Microbial taphnomy of archaeological bone

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Microbial Taphonomy of Archaeological Bone

Author(s): A. M. ChildReviewed work(s):Source: Studies in Conservation, Vol. 40, No. 1 (Feb., 1995), pp. 19-30Published by: International Institute for Conservation of Historic and Artistic WorksStable URL: http://www.jstor.org/stable/1506608 .

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2/13

MICROBIALTAPHONOMY

OF

ARCHAEOLOGICAL

BONE

A.M.

Child

Summary-Taphonomy

is the

study

of

all

changes

that occur within an animal or

plant following

death. Bone

is

the

predominant

material

of

animal

origin

to

survive within the

archaeological

environment.It

is the

source

of

a

wealth

of information concerning

the

relationships,

diets,

disease and

ages

of people

(and

animals)

in

past

cultures. For this

information

to

be extracted

and

interpreted,

there

is

a need

for

conservators

and

archaeological

scientists to be aware

of

the

processes

of

taphonomic change

which

may

occur

in

bone.

These

changes

are

legion;

this

paper

attempts

to

define

some

of

the

major changes

seen in bone

which

may

be

attrib-

utable to the various

actions

of microorganisms.

Introduction

Taphonomy

is the

study

of all

changes

occurring

within

a substrate

following

death. These

changes

are many and varied, and it is not the aim of this

article to

present

a

comprehensive

list. The main

factors involved

in

the

taphonomic

changes

of

bone

in

the

burial environment

will be discussed

below.

Bone is the

predominant

material of animal ori-

gin

to survive within

the

archaeological

environ-

ment. It

is

a

composite

material,

comprising

both

inorganic

and

organic

fractions. Its

study

reveals

considerable

information to

the

conservator,

the

bone

specialist

and the

archaeological

scientist. For

this

information

to be

retrieved,

the conservator

must be aware of

the

likely

mechanisms

of

decom-

position

and

the

state of

preservation

of

the exca-

vated bone.

One

of the

major

problems

confronting analysts

and

conservators of

archaeological

bone is the

assessment of its

integrity,

since

conservation

treat-

ments will be

dictated

by

degree

of

preservation.

Both the

inorganic

and

the

organic phases

of bone

can be

changed

by taphonomic

processes,

and esti-

mation of the

degree

of

preservation

of

these

phases

based

upon

external

morphology

has

been

proved

to

be unreliable

[1].

Definitions

For

clarity,

some

of

the

terms

used

in

this

paper

have been defined here. Other definitions exist,

however;

the

definitions

given

here have

been

selected because

they

represent

the

most common

usage.

Biodegradation:

any

change

in the

properties

of

a

material

caused

by

the

activities of

organisms.

Received

30

June 1993

Received in revised

orm

22

August

1994

Studies in

Conservation40

(1995)

19-30

Biodeterioration:

any

undesirable

change

in

the

properties

of a

material

caused

by

the activities

of

organisms [2].

Biostratinomy:

changes

occurring

in an

animal

or

plant

after death

but before

burial

[3].

Diagenesis:

changes occurring

in

the

animal or

plant following

death

and

burial

[3].

Microbial

decomposition:

deleterious

changes

to

a

substrate

due to

the action of

microorganisms,

their

metabolic

by-products

and their

enzymes.

Bone structure

Bone is a highly specialized composite material

comprising

both

inorganic

(mineral)

and

organic

(mostly

protein) phases.

Its

inorganic

fraction

(90 )

is calcium

hydroxyapatite.

The

remaining

10

is

organic,

made

up

of

collagen,

non-collagenous

pro-

teins

(NCP),

lipids,

mucopolysaccharides

and

other

carbohydrates

[4].

There are

two

types

of

bone,

cancellous

bone

(spongy

bone)

and

compact

bone

(cortical

bone),

and their

distribution within

the

body

is dictated

by

biomechanical

considerations.

Both

compact

and

cancellous bone

are

formed

by

the

deposition

of

collagen

fibrils

in

layers.

In

com-

pact

bone,

these

layers

are laid

down in

rings

around the osteone, their alignment changing by

approximately

90? in

each

layer [5].

Bone mineral

Hydroxyapatite, Ca10(PO4)6(OH)2,

s the

basis of

the

inorganic component

of

bone.

Bone

mineral

is

a

complex

substance,

not

stoichiometric with

respect

to

hydroxyapatite.

Its

calcium to

phosphorus

ratio

is

significantly

less than

10:6,

so

that the

apatite

requires,

in

addition to its

calcium,

phosphate

and

hydroxy

ions,

substantial

amounts of

carbonate and

19


3/13

A.M.

Child

lesser

quantities

of

pyrophosphate, magnesium,

sodium and

potassium

[6].

Strontium and

lead,

ingested

as

part

of the diet of

an

individual,

are

stored in the

skeleton. Fluoride ions have a

high

affinity

for

bone

mineral,

converting hydroxyapatite

to

fluorapatite,

and

fluoride

analyses

have been

used to check

provenance [7].

Bone

proteins

The

organic

fraction of bone

comprises

10-15

of

the total bone

weight.

There is one

major protein

component,

collagen,

and

a

group

of

proteins

termed

collectively

non-collagenous protein

(NCP).

Collagen

The

collagen

triple-helix

is

present

in

abundance

in

a

range

of

different

vertebrate tissues. Thirteen dif-

ferent

collagen

types

have been

described,

varying

in their chain composition and amino acid sequence

[8].

The

collagen present

in bone is

Type

I,

which

comprises

two

chains of one

composition,

al(I),

and one of

another,

a2(I).

Bone

collagen

has

an

average composition

of 30

glycine,

14

proline

and 11

hydroxyproline,

the remainder

making up

less than half the

total amino acids

[9]. Collagen

is

the

only

human

protein

in

which

hydroxyproline

occurs

in

significant

amounts;

this amino acid

restricts the

action of

proteases

[10].

In

the

Type

I

collagen

molecule,

the

protein

chains are

approxi-

mately

1000 amino acids

(residues) long

with

glycine

(the

smallest amino

acid) occurring every

third residue to give the dominant sequence:

glycine-proline-hydroxyproline

Because

of

the

presence

of

glycine

residues,

the col-

lagen

chains

are

tightly

twisted;

chains

which

are

tightly

twisted have an

increased resistance

to the

action of

proteolytic

enzymes.

The chains contain

hydrogen

bonds between

the amide

nitrogen

of

glycine

in

one chain

and the

non-glycine

carboxyl

oxygen

in

an

adjacent

chain

[11].

Mature

Type

I

collagen

is further

strengthened by

a covalent bond

which forms between

lysyl

amino acid

[12].

This

covalent bond

is

peculiar

to bone

collagen,

and

makes the molecule even more resistant to enzy-

matic

cleavage

and

laboratory

extraction

proce-

dures.

The

presence

of the

mineral,

which is

in

intimate association

with the

collagen,

further

inhibits the

access and action

of

enzymes.

Non-collagenous

proteins

Non-collagenous proteins

(NCP)

are a

complex

group.

Bone contains

two sources of

NCP,

one

arising

from outside the bone

(i.e.,

a-2HS-glycopro-

tein and

albumin)

and

the other from the bone

itself

(i.e.,

osteonectin

and

osteocalcin).

Osteonectin

is

thought

to be

involved

in the

deposition

and

development

of

primary

bone

tissue,

and osteocal-

cin is concerned

with

the

remodelling

and

develop-

ment of

secondary

bone tissue. The

bone-specific

NCPs

are

high

in

aspartic

and

ycarboxyglutamic

acid

residues

[13].

Bone as

an

archaeological

resource

As stated

above,

bone

is

the

predominant

material

of animal

origin

to survive

within the

archaeologi-

cal environment.

Its

study

reveals considerable

information

to the

conservator,

the bone

specialist

and the

archaeological

scientist.

Archaeological

skeletal materials

provide

two avenues

of

investiga-

tion:

palaeopathology

and

archaeological

science.

Palaeopathology

The gross morphology of archaeological bone can

be

changed

by

the burial

environment,

but

physical

measurements of skeletal bone

dimensions will still

yield

information on

growth,

health and incidence

of some diseases

within human

populations.

Similar

examination

of animal bone

produces

information

relating

to

butchering

methods,

diet,

hunting

and/or

farming techniques practised by

ancient

populations

[14]. Microscopic

examination of

archaeological

bone

(and

the related

surviving

soft

tissues)

will

yield

more information

on

disease

[15].

Archaeological

science

The preservation of bone depends not upon the

length

of burial

but

upon

the environment

of bur-

ial.

If

bones

are

sufficiently well-preserved,

they

still

contain

indigenous organic

macromolecules.

These

can be used

to

provide

information

on:

-the

age

of

the bone

by

radiocarbon

dating

[16-18]

or

by

amino acid racemization

[19-22];

-the diet of

the

animal

by

stable

isotope

analysis

[23-26];

-genetic relationships

[27-29].

This information

is

only

reliable where

the

proteins

used have survived unchanged by biological, physi-

cal or chemical

degradative

processes [30].

Survival

of

proteins

in

archaeological

bone

This

article is restricted

to a discussion of

the

effects

on bone

of the microbes within

terrestrial

environments.

It

is assumed here

that the bone

(with

or

without

flesh)

enters

the burial environ-

ment

in

direct

contact

with the

soil,

and is not

buffered

by

the

presence

of a

coffin,

shroud

or

other

protective layer.

Studies n Conservation0 (1995)19-300


4/13

Microbial

aphonomy

f

archaeological

one

Bone

protein

is

unusual

in that

it survives over

archaeological,

even

geological,

time scales.

Generally, protein

loss

from

unmineralized sub-

strates

is

considered to be

exponential,

so that the

amount

which

survives

depends

upon

the initial

concentration

[31].

The

protein

loss within archaeo-

logical

bone does not follow this

pattern

and,

as a

result,

archaeological

bone can contain

only

1-2

nitrogen

[26].

Indeed,

the

carbon to

nitrogen

ratio

in

archaeological

bone

collagen

does

not

change

from that of modem bone

until almost all

of

the

collagen

(97 )

has been lost

[23].

Where 98

of

the

original

bone

protein

has been

lost,

the amino

acid content is

enriched

in

aspartic

and

glutamic

acid residues

[26,

32].

The

original

source of

these

residues is

uncertain; however,

three

possibilities

exist:

-contamination of the bone

proteins

with soil

pro-

teins [33];

-survival of

the

NCP,

rich

in these

residues,

whose

presence

is

masked

when

collagen

survives

well

[32];

-selective

decomposition

of the

collagen

helix,

to

leave acidic amino acids of

collagen

origin.

The

degree

to which

proteins

survive intact in

bones

from

archaeological

contexts

can be

mea-

sured

using

immunochemical means.

Although

these

methods are well

established

for

application

to

modem

materials,

they

have

only recently

been

applied

to

archaeological

ones

[34-39].

Results must

be viewed with caution [40]; however,

they

show

that

indigenous proteins (i.e.,

apohaemoglobin

and

osteocalcin)

survive in

an

immunologically recog-

nizable form in bones.

Proteins

(both

collagens

and

NCP)

are

thought

to

survive

in bone

because

of

their close association

with the mineral

phase,

which

protects

against

enzyme

attack.

The

collagens

are

further

preserved

because

of their

innately protective

structure and

chemistry

(see

'Collagen'

above).

Dissolution of the

bone mineral

The response of the mineral and organic phases of

bone to

the

burial

environment

will

differ.

Janaway

[41]

suggested

that

decomposition

of

bone

probably

occurs in the later

stages

of

the

decomposition

of a

corpse,

but this

is

not

likely.

Deterioration,

both

biochemical

(autolysis)

and

microbiological,

of tis-

sue

begins

immediately

after

death,

the

soft tissues

usually

showing

the

effects

first. If the

microbiologi-

cal

decomposition

of bone is inhibited

by

the

con-

tinued

presence

of the mineral

phase,

then the

degradation

of

collagen

will

proceed by

chemical

means

(see

below).

Dissolution due to

the soil

chemistry

The chemical and

physical

deteriorationof buried

bone

depends

solely

upon

the

chemistry

and

bio-

chemistry)

of the

surrounding

burial

environment.

Acidic

soils

will

dissolve

hydroxyapatite,

but the

thresholdof acidity required s not clear; at pH

<

5,

demineralization

s

promoted[42].

The rate of

dissolution

will

depend

on the

pH

of

the

soil,

the

concentration f

chelatingagents

and the

degree

of

water

percolation.

Bone tends to survive better in

soils with a

neutral or

very

slightly

alkaline

pH.

This

is

because

the bone mineral is

less

likely

to

dissolve,

but it must be

noted that soils low in

phosphate

will also

promote

demineralization

43].

Calcium

hydroxyapatite,

Ca10(PO4)6(OH)2,

can

be

changed

both

by

dissolution

and

recrystallization

and

by

hetero-ionic

substitution

(e.g.

Fe2+,

Ca2+).

Vivianite

(Fe3(PO4)2

8H20),

brushite

(CaHPO4)

and

calcite (CaCO3)will form within archaeological

bones

depending

upon

the

pH

and

chemistry

of

the

soil matrix.

Soils

high

in

Ca2+will

lead

to

the

break-up

of the

bone,

because

CaCO3,

ormed in

high-calcium

oils,

occupies

a

larger

space

than the

crystals

of

Ca10(PO4)6(OH)2.

The

presence

of

CaCO3

exerts internal

stresses within

the

bone

which will

result in

embrittlement and

cracking.

Internal

stresses

which

cause

damage

to the

gross

morphol-

ogy

of bone are

induced when bone is

exposed

to

alternating

wetting/drying

and

freezing/thawing.

These stresses are

aggravatedby

the

presence

of

soluble salts

[44].

Dissolution due

to microbial

decomposition

Generally,

n

well-aerated

oils,

the

decomposition

of

organic

matter is

rapid.

The

high

mineral

con-

tent of

bone,

however,

will

initially

inhibit the

microbial

decomposition

of

collagen.

This decom-

position

will

be

affected

by

the relative

numbers

constituting

the

microbial

population

of

different

soils.

The

composition

of the soil

microflora is

highly

dependent

upon

soil

pH,

and the

metabolic

activity

of

microorganisms

will

influence he

pH

of

the

soil,

especially

those

activities that involve

redox reactions[45]. The conditions which affect

the survivalof bone in the

archaeological

nviron-

ment

[46]

are

also

those

which will affect the

growth

and

survival

of

microorganisms

n the soil

[45,

47].

Microorganisms

will

penetrate

into bone

using

natural

spaces(e.g.

blood vessel

and nerve

acunae),

releasing

heir

enzymes

and

depositing

amino acids

[48, 49]. They

will

grow

using

the

surrounding

unmineralized

issues as a food

source and excrete

their

secondary

metabolites. The

nature of these

products

of

microbial

metabolismwill

dependupon

Studies n Conservation0 (1995)19-30 21


5/13

A.M.

Child

the

environment

in

which

the

decomposition

occurred.

Anaerobic and aerobic

decomposition

Microorganisms

can be

divided into three cate-

gories, depending upon

their metabolism:

they may

be either

obligately

aerobic,

facultatively

anaerobic

or

obligately

anaerobic

[50].

In

aerobic

environments,

complete

decomposition

of the

protein

to

carbon

dioxide,

water,

nitrogen

dioxide and

sulphur

dioxide

occurs without

a

con-

siderable reduction

in

pH;

therefore the

proteins

in

bone are more

likely

to survive. All the

fungi

and

some of the bacteria have an

obligately

aerobic

metabolism,

degrading complex proteins

with

prote-

olytic

enzymes. Rapid

aerobic

growth

will

promote

anaerobic

conditions;

this

occurs when

the rate

of

oxygen consumption

exceeds the rate of

oxygen

dif-

fusion

into the

system.

When

anaerobic conditions

have been

achieved,

the

obligate

aerobic

microor-

ganisms

will

stop growing,

but the bacteria that

have the

facility

for both anaerobic and aerobic

decomposition

will switch

to

anaerobic

pathways.

The

obligate

anaerobes

will

start to flourish.

Anaerobic fermentation

uses

substances other

than molecular

oxygen

as a terminal electron

accep-

tor

[50].

Microbial

decomposition

of

protein

under

anaerobic conditions

leads,

initially,

to a

drop

in

pH.

This is due

to

the

generation

of

acidic com-

pounds

such as low molecular

weight

fatty

acids

and amino

acids,

which are

by-products

of

anaero-

bic

metabolism.

Their

presence

will result in the

demineralization of

hydroxyapatite

and the

expo-

sure

of

collagen

to

collagenolytic enzymes.

Extra-

polating

from work on dental caries

[51],

an

increase

in

proton

concentration

will

result

in the

partial

demineralization of

hydroxyapatite,

because

it will

buffer

the

changes

in

pH

at its own

expense.

Microbial fermentation of

bone

proteins by

anaerobic

pathways

could lead to the total loss

of

the

organic

fraction of bone since the

products

of

the reaction

perpetuate

the

reaction;

total

loss

of

the

organic

fraction, however,

is

not

likely.

As the

rate

of anaerobic

decomposition

slows due to the

lack of

metabolites,

oxygen

will

diffuse into the

sys-

tem. Eventually, the rate of anaerobic degradation

will be

surpassed

by

the rate

of

oxygen

diffusion

and

the

system

will return to its

aerobic

state,

when

the rate

of

aerobic

degradation

will

be determined

by

the

concentration

of

bio-available

organic

mat-

ter

[52].

Catalyzing

bone

decomposition

To

decompose

bone,

a

microorganism

must be

able

to

obtain

energy

from the

collagen

and mineral.

For the microbial

enzymes

to

gain

access to the

col-

lagen,

the microbe must also

be able to

demineral-

ize the

bone,

or

grow

in

an environment

where

demineralization occurs

(e.g.

a

low-pH soil).

Enzymes

Enzymes

are

proteins

which

can increase

the rate

of

(catalyze)

biological

reactions between

106-

and 108-

fold. As well as

increasing

rate,

enzymes

are

specific

in the

reactions which

they

will

affect.

An individ-

ual

enzyme

will

catalyze

a

specific

reaction with a

unique

substrate

or

group

of substrates.

Enzymes

that

disrupt

proteins

are called

proteolytic enzymes

or

proteases,

and

enzymes

that are

unique

in

their

ability

to

hydrolyze efficiently

the

triple-helical

regions

of

collagen

under

physiological

conditions

(i.e.,

moderate

temperature

and around neutral

pH)

are called

collagenases. Collagenases

are

complexes

of several different enzymes, each of which has a

different

catalytic

function.

There are two

accepted

types

of

collagenase [53].

Collagen degradation by

vertebrate

collagenases

Vertebrate

collagenases

(tissue collagenases)

disrupt

unmineralized

collagen

into

only

two

fragments by

action at a

specific

site within the

al(I)-chain,

a sin-

gle

glycine-isoleucine

bond between residues 772

and 773

[54].

The

enzyme appears,

therefore,

to rec-

ognize

the whole

collagen

molecule

configuration.

Collagen degradation by

microbial

collagenases

On the other hand, microbial collagenases recognize

small

amino acid

sequences.

All

microbial

collage-

nases examined so

far

appear

to have

the

same

amino

acid

sequence requirements

for

cleavage

[55-59];

hence examination of the action of a

well-

characterized bacterial

collagenase

can be included

here.

The anaerobic

bacterium Clostridium

histolyticum

produces

a

collagenase

complex

of

six

enzymes.

All

are

highly

active

against

collagen

and

devoid

of

other

proteolytic

activities.

Study

of their amino

acid

sequence requirements

for

cleavage

is

challeng-

ing,

since

purification

of

these

enzymes

is

difficult;

however, the requirement for one of them (,3-colla-

genase)

has been demonstrated

[60].

Unlike verte-

brate

collagenases, 3-collagenase

does not have

a

rigid sequence requirement,

but will

cleave

between

the

X- and

the

glycine-

residues in

any

of the

sequences

below:

-glycine-Y-X-glycine-proline-hydroxyproline-

-glycine-Y-X-glycine-alanine-arginine-

-glycine-Y-X-glycine-Z-alanine-

where

X-,

Y-

and Z- can be

any

of

the amino

acids

which constitute

collagen, except glycine.

Microbial

Studies in Conservation40 (1995) 19-302


6/13

Microbial

taphonomy of archaeological

bone

collagenases,

therefore,

have

active sites

along

the

length

of

the

collagen

molecule which

are

dictated

by

amino acid

sequence

rather than whole

molecule

configuration.

Like

tissue

collagenases,

they

are

metalloproteinases

containing

a

zinc ion at the

active site

[61, 62].

Microbial

collagenases

cleave the

collagen

helix

into

a

range

of

short

peptides

by hydrolysis

at

mul-

tiple

sites

along

the

triple

helix.

The

major

product

following

treatment

of

Type

I

collagen

with the

col-

lagenase

complex

from

Cl.

histolyticum

is the

tripeptide

glycine-proline-hydroxyproline, although

many

other

products

are

generated.

Like vertebrate

collagenases,

microbial

collagenases require

that the

collagen triple-helix

be

demineralized

before the

enzyme

can

gain

access

[63].

Collagen

degradation

by proteases

Microorganisms

that

produce

collagenase may

vary

in the rate at

which

they

will

decompose

the

bone.

The

microbial

by-products

of

metabolism and

the

presence

of

other

enzymes (e.g.

proteases)

can

also

affect

the

collagen.

Proteases

appear

to have no

action

on

the

collagen

triple-helix;

it could be

argued

that their

only

value in

collagen

decomposi-

tion is

that

of

reducing

the

short

peptides

released

by

collagenase

action to

single

amino acids.

It

was

suggested

above

(see

'Collagen degradation

by

microbial

collagenases')

that the

only enzyme

sys-

tem

capable

of

cleaving

the chains

of

Type

I

colla-

gen

in their

helical

regions

is

the

collagenase

system. This

may

not be the case.

One

protease, chymotrypsin (enzyme

classifica-

tion number: E.C.

3.4.21.1),

has been shown to

have

collagenolytic

activity.

Classically,

chy-

motrypsin

should

have

little effect

upon

collagen,

since

peptide

bonds

which

involve

hydroxyproline

are resistant to its action

[10].

Chymotrypsins

from

the

mid-gut

of

shrimps

(Penaeus

monodon,

P.

japon-

icus

and

P.

penicillatus)

have been

shown

to

have

some

collagenolytic activity [64].

The

shrimp chy-

motrypsins

are resistant to the

standard

a-chy-

motrypsin

inhibitors,

and some have been shown

to

have a

higher

affinity

for

collagenolytic

activity

than for other proteolytic activities [65].

Collagenolysis

by non-specific

proteases

has been

promoted by

low

pH

[66].

Pepsin

(E.C.

3.4.23.1)

in

acetic

acid

is

the standard

laboratory

method

by

which

Type

I

collagen

is

removed

from demineral-

ized

bone

[67].

This

enzyme

is

used to cleave

the

covalent

bonds

holding

the

triple-helix together.

Unlike

collagenase,

it

has no action

upon

the

helices themselves.

Indeed,

low

pH

could be the

mechanism

by

which

some

fungi produce

'tunnels'

(see 'Diagenetic changes'

below)

within

archaeologi-

cal

bone

[68].

Although

no

microbial

proteases

capable

of

cleaving collagen

in

its helical

parts

have

yet

been

described,

the

possibility

of this

decomposition

pathway

cannot be ruled out.

The taphonomyof bone

Taphonomy

was defined above as the

study

of

all

changes

which occur within

an animal

after

death

[3].

The

taphonomy

of bone is influenced

by

the

physical

and chemical

characteristics

of

the sur-

rounding

environment

and

the destruction

of the

surrounding

soft tissue. There are two

main

processes by

which

bones and soft

tissue become

degraded,

both of which affect the

stability

of the

associated

collagen.

These

processes

are

enzymatic

(which

includes

both

autolysis

and

microbial

decomposition)

and

chemical;

both are

affected

by

temperature.

Limitations

of

burial

temperature

The

range

of soil

temperatures

is dictated

by

the

air

temperature

and the soil

depth.

In

Britain,

at

a

depth

of

300cm,

the

temperature

range

is 10.5

?

2?C

[69].

Published

studies

of the

action

and char-

acteristics of microbial

collagenase

have been car-

ried out

at

28?C

and

37?C. The

microorganisms

that will

produce collagenases

at soil

burial

temper-

atures

cannot

be

extrapolated

from this

literature,

since

a

significant temperature

reduction of 18-

27?C will severely restrict many different species,

including

Clostridium

histolyticum [70].

Microbial

studies at low

temperatures

are

required.

Although only

a few

studies of this

nature have been

undertaken,

results

so far

indicate

that certain

types

of

bacteria

and

fungi

can

be

iso-

lated

repeatedly.

The bacteria

and

fungi

able to

produce collagenases

at low

temperatures

are

pre-

sent

in

archaeological

bone and

associated

soils

in

very

low numbers

when

compared

to the

total

microbial

population.

The

bacteria

and

fungi

are

obligate

aerobes,

able to

grow

over

a

wide

range

of

temperatures (4-39?C)

and

pH

values

(3-6-9-0)

[68,

71]. The bacterial species isolated (Pseudomonas,

Aeromonas,

Xanthomonas)

produce

metabolites

which are

toxic to

fungi

and therefore

can

compete

successfully

with a wide

range

of

fungal

species

(see

'Microbial interactions'

below).

Autolytic changes

As a

cell

dies,

it

releases a

mixture

of

enzymes

from

the

lysosomes.

These

enzymes

have an

autolytic

(self-destructive)

function

and

usually

take the form

of

proteinases

and

DNAses,

which

speed up

the

destruction of the

tissue and

its

component

cells.


23


7/13

A.M. Child

Autolysis usually

follows

very rapidly

after

death;

even

in

the

short

interval

between death and

burial,

the

osteocytes,

marrow and

neuro-vascular

bundles

undergo

autolysis

[72]. Autolysis

is

self-limiting

and,

after a certain

stage

is

reached,

the

cells

remain stable for long periods of time [73]. The

majority

of

changes

in

bone, however,

affect the

extracellular matrix

proteins

(i.e.,

collagen)

which

are not amenable to

autolysis.

Microbial

changes

Augmenting

the

processes

of

autolysis

is microbial

decomposition:

autolysis

opens up

the

soft

tissues,

thus

increasing

access for

microorganisms.

Decomposition

of whole

bodies involves the loss

of

both soft and hard

tissues,

and both

aerobic

and

anaerobic environments

will

be

achieved within the

rotting

flesh.

Due to the

presence

and

high

numbers of

gut

flora

(which

include some

microorganisms

capable

of

inducing

dental caries

[74]),

the bones

within the

abdomen

and thorax

will suffer the

demineralizing

effects

of

putrefaction

for

longer

than the

long

bones

or the skull. This is

borne out

in

some

stud-

ies,

but not

in

others

[75,

76]. Laboratory

studies

using

bones

inoculated

with

microorganisms

[77]

support

the

premise

that

soft tissue destruction

aug-

ments hard tissue

loss.

Biostratinomic

changes

Autolytic

and

microbial

changes

appear

very

soon

after death. Autolytic destruction can occur within

10

seconds

following

death of

the

cell

[73].

The

types

and

degree

of microbial

destruction

will be

dictated

by

the environment

in which

the

corpse

lies.

If burial does not

immediately

follow

death,

the

growth

of

microorganisms

which would

nor-

mally

be

prohibited

by

the low burial

temperatures

will

be

promoted

(e.g.

Cl.

histolyticum).

Once the

body

has

cooled,

these

microorganisms

are

unlikely

to

grow,

but it

is

possible

that the

temperature

of

the

decaying

body

may

increase

sufficiently

to

allow

for their

growth.

It

is

probable

that Cl.

histolyticum

has its most

important role in the breakdown of collagen in the

pre-burial

stage. Collagen

breakdown

will

occur as

the

bones and associated

tissues

lie on

(or slightly

below)

the surface

of the soil.

Cl.

histolyticum

is

present

in the

soil,

but also

in

the

gut

of humans

and

some animals

as normal flora.

Diagenetic changes

Janaway

[41]

considered that

soil was

less

impor-

tant

in the

initial

stages

of

decomposition, arguing

that the

body

will

create

its own

environment

which

in turn

will

modify

the effects

of the

surrounding

soil.

Studies

in

soil

microbiology

have

shown that

this

is not

likely.

Microorganisms

are

introduced

into

farming

soils so that certain

growth

character-

istics of the

soil

may

be

improved.

These micro-

organisms

have been

genetically manipulated

to

give required

characteristics.

In

almost all

cases,

these introduced microorganisms die out; they do

so as

a

result

of

competition

with

the

normal

soil

flora,

although

some success has been achieved

by

genetically manipulating

soil isolates

and

re-intro-

ducing

them into the same

environment

[78].

If a substrate

(i.e.,

a

corpse)

which contains

microflora

is

added to

soil,

its own microflora

will

be

destroyed by competitive

interactions

with the

normal soil flora.

This

may

take

only

a few

months

or

years,

but the

microorganisms

present

within

the

corpse

at death

may

induce

diagenetic changes

before

the

indigenous

population

is

destroyed.

Overall,

the initial

taphonomic changes

induced

in

a corpse may be the result of autolytic mechanisms

and inherent microbes.

The most

significant diage-

netic

changes

seen

in

a

corpse

are more

likely

to be

due

to the action of

soil macro- and microflora.

A

particular type

of

diagenetic change

is that

seen

in

microscopical

focal destruction

(MFD).

These are

localized 'tunnels'

thought

to

be

pro-

duced

in

bone tissue

by

the action

of

microorgan-

isms

(for

a

review,

see Bell

[79]).

The dimensions

of

the

MFD

are identical

with the dimensions

of

microorganisms;

the

microbial metabolites

cannot

diffuse

far into the

dense bone tissue.

In some

types

of

MFD,

mineral

redeposition

along

the

interior

walls of the 'tunnels' has been noted. It is thought

that this is

due to the death of

the

microorganisms:

when a

microorganism

dies,

the

pH

of the

sur-

rounding

medium rises

due to the release of

ammo-

nia and

other microbial

metabolites.

This rise

in

pH

will

promote

the

re-precipitation

of dissolved

hydroxyapatite.

An

unexpected

alteration

of bone

protein by

the

bacterium

Pseudomonas

fluorescens

has been

shown

[77]. Decomposition

studies

using

various

strains of

this

bacterium,

both

singly

and

in

concert,

has

shown

that the bacterium

has

the

facility

to

change

the rate

of racemization

of the

aspartic

acid

residues in the insoluble collagen of bone.

Post-excavation

Microbial communities

are

affected

by

environmen-

tal disturbances.

The inherent

microbial

population

within

archaeological

bone

probably

receives

its

greatest

disturbance

on excavation.

The

problems

created

by

the sudden

alteration

of redox

potential

will

be

exacerbated

by storage

in

a

warm

'finds

tent'.

The microbial

loading

within

the bone

will

already

be

significant;

alteration

of

temperature

alone will increase

microbial

growth

rates

since,

generally,

enzyme

reaction

rates are doubled

for

Studies

in Conservation40

(1995)

19-30

4


8/13

Microbial

aphonomy

f

archaeological

one

every

10?C rise in

temperature

[80].

The

con-

comitant

ncrease

n

oxygen

concentrationwill

pro-

mote

rapid

aerobic

decomposition

of

organic

components.

Fast-growing

ungi, previously

held

in

check

by

burial

conditions,

tend to

flourish at the

expenseof other organisms.With the presenceof

metabolites

rom

fungal

action,

the

rate

of

decom-

position

of the

bone

will,

to a certain

extent,

be

controlled

by

the interactionof the inherent

micro-

bial

species

(see

'Microbial interactions'

below)

until

physical

conditions

(e.g.

storage

at

controlled

relative

humidity)perform

his

function.

Chemical

hydrolysis

If

the mineral

phase

of bone retains its

integrity,

then

collagen

can

only

be lost

following

chemical

alteration.

A

number of chemical transformations

will occur and will continueafter

death,

such

as the

processof non-enzymiccrosslinking,but the most

significant hange

will

be

hydrolysis

of

the

peptide

bond.

A

simple,

conceptual

model

of

collagen

peptide

bone

hydrolysis

has

been

suggested.

This model still

requires

much work before

it can

be

usefully

applied

to mineralized

ollagen,

but

it

fits

well with

many

observed

phenomena

81].

The rate of colla-

gen

loss from the

bone will

depend

upon

the rate of

peptide

bond

hydrolysis,

the

rate of

diffusion of

these small

fragments

out of the

bone

and

the

rate

of

post-mortem rosslinking.

Crosslinkswill form as

a

result

of the

continued

non-enzymic process. 'Vegetabletannates' in the

burial environment

will

bond to

the

collagen

[82].

The

degree

of

bonding

by

these

tannates

will

depend upon

the

rate of diffusion

of

these chemi-

cals into the bone. This diffusion

will be

enhanced

following

demineralization f

the bone.

It

is

likely

that

the

presence

of these

compounds

n the

bone

will

physically

inhibit

the

action

of

collagenolytic

enzymes.

Limitations

of microbial

decomposition

Once microbialdecompositions stopped,the only

degradation process

occurring

is chemical. The

causes of the

reduction

n

microbial

activitymay

be

multifactorial. f

any

one or a combinationof these

conditions is

met,

then microbial

decomposition

may

slow

or

even

stop,

but chemical

hydrolysis

will

continue.

Burial

temperature

As

the

rate of

decomposition

of

the soft tissue

slows,

due to

its

removal

by

aerobic

and anaerobic

decomposition,

he

temperature

f

the

decomposing

matrix will fall.

The

fall

in

temperature

will

slow

microbial

activity.

This

slowing

will

have a

positive

effect on the fall in

temperature,

ntil

the

tempera-

ture of

the

decomposing

material

reaches that

of

the

surrounding

oil.

Alkaline and acid

soils

Alkaline

soils

will

buffer

the microbial

acids and

inhibit

the dissolution of the

bone mineral.

Conversely,

acidic soils will

tend

to

promote

bone

decomposition,

although

this is

only

a

generaliza-

tion.

The rate of

hydrolysis

of

peptide

bonds

is

increasedn both acid and alkaline

environments.

Microbial interactions

Many

bacteria and

fungi generate by-products

of

metabolism that

are

antagonistic

to other

micro-

organisms i.e., antibiotics). These chemicalshave

a

suppressant

ffect

both on membersof the same

genus

and on

completely

different

genera

83,

84].

Generally,

competition

between

microorganisms

tends to

prolong

the

life of the substrate.

The

greater

he

variety

of

microorganisms

within

a

sub-

strate,

the

longer

it is

likely

to

survive,

since

some

microbial

energy

must be directed owardscontrol-

ling

other

microorganisms

rather than substrate

digestion

alone.

Non-microbial

inhibitory

substances

Burial

environments

may

inhibit

the

growth

of

microorganisms apable of degradingbone. The

presence

of

copper(I)-but

probably

not

iron(II)-

ions

as well

as

other

inhibitory

substances

(e.g.

vegetable

tannates)

may

promote

the survival

of

both

the

soft and

the hard

tissues

[85].

Extremes

of temperature

Extremesof

temperature

an

promote preservation

of both the soft and the hard tissues:

they

are

known to survive

n

desertsand

permafrost

egions

[37].

Desiccation

will

reduce microbial

activity

and

hence the

only

mechanism or

collagendegradation

is chemical hydrolysis,the rate of which would

depend

upon temperature.

Enclosed

environments

Enclosed environmentswhere there

is

little

or no

leaching

will

promote

the

preservation

f

bone.

An

equilibrium

will be reached

between

the

microbial

products

of metabolism and

the

bone mineral

buffering

capacity.

The

presence

of

the microbial

metabolites

will inhibit

further microbial

growth,

since

they

are toxic to the

microorganisms,

and

thus

the

microbial

populations

will

slowly

decline.

Studies n Conservation0 (1995)19-30 25


9/13

A.M. Child

Conclusion

The various

pathways

for the

microbial

taphonomy

of

archaeological

(and

historical)

bone have been

defined here

and,

even

though

an exhaustive list has

not been

given,

it can be seen that

a

bewildering

range

of reactions is

possible.

These include dem-

ineralization of the

bone,

damage

due to

the

action

of

microbial

enzymes

(both

collagenases

and

pro-

teases),

the microbial

production

of MFD

(micro-

scopical

focal

destruction)

and the alteration

of

the

proteins

following

decomposition.

Acknowledgements

The author

would like

to thank Professor R.D.

Gillard,

Dr

Matthew

Collins,

Dave

Watkinson,

Susan

Hardman and

Naomi Earl-Turner for their

helpful comments during the preparation of this

manuscript.

Thanks are

also due

to

Dr

J.

Morgan

and

the

staff of the Cardiff

Royal Infirmary,

Bacteriology

Department,

for

their

kind

permission

to use

the

laboratory

facilities for the microbial

iso-

lation work.

The

project

was funded

by

the Science

&

Engineering

Research Council

(Science-Based

Archaeology

Committee).

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M.

K.,

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J.,

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J. J.

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lsevier,

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EFREMOV,

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Author

ANGELA

M.

CHILD

studied

microbiology

at the

University

of London and

worked for several

years

within the

National

Health Service. She

went

on

to

study archaeological

conservation at the

University

of Wales

College

of

Cardiff,

graduating

in

1988.

Her

special

interest was the

decomposition

of

organic

materials and she

completed

her PhD

on

aspects

of bone

decomposition

in

1992,

followed

by

a

two-year

SERC

(SBAC)

post-doctoral fellowship

which continued the

investigation

of

the interaction

of

microbial

populations

in

the

decomposition

of

archaeological

bone.

She is

currently

working

in

the

field of ancient

biomolecules,

on the interaction

of

osteocalcin with hydroxyapatite. Address: Fossil

Fuels & Environmental

Geochemistry,

NRG,

Drummond

Building, University of

Newcastle,

Newcastle

upon

Tyne

NE1

7RU,

UK.

Resum--La

taphonomie

est

l'etude

de tout

changement qui

intervient sur un animal ou sur

-une

plante

apres

sa

mort. Les os

sont les

principaux

materiaux

d'origine

animale

d

survivre

dans

l'environnement

archeologique.

C'est une source

importante

d'informations

sur

les

relations,

les

regimes,

les

maladies et les

dges

des

peuples

(et

des

animaux)

des anciennes

cultures. Pour trouver et

interpreter

ces

informations,

il est

besoin

de

conser-

vateurs

et

d'hommes

de sciences

archeologues qui

connaissent les

processus

de

changements

taphonomiquesqui

peuvent

advenir

aux os. Ces

changements

sont nombreux:

ce

papier

tente de

definir quelques-uns

de ces

princi-

paux

changements, qui peuvent

etre

attribues a l'action

variee

des

microorganismes.

Zusammenfassung-Taphonomie

bezeichnet das Studium aller

Veranderungen

an Lebewesen und

Pflanzen

nach

dem

Tod.

Knochen sind das

vorherrschendeMaterial lebenden

Ursprunges,

das sich im

archaologischen

Rahmen erhalt. Sie

sind die

Quelle fir

eine

Vielzahl

von

Informationen

uiber

Beziehungen, Nahrung,

Krankheiten und

Alter von Menschen

und Tieren

vergangener

Kulturen. Damit

diese

Informationen

gesammelt

und

interpretiert

werden

k6nnen,

miissen Konservatoren

und

archdologische

Wissenschaftler

ein

Bewujftsein

ur

taphonomische

Veranderungen

entwickeln,

die sich

in

Knochenmaterial

abspielen

konnen.

Die

vorliegende

Arbeit

versucht,

wichtigste

in

Knochen

feststellbare

Veranderungen

zu

definieren,

welche den

zahlreichen

Aktivitdten

von

Mikroorganismen

zuzuschreibensind.

Resumen-Tafonomia

es el estudio de todos los cambios

efectuados

en

las

sustdncias animales

y vegetales

despues

de la muerte

de

las mismas.

De los restos de

origen

animal,

el

hueso

es el

principal

material

per-

Studies in Conservation40 (1995) 19-30 29


13/13

A.M.

Child

durable dentro

del dmbito

arqueol6gico.

El hueso es una abundante

uente

de

informacion

acerca de las

rela-

ciones

sociales,

dietas,

enfermedades y

edades

de

gentes

(y

animales)

de culturas

pasadas.

Para

extraer

e

interpretar

esta

informacion,

es necesario

que

los conservadores

y

cientificos

arqueol6gicos

esten conscientes

de

los

procesos

de

cambio

tafonomicos

que

pueden

ocurir

en el

hueso. Estos

procesos

de cambio son numerosisi-

mos.

Este

trabajo

propone

la

definicion

de

algunos

de los

principales

cambios observados

en hueso

y

atribuibles

a las acciones varias

de los

microorganismos.


Microbial taphnomy of archaeological bone

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Transcript of Microbial taphnomy of archaeological bone