (i) easily-degradable substrates associated with the dominance ofthe acidic CSTR, (ii) moderately-degradable substratesassociated with the dominance of the enzymatic PFR,and (iii) slowly-degradable substrates associated withthe dominance of the microbial CSTR.

– The ‘horse sub-structure’: acidic CSTR – enzymatic PFR–microbial batch reactor and microbial CSTR in series, isfound in those species of birds and mammals havingeasily-, moderately- and mainly slowly-degradable diets

Human-like structure

Cow-like structure

Ant-like structure

Termite-like structure

Fig. 3 The four basic digestive structures. The Squares, horizontalrectangles and the series of vertical rectangles correspond to, respec-tively, CSTR, PFR, and CSTRs in series. Black, grey and whitecorrespond to, respectively, basic, neutral and acid pH conditions.Dark grey spots indicate presence of large microbial communities

Bioenerg. Res. (2013) 6:1063–1081 1071

(Table 1 and Fig. 4b). The difference from the previousostrich sub-structure is its larger microbial batch reactorcompared to the microbial CSTR in series. The thresholdwe adapted to distinguish between these two sub-structures was a volume of the microbial batch reactorgreat than 10 % of the total digestive-tract volume andgreater than that of the microbial CSTR in series. It isinteresting to note that two birds belonging to the ratitegroup (ostrich and rhea) and sharing the same diet havetwo different sub-structures (ostrich and horse sub-structures, respectively). The horse sub-structure is mainlyobserved in glires (rodents and lagomorphs) and Equidae.The size of the acidic CSTR varies from 4 % (capybara,horse) to 81 % (dugong), the size of the enzymatic PFRfrom 2 % (capybara) to 35 % (bandicoot) and the size ofthe microbial batch reactor from 9 % (dugong) to 92 %

(capybara). The relative size of the three compartments,similar to the ostrich sub-structure, is related to the food:(i) easily-degradable substrates associated with the domi-nance of the acidic CSTR (sea grass with dugong), (ii)moderately-degradable substrate associated with the dom-inance of the enzymatic PFR (omnivorous with bandi-coot), and (iii) slowly-degradable substrate associatedwith the microbial batch reactor (grass with capybara).Moreover, this last sub-structure includes lagomorphs androdents, which are also characterized by an additionaldigestion phase through their ability to re-eat feces(described in section “Recycling”).

To sum up, the process based on the human-like structurecan be described by the sequence of reactors (acidic CSTR –enzymatic PFR – microbial reactor), while the relative size of

80%

60%

40%

20%

0%

Microbe 100%

80%

60%

40%

20%

0%

Microbe 100%

80%

60%

40%

20%

0%

Microbe 100%

d

b

80%

60%

40%

20%

0%

Microbe 100%

c

a

Fig. 4 The three-componenttriangles show the relative sizeof the reactor-like organscorresponding to the three typesof hydrolysis (enzyme, pH ormicrobial). a Shows resultsaccording to the degradabilityof the food: pictograms of leaf,wheat and ham correspond toanimals that eat slowly-digested, moderately-digested,or easily-digested foodrespectively. b Shows resultsaccording to the different typesof digestive structure : animalsilhouettes correspond to thetype of structure or sub-structure (bovid, termite, ant,mole, sea-chub, ostrich andhorse; sections “From reactor toprocess: definition of basicstructures” and “Structure andbiodegradability of substrates”).c Shows the results according tothe different sizes of animals:white circles correspond toweight below 1 g, the blackcircles increase in size tocorrespond to higher weights:30 g to 1 kg, 1–10 kg, 10–100 kg, 100–500 kg, up to500 kg. d Shows the resultsaccording to the phylogeny ofeach animal (insect, fish,reptile, bird, monotreme ormammal) represented bycorresponding pictograms:butterfly, fish, gecko, finch,platypus and rabbit

1072 Bioenerg. Res. (2013) 6:1063–1081

the microbial compartment is correlated to the degradability ofthe substrate. In this structure, the microbial compartmenttakes the form of either a CSTR, CSTRs in series, or a batchreactor.Within the human-like structure, the different stages ofsubstrate breakdown are: (i) grinding to improve the sub-strate’s accessibility to enzymes, (ii) acid and host-enzymatichydrolysis in an acidic CSTR, (iii) host-enzymatic hydrolysisin a PFR, and (iv) microbial enzymatic hydrolysis in a micro-bial reactor (CSTR, CSTRs in series, or batch reactor). In thehuman-like structure, it is important to note that both acidand host-enzymatic hydrolyses take place prior to microbialenzymatic hydrolysis.

Bovid-like Structure

The basic sequence is microbial reactor – acidic CSTR –enzymatic PFR – microbial CSTRs in series. This structureis common to several groups of vertebrate. Most of thembelong to some phylogenetically-related family of theArtiodactylamorpha groups (ruminant, pseudo-ruminant,cetaceans, and hippopotamus) [24]. This structure is alsoreported for one bird genus (hoatzin) and for several otherfamilies of mammals including some Marsupials, Folivora,and Primates not groupedwithin the vertebrate phylogeny. Thisstructure suggests it might also be suspected in other animalssuch as the ostrich [25]. It is consequently a convergence during

evolution, driven only by the diet of slowly-degradable food-stuffs usually rich in cellulose, hemicelluloses, or chitin.

The main variation between the digestive tracts of theseanimals is in the shape of the different compartments of themicrobial reactor developed from the ‘classic’ human-likestructure (Fig. 6). Two types can be loosely identified: withinthe first type, which includes the Artiodactylamorpha groups(bovid, camel, grey whale, and hippopotamus) and a bird(hoatzin), the microbial upstream reactor is mostly a CSTR(Fig. 6). The second type includes the other mammals(kangaroo, colobus, and sloth) in which the microbial upstreamreactor is mainly CSTRs in series (Fig. 6).

This remarkable convergence within the evolutionaryprocess encompasses very different animals. However, inaddition to the type of microbial upstream reactor, somespecific features of the digestive process remain associatedwith phylogeny. For the South America hoatzin, the grind-ing phase occurs inside the bacterial CSTR (modified crop),as is the case for most birds (Fig. 6). The well-knownruminant (bovids and relatives) and pseudo-ruminant (llama,camel, and relatives) groups present an additional feature: theability to recycle digested food through the first microbialcompartment (described in section “Recycling”). It is alsointeresting to note that some whales, generally considered as

Mole sub-structure (mole example)

Sea chubs sub-structure (sea chubs example)

Ostrich sub-structure (rhinoceros example)

Horse sub-structure (rhea example)

Fig. 5 The sub-structures characterized in a human-like structure. Thein-line rectangles, unaligned rectangles, horizontal rectangles and theseries of vertical rectangles correspond to, respectively, CSTR, batchreactor, PFR, and CSTRs in series. Black, grey and white correspondto, respectively, basic, neutral and acid pH conditions. Dark grey spotsindicate presence of large microbial communities

Hoatzin example (bird)

Cow example (ruminant)

Kangaroo example (marsupial)

Colobus example (monkey)

Grey whale example (cetacean)

Fig. 6 Five examples of the bovid-like structure. The in-line rectan-gles, unaligned rectangles, horizontal rectangles and the series ofvertical rectangles correspond to, respectively, CSTR, batch reactor,PFR, and CSTRs in series. In the bovid example, diagonal linesindicate a solid–solid size separator (reticulum) and black and whiteshade-off indicates organ dedicated to water absorption (omasum).Black, grey and white correspond to, respectively, basic, neutral andacid pH conditions. Dark grey square spots indicate presence of largemicrobial communities. Black circles indicate the location of grindingin a microbial CSTR

Bioenerg. Res. (2013) 6:1063–1081 1073

carnivorous animals, belong to this structure, which is to beexplained mainly by the importance of the composition oftheir food. For example, the grey whale eats shrimp which arevery rich in chitin, a slowly-degradable carbohydrate whosedegradability potential makes it more similar to plants than tomeat [26].

To sum up, the process involving the bovid-like structurecan be described as using first a microbial reactor [CSTR(e.g., bovid), CSTRs in series (e.g., colobus) or batch reactor(e.g., hippopotamus)] where any easily-degradable sub-strates can be processed directly by the host. The firstmicrobial reactor generates microbial by-products, such asVFA, which are up taken by the host, and microbial biomasswhich is then partially digested in the final stage at the endof the structure (see human-like structure). In the bovid-likestructure, microbial enzymatic hydrolysis occurs before acidand host-enzymatic hydrolysis. The bovid-like structure is ahuman-like structure with an additional upstream microbialCSTR.

Ant-like Structure

The basic sequence is enzymatic PFR –microbial CSTR. It isobservedmainly in the class of hexapods including the termite(Isoptera), but it does not concern this entire insect group [13].For example, within the Isoptera order (termites), only lowertermites (all families), and higher termites from theMacrotermitinae family have this structure. Some higher ter-mites have also introduced a kind of symbiosis with a funguswhich they maintain as a pure culture in their nest (see belowsection “Termite-like structure”). In hexapods, grinding iscarried out by mandibles (instead of mammalian teeth). Theant-like structure can be summarized as being a human-likestructure without the acidic CSTR step.

Termite-like Structure

The basic sequence of this structure is enzymatic PFR – alkalineCSTR – microbial CSTR.

This structure is reported for the Isoptera order, but onlyin three families of higher termites (Apicotermitinae,Nasutitermitinae and Termitinae) [27]. An alkaline CSTR(pH ranging from 10 to 12.5) [15] and a large microbialCSTR are the main distinguishing features. All these ter-mites eat soils which are slowly-degradable substratescontaining micro-organisms and humic acids.

As with the human-like structure, the termite-like struc-ture starts with a host-enzyme hydrolysis and finishes withmicrobial hydrolysis. But it differs in that the enzymaticPFR precedes the pH CSTR, and the pH in this reactor isbasic instead of acid. Little information is available aboutthe presence or absence of host enzymes within the basicCSTR reactor.

Other 'Improvements'

Animals have thus developed original processes for opti-mizing the digestion of organic matter and its assimilation.In addition to the structures presented above, some speciesor groups of species have improved their digestive processwith new (modified) or additional organs, or new ‘processmanagement’. Some of these known improvements arepresented below.

Recycling

Within the bovid-like structure, substrates (mostly grass andleaves) are first ground and then digested in a microbial CSTR.In the following stage, the ruminants and pseudo-ruminants,represented by the bovid and the camel respectively, haveadopted an original strategy for optimizing their digestiveperformance. A specialized organ (called the reticulum) playsthe role of separator in which particles are sorted according totheir size: small digested particles are sent to the next reactor,whereas the biggest solids are regurgitated to the mouth to bereground again (Fig. 7). Thus, particles are sent to the follow-ing acidic CSTR (Fig. 7) only when their size is small enough.In the ruminant group, an additional compartment (called theomasum) provides re-absorption of water contained in themicrobial CSTR before entering the acidic stage (Fig. 7).

Recircularisation (cow (ruminant) example)

External reactor (termite example)

Ceacotrophy (rabbit example)

Fig. 7 Examples of improvement within the digestive process. The in-line rectangles, unaligned rectangles, horizontal rectangles and theseries of vertical rectangles correspond to, respectively, CSTR, batchreactor, PFR, and CSTRs in series. Diagonal lines on a square indicatea solid–solid separator. Black, grey and white correspond to, respec-tively, basic, neutral and acid pH conditions. Dark grey spots indicatepresence of large microbial communities. The water re-absorptioncompartment present in ruminants but absent in the pseudo-ruminantsis indicated with a star. Small white circles correspond to recirculatedrumen large particles. Small black circles correspond to wet feces(ceacotrophs)

1074 Bioenerg. Res. (2013) 6:1063–1081

This re-absorption compartment is, however, absent in thepseudo-ruminant groups (camelid group: camel, dromedary,and llama). This absence can be explained by the fact that inanimals from desert environments the microbial CSTR reactoruses less water than in ruminants.

Another original adaptation for optimizing digestive per-formance found in some animals is coprophagy or the re-ingestion of feces (Fig. 7). It occurs in several unrelatedmammals feeding on slowly-degradable substrates. The listincludes lagomorphs (rabbits, hares), some rodents (guineapig, rat, beaver, etc.), the folivorous lemur (Lepilemurmustelinus) and the ringtail possum [28]. Ingestion of fecesis also usual in all termites [29]. In the lagomorph group, thisadaptation includes the re-ingestion of a special type of fecestermed caecotrophs: in contrast to normal dry feces,caecotrophs are moist [12].

This structure presents features similar to those of thebovid-like structure, in that bacterial biomass grown onslowly-degradable substrates during their first transit throughthe digestive tract is then digested during its ‘recycling’ withinthe same process. This recycling facility allows animals torecover nutrients produced in the hindgut. Interestingly, mainlythe bovid structure not the rabbit structure occurs in largeranimals. In fact, small animals need more energy per unit ofbody mass. Per kg of body mass, a 25-g vole, for example,needs about 12 times more energy than a 650-kg horse[30, 31]. With the same type of food and the same rate offermentation, a vole has to eat 12 times more food. As aconsequence, if a constant ratio of animal mass to size ofdigestion track is assumed, the retention time of the foodsubstrate decreases drastically. Since a large reactor such as arumen is not possible for small animals (it would need to be 12times larger for the same retention time), some small herbivo-rous animals have to combine host-enzymatic digestion andmicrobial fermentation.

Isoptera, especially Macrotermitinae termites fed on fo-liage [27], display yet another original adaptation for opti-mizing the digestion of dietary compounds: they use anexternal reactor for digesting plant material (Fig. 7).Termites inoculate the substrate with one species of symbi-otic fungus, Termitomyces, in pure culture conditions.During this process, leaves are first cut by termites into‘confetti’ and then ingested. After a first fast transit, termitesplace feces-like deposits on the top of the fungus comb. Thefungi digest the matter before it is eaten again with the fungiby the termites. It is interesting to note that this externalreactor is bigger than the termite digestive tract alone, andthus permits a longer retention time. This adaptation in thetermite, which enables it to maintain a pure fungus culture,is not fully understood. Some authors suggest that termitemounds can also be considered as an external rumen [32].

Two other insects, the leaf cutter ant and the ambrosiabeetle, have also developed a fungus-based process to pre-

digest the substrate in an external reactor. Leaf cutter antsactively cultivate their fungi, which belong to the Lepiotaceaefamily, feeding them with freshly-cut plant material and keep-ing them free from pests and molds. Fungi, cultivated by theadults, are used to feed the ant larvae [33]. Ambrosia beetles,belonging to the weevil subfamilies Scolytinae andPlatypodinae (Coleoptera, Curculionidae), colonize and feedon the xylem (sapwood and/or heartwood) of dying orrecently-dead trees. They excavate tunnels in dead trees andcultivate fungal ‘gardens’, their sole source of nutrition. Thefungi penetrate the plant’s xylem tissue, which will bedigested and concentrate the nutrients on and near the surfaceof the beetle gallery. Beetles and their larvae graze on themycelium exposed on the gallery walls [34].

Very Long Small Intestine

In relation to slowly-degradable substrates, some animals havea very long small microbial PFR which seems to replace the n-series of CSTRs. This organ is found in the digestive tract ofthe Atlantic white side dolphin (Lagenorhynchus acutus), theblack bear (Ursus americanus) and the dugong (Dugongdugong) [12]. These three species have different feedinghabits; their food ranges from easily- to slowly-degradablesubstrates. The role of this organ remains unknown.

Other Original Features

Other original features can also be found in digestive tracts,without an obvious explanation of their role or impact on thedigestion process.

Within the monotreme group, platypus and echidna, andin some fish, the first CSTR reactor corresponding to thestomach of most of the placental mammals is not acidic, butpresents a neutral pH with enzymatic activity.

The common marmoset (Callithrix jacchus) and somespecies of gum-eating primates have a developed caecum.Fluid digesta are separated from large particles in the prox-imal colon and selectively retained in a caecum.

Cyclorrhaphous flies have a very acidic section in themiddle of the midgut [35]. Lysozyme active at low pH issynthesized in this section, and permits the digestion ofingested bacteria.

The caecal appendix is a blind-end tube located near thejunction of the small intestine and the large intestine in humans;depending on the animal, this small caecum varies greatly insize and shape. It is widely present in the Euarchontoglires, andhas also evolved independently in the diprotodont marsupials[36]. It is generally considered to be a vestigial organ, the finaltransformation of functional caeca over time resulting fromnatural selection. However, some authors assume that thisappendix can serve as a "safe house" for useful bacteria if, forexample, certain diseases flush digestive bacteria from the rest

Bioenerg. Res. (2013) 6:1063–1081 1075

of the intestines [37]: in theory, small caeca can facilitate the re-inoculation of the colon if the contents of the intestinal tract arepurged following exposure to a pathogen.

Operating Conditions

Retention Time

Little data is available on the retention time within thedigestive process. Table 2 presents data for fluid andparticle retention times. In a general perspective, theretention time in poikilothermic animals (cold-blooded) ismuch lower than in homeothermic animals (warm-blooded).For homeothermic animals, the average retention time isfrom 0.5 to 3 days, and does not seem to be correlatedto the type of food or the size of the animal [38]. Someextreme exceptions exist, such as the tiny hummingbirdwith liquid nectar (1.2 h), or the koala which displayslow metabolic activity (100 h), as does the cold-bloodediguana which needs more than 15 days for particles topass through its digestive tract. In addition, the retentiontime within a species can be flexible: for an example, it isshorter for lactating female koalas in the wild (39 h) than forcaptive koalas (100 h) [39]. Also passage times for the giantpanda are faster for shoots (7.9 h) and stems (10 h) than forleaves (13.8 h) [40].

Moisture Regulation

The flux of water is carefully regulated throughout thedigestive process. On the one hand, microbial activity re-quires water but, on the other, water is often a limitedresource and the amount of water involved increases thesize of the reactor. In mammals, water is added to the food inthe mouth from the salivary glands during chewing andgrinding. Saliva is secreted in large amounts (e.g., 1–1.5 liters/day in humans, 160–180 liters/day in bovids).For the bovid, water is partially removed in a specializedorgan before passage to the acidic CSTR (stomach).Depending on the availability of water in their environment,many animals remove water from their feces thanks to apress-like organ. Moisture content within the feces variesfrom 80 %- 90 % in cattle dung, 75 % for a normal humanstool to 27 % for a desert rodent (Dipodomys merriami)[41]. The water content of the rabbit colon and rabbit cae-cum is between 77 % and 80 %, whereas the bovid rumen isbetween 93 % and 95 %. Anaerobic reactors are classed , onthe basis of the moisture level of the organic substrate, aswet (<10 % total solids, TS), semi-dry (10–20 % TS) anddry (>20 % TS) [42, 43]. The solid content of both the colonand the caecum of the rabbit corresponds to a dry anaer-obic digestion process, while that of the bovid rumen ischaracteristic of wet anaerobic digestion.

Link between Type of Structure, Animal, Size and Diet

The relationship between, or limitations due to size and dietmainly depends on physiological factors, e.g., body temper-ature, with no direct link to the type of digestive structure.Within the homoeothermic category, the smallest vertebratesof about 2 g use easily-degradable substrates, such as nectarfor the bee hummingbird (Mellisuga helenae) or insects forKitti’s hog-nosed bat (Craseonycteris thonglongyai) and theEtruscan shrew (Suncus etruscus). Among eaters of slowly-degradable substrates (grass), the human-like structureranges from the vole up to larger-sized such as the elephant(weight between 0.03 kg and 14,000 kg) whereas in thebovid-like structure the range found is smaller : the royalantelope (Neotragus pygmaeus), with a weight of 1 to 3 kg,up to the grey whale (about 36,000 kg). A closer correlationcan be observed between the type of diet and the size of thedifferent compartments. For animals eating easily-degradablesubstrates, the size of the acidic CSTR is the largest digestiveorgan, in the common mole more than 80 % of the totalvolume of the digestive tract (Table 1 and Fig. 4b). In contrast,in animals digesting slowly-degradable substrates, the micro-bial compartment predominates in size, whatever its type: themicrobial compartment represents up to 90% of the volume ofthe digestive tract in the horse, bovid, hoatzin, and termites(Table 1 and Fig. 4b).

Allometry has been demonstrated between body massand moisture content in the entire gastrointestinal tract ofherbivores, and between body mass and total intestinalvolume (caecum excepted) in various mammals [44].However, there is no obvious correlation between the massof an animal and the retention time [38], or between themass of an animal and its digestive performance [45](Fig. 4c).

Evolutionary Aspects

Evolution of Digestive Structure and Phylogeny

According to the concept of bio-mimicry, it is relevant tocompare different structures for their similarity. In this paper,we have analyzed two structures, independent in terms ofevolution: one from a vertebrate, the other from an arthropod.Despite different evolution, the two show great similarity(Fig. 4d). First, the structure of the digestive tract has hardlyvaried throughout evolution. However, while having the samestructure, the function of an organ can change from onephylogenic group to another, thus permitting a focus on evo-lutionary convergence. In fact, the correlation between suchevolutionary convergence and feeding habits strongly indi-cates the efficiency of the shared feature, and can be a usefulsource of understanding.

1076 Bioenerg. Res. (2013) 6:1063–1081

The following examples provide clear evidence:A first example is the importance of grinding (but not

cutting) slowly-degradable substrates. The grinding functioncan be carried out by teeth, mandibles or the gizzard in,respectively, mammals, arthropods, or birds. Grinding is onlyabsent in some carnivores (e.g., snakes).

The size of the microbial reactor is correlated with thedegradability of a substrate whatever the structure or thelineage. Thus, the size of the microbial reactor goes up to90 % of digestive tract volume in the horse, bovid, hoatzin,and termite.

To hydrolyze food, reactors with extreme pH are presentin both the vertebrate and arthropod lineages. However,each lineage displays a contrasting feature: acid pH forvertebrate lineage, whereas arthropod lineages use basic pH.

A noteworthy evolution is the presence of a microbialCSTR upstream of the tract, corresponding to slowly-degradable substrates. Most of the bovid-like structure isphylogenetically clustered within the Cetartiodactyla clade(pseudo-ruminant, ruminant, and hippo) [24]. Molecularphylogeny shows that the whale is phylogenetically closeto Artiodactyla [24]. However, some animals sharing thisstructure are phylogenetically distant, including the kangaroo(marsupial) and the colobus (mammal, a monkey).

‘Domestication’ of Microbes (Origin, Evolution, Inocula)

Throughout evolution, animals and micro-organisms have de-veloped a symbiotic relationships [17, 46]. For ‘domesticated’micro-organisms, a digestive tract provides some nutrients, an

Table 2 Retention times for fluids and particles of different animals

Species Common name t°C* Weight (kg) Degradability Retention time (hour) References

fluids particles

Reptiles

Chrysemys picta Painted turtle P 0.5 easily 35 56–60 [56]

Caiman crocodilus Common caïman P 40 easily 41 162 [56]

Geochelone carbonaria Red-footed tortoise P 28 slowly <48 170–363 [56]

Iguana iguana Green iguana P 9 slowly <48 207–386 [56]

Birds

Anser anser Goose H 3.3 slowly 5.7 ND [57]

Dromaius novaehollandiae Emu H 38 slowly 3.9 4.7 [58]

Struthio camelus Ostrich H 100 slowly ND 48 [59]

Selasphorus platycercus Broad-tailed hummingbird H 0.003 easily 1.2 - [60]

Lagopus muta Rock ptarmigan H 0.4 moderate 9.9 1.9 [12]

Phoebetria fusca Sooty albatross H 2.5 easily 6.3 15 [12]

Eudyptes chrysocome Rock hopper penguin H 2.5 easily 3.8 17 [12]

Mammals

Macropus giganteus Eastern grey kangaroo H 66 slowly 14 30 [61]

Ovis aries Sheep H 100 slowly 35 50 [62]

Llama glama Llama H 200 slowly 36 52–60 [63]

Bos taurus Cow H 800 slowly 20 55–66 [64]

Camelus dromedarius Dromedary camel H 800 slowly 6.1 55.5 [65]

Equus caballus Horse H 500 slowly 18-22 23–27 [64, 66]

Vombatus ursinus Wombat H 30 slowly 36 62 [67]

Rattus norvegicus Rat H 0.3 moderate 12-35 13–35 [68, 69]

Cavia porcellus Guinea pig H 1 slowly 13-23 13–31 [68, 70–72]

Trichosurus vulpecula Common brushtail possum H 2.3 slowly 36-64 33–71 [73–75]

Oryctolagus cuniculus Rabbit H 2.1 slowly 17-91 17–91 [69, 75, 76]39-197 39–197

Pseudocheirus peregrinus Common ringtail possum H 2.3 slowly 63-210 [75]

Petauroides volans Greater glider H 1 slowly 23 50 [73]

Phascolarctos cinereus Koala H 6 slowly 100 213 [77]

* P corresponds to poikilothermic animals (cold-blooded) and H corresponds to homeothermic animals (warm-blooded)

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appropriate environment, and protection against othersmicroorganisms. For the host animals, micro-organisms pro-vide nutrients, mostly in the form of organic fatty acids.Micro-organisms can also become a nutrient for the animal,when the microbial fermentation takes place at the beginningof the structure (bovid-like structure) and, also, in the case ofcaecotrophy (rabbit) or an external reactor (termite).

Nevertheless, the microbial communities involved arevery complex: more than 1000 species are found in humanfeces [41, 47], horse feces [48], rabbit caecotrophs [49],bovid rumen [50], giant panda feces [51], or termite gut[52]. A co-evolution seems to have occurred between mi-crobial communities and each animal species, but the levelof such specificity has not yet been clearly defined. In thethoroughly-characterized microbiota of the human colon, acore of specific species is present in all the members, thoughthe ratio between the dominant bacteria varies [53].Moreover, the structure of the microbial community is stableover time [54]. Specific microbes must be associated to themechanism of vertical transmission from one animal gener-ation to the next. Inoculation from the ‘microbiome’ ofparents may occur just after birth, since intestines beforebirth are sterile. As an example, young elephants, pandas,koalas, mole rats, and hippos eat the feces of their mother,probably to obtain the bacteria required for effective diges-tion. Some organs, such as the appendix, can also facilitatethe re-inoculation of specific commensal bacteria in thecolon (see section “Other original features”).

Conclusion

By the way of conclusion, we propose to focus on the originalfeatures of animal guts and on major discrepancies betweenanimal guts and an anaerobic digester, in order to suggestsome possible improvements to the industrial processes inthe light of this survey.

Original Features in Animal Guts

To deal with the huge diversity of organic material used as food,digestive tracts display an extraordinary variety of organ shapes,functions, and structure. However, within this diversity, almostall the digestive processes converge into twomain configurationsrelated to the level of substrate degradability (Fig. 8).

In the first configuration, for the digestion of easily- ormoderately-degradable substrates (roughly speaking, thosenot protected by cell walls), the most efficient process evolvedis made up of acid and enzymatic hydrolysis in a CSTR,followed by an enzymatic PFR which facilitates the uptakeof the nutrients. A microbial community in a CSTR can carryout the digestion and uptake of organic fatty acids, dependingon substrate degradability.

In the second configuration, to cope with substrates whichare not easily degradable (roughly speaking, those protectedby cell walls), the most prevalent process evolved first grindsand moistens the organic substrate for transformation to or-ganic fatty acids and microbial biomass inside a microbialCSTR. After this stage, the microbial biomass becomes amoderately-degradable substrate and can be digested as de-scribed previously above. To this end, some animals have alsodeveloped additional upstream microbial digestion by using(i) a ‘new’ upstream organ, as in the bovid (mammals) orhoatzin (bird), (ii) coprophagy, as in the rabbit or guinea pig,or (iii) an external reactor with fungi, as in the termite.

Other lessons are provided by animal digestive tracts:

& The use of regulated mesophilic temperature (around37 °C) decreases retention time

& The importance of extreme pH hydrolysis (acid forvertebrates, basic for insects)

& The role of grinding rather than cutting& Enzymes from both organisms and microbes may be used

but not in the same reactor. The parameters of the process,such as a short retention time in PFR or an extreme pH

Fig. 8 Schematic view of therelative size of the reactorsdepending on the degradabilityof the substrate

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within a CSTR, avoid the use of enzymatically-degradedsubstrates by the microbes

& The size of the microbial reactor within the process iscorrelated to the level of substrate degradability.

Basic Discrepancies between ‘Artificial’ AnaerobicDigesters and Animal Guts

The goals of the two types of digestion are different. Thepurpose of animal guts is to produce and ingest smallcompounds such as acetate or sugars while avoiding meth-ane production, which represents a loss of carbon for theorganism. The aim of engineers is essentially only methaneproduction from an organic carbon source. These differentobjectives correspond to a difference in the basis of thedigestive structures. In the animal context, the structure isbuilt around an enzymatic reactor, as evolution shows inprimitive organisms. The acetate or sugars produced aretaken up from this organ and the microbial reactor appearsas a complement to the enzymatic reactor. In the artificialanaerobic context, the structure is built around the microbialreactor, and methane is taken up from the process.

The two types of digester also differ in the structure of theirmicrobial communities. Each animal gut contains its ownmicrobial community, which is transmitted from generationto generation and has co-evolved over millions of years [55].In contrast, each new artificial digester is empirically seededwithout any selection or conservation of efficient microbialcommunities.

Their natural cycles, including competition and predation,impose on animals several specific constraints that engineersmay consider as negative for efficient anaerobic digestion. Infact, an animal’s mobility, necessary for catching food andrunning away from predators or to be an efficient predator,imposes a relatively small reactor size. As a consequence,solutions evolved by animals are the low water content andshort retention time of organic matter inside the body.Moreover, competition for food has led to many animals be-coming food specialists, and omnivores such as humans andbears are the exception. In contrast, the tendency for artificialanaerobic digesters is to diversify their sources of biomass.

Lessons for Improving Artificial Anaerobic Digesters

First of all, digestion must be conceived as a global processwith several stages, and not as a digester with occasional pre-or post-treatment. Among such stages in the process, theimprovement of substrate accessibility, provided by grindingand extreme pH, seems very important. For most efficientdegradation of substrate, microbial communities should becarefully selected and re-used from one digester to anotherover a long time, in imitation of the selection which occurs inanimal guts. This microbial selection needs to be done with

one type of substrate within an appropriate whole-processconfiguration. A short retention time and high-solids digestionare compatible with a high level of degradation, an advanta-geous combination in process intensification. Enzymatic treat-ment can be applied to easily-degradable molecules withoutcompetition from microbes, which can be reserved for slowly-degradable substrates. In animal guts, competition with mi-crobes has been avoided by process features such as shortretention times, or extreme conditions such as acid or basic pH.

Acknowledgments The authors are grateful for financial supportfrom the Agence Nationale de la Recherche (ANR), France, undergrant No. ANR-09-BIOE-06 (DANAC project).

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