Vol. 16, No.

3, 131–140, 2001

Minireview

The Soil Microbial Biomass: Concept, Measurement and Applications

in Soil Ecosystem Research

PHILIP BROOKES1*

1 Agriculture and Environment Division, IACR-Rothamsted Experimental Station, Harpenden, Herts AL5 2JQ,
UK

(Received May 9, 2001—Accepted July 6, 2001)

For many purposes the soil micro-organisms can be considered as a single pool of living soil organic matter
(the soil microbial biomass). Current methods to measure the microbial biomass are described and their merits
and demerits discussed. The concept of the microbial biomass as a living soil organic matter pool is illustrated by
reference to it as a labile reservoir of potentially plant-available nutrients. An estimate of the turnover times of
biomass carbon (C) of 0.94 years and of biomass phosphorus (P) of 0.39 years shows that the turnover rates of
nutrients within the biomass may be quite different. An understanding of the dynamics of biomass P is important.
The lack of P availability in many tropical agricultural soils has been described as ‘the bottle-neck of world
hunger’. Even if P is supplied it may be rapidly and irreversibly fixed in these, usually, strongly P-fixing soils.
By adding small rates of animal manures with the fertilizer, more biomass P is formed. During the process of
biomass turnover, this P may be released slowly and taken up by the crop more efficiently. Thus, in a Kenyan
P-fixing soil, crop yields were much larger when both manure and fertilizer P were given than when either were
applied singly.

Key words: Microbial biomass measurements, microbial biomass P and C

The soil microbial biomass comprises all soil organisms soil organic matter.
with a volume of less than about 5´103 mm3, other than The biomass, although comprising only about 1 to 4% of
living plant tissue, and can thus be considered as the living total soil organic matter, is an important labile reservoir of
part of soil organic matter. Jenkinson15) eloquently described essential plant nutrients, e.g. nitrogen (N), phosphate (P)
it as “the eye of the needle through which all the organic and sulphate (S). In arable Northern European soils it can
materials must pass” as they are broken down to simple easily contain 100 kg N ha-1 and up to 2 or 3 times more in
inorganic components including water, carbon dioxide, grassland or woodland soils. It is now widely accepted that
nitrate, phosphate and sulphate, that plants can use again. the fertility of both natural and agricultural ecosystems fre-
Because it is living, the microbial biomass responds quently depends upon the nutrients being very efficiently
much more quickly to changing soil conditions, particularly cycled within the organic pools of plants, microbes and
decreases or increases in plant or animal residues, than does organic matter. The biomass is thus both a sink and source
soil organic matter as a whole. Measurable changes in mi- of nutrients, which become available during the process of
crobial biomass may thus reflect changes in soil fertility, biomass turnover.
due, for example, to changing soil management, long before
such changes are reflected in changes in the total pool of The microbial biomass concept
There have been many studies of individual soil micro-
* Corresponding author; E-mail: philip.brookes@bbsrc.ac.uk, Tel: organisms grown either in vitro or in soil under axeinic con-
+44–15827–63133, Fax: +44–15827–60981 ditions. However, in terms of studying soil nutrient dynam-
132 BROOKES

most certainly increase our understanding of microbial sur-

vival in soil, and of the factors controlling specific process-
es. However, for ecosystem studies and investigations into
carbon or nutrient flows that result from large consortia of
microbes processing a wide range of substrates, the ‘black
box’ approach, in which the microbial population is treated
as an undifferentiated whole, still has much to offer.
Here I outline some of the methods that we have devel-
oped to measure the soil microbial biomass. I also attempt
to show what these methods have revealed about some of
the characteristics of the biomass and about its role in the
maintenance of soil fertility.

Measuring the soil microbial biomass

Direct microscopic counting
Microscopic counting is still the most direct method of
estimating the amount of microbial biomass in soil, but is
technically difficult and completely unsuitable for routine
use. Thin films are prepared from an agar-soil suspension,
mounted on microscope slides and then treated with an ap-
propriate stain. Phenolic aniline blue is often used as it
stains protein and is thus considered to give an estimate of
the entire population. The numbers and sizes of spherical
organisms and the lengths and diameters of fungal hyphae
are measured and converted to total biomass by using con-
version factors for specific gravity, percentage carbon con-
Fig. 1. The role of the microbial biomass in the cycling of plant tent and percentage dry matter, obtained from micro-organ-
nutrients. isms grown in vitro. Other stains, especially fluorescent
ones like fluoroscein isothiocyanate or acridine orange, are
ics, work with single species, or even a cluster of species of much easier to count with but do not stain the full range of
micro-organisms has not been generally useful. Exceptions organisms. This method is discussed in more detail by
include mycorrhizae and Rhizobium which have very speci- Jenkinson et al.20) and Jenkinson and Ladd16).
alised functions. Part of the problem is that so few soil mi-
cro-organisms have yet been identified and many cannot be Fumigation-Incubation method
cultured. Another problem is that soil microbial activity re- Jenkinson and Powlson19) showed that more CO2 was
sulting, for example, in carbon dioxide (CO2) evolution, N evolved from a soil fumigated with chloroform, following
mineralization or immobilization, is the net result of com- fumigant removal and aerobic incubation, than from a simi-
plex interactions between the many thousands of microbial lar non-fumigated soil. They subsequently showed that this
species and the many thousands of organic compounds, extra CO2 (the CO2 flush) came from the microbial cells,
which, together, comprise soil organic matter. killed by CHCl3, as they were decomposed by the subse-
As with organic matter itself, using a ‘black box’ ap- quent recolonizing population. They suggested that mea-
proach—measuring the microbial biomass as a single, un- surement of the CO2 flush could provide an estimate of the
differentiated unit19)—has proved surprisingly useful in amount of biomass in soil.
studying soil organic matter dynamics. I hope to illustrate The standard Fumigation-Incubation (FI) method uses
these concepts further in this paper. In doing this, I am soil, first incubated at 40–50% water-holding capacity
mindful of the development of powerful techniques in mo- (WHC) for 7–10 days at 25°C, then given a 24 hour fumiga-
lecular biology which will enable us to study the survival tion with ethanol-free CHCl3, followed by fumigant remov-
and biology of single microbial species in whole soil, with al and a 10 day aerobic incubation of the soil following re-
its full suite of organisms intact12). This approach will al- adjustment to 50% WHC. Biomass C (Bc) is then calculated
Soil Microbial Biomass Dynamics 133

from: Bc=Fc/kc where Fc=[(CO2-C evolved from the fumi- inorganic P (Pi) made extractable to 0.5 M NaHCO3.
gated soil)] minus [(CO2-C evolved from the non-fumigated Brookes et al.8) reported that biomass P could be estimated
soil)], both over the 10 day period. The constant kc is taken from measurement of this increase in Pi, with a correction
to be 0.45 under these conditions, on the basis that approxi- made to account for incomplete extraction of Pi due to fixa-
mately 45% of the carbon in micro-organisms added to tion on soil colloids etc. and assuming that about 40% of the
soils, followed by fumigation and incubation as described P in the soil microbial biomass is extracted by 0.5 M
above, is evolved as CO2 in 10 days. This method has been NaHCO3 following CHCl3-fumigation.
widely used since its introduction and, provided the soils are Sulphur is also released from the biomass during CHCl3
first incubated as above, results are in quite good agreement fumigation and its measurement after extraction can also be
with measurements obtained by direct microscopy20,39). Bio- used to estimate biomass S10,33).
mass C measurements can be converted to total biomass by The FE method offers some considerable advantages
assuming that the biomass contains 45% C on a dry weight over FI. Biomass measurements can be made across the
basis. whole pH range39), in soils containing actively decomposing
The FI method cannot be used in soils that have recently substrates, e.g. cereal straw, both in the laboratory24) and in
been air-dried. Air-drying both kills some of the biomass the field25) and in freshly sampled soils, all conditions where
and renders some non-biomass C decomposable36). In addi- FI is unreliable. Reliable biomass measurements by FE have
tion, FI measurements are unreliable in soils which contain also been reported in paddy (i.e. waterlogged) soils14).
much free CaCO3, soils which have recently received fresh As with FI, the FE method is suitable for use with isotope
substrates23), waterlogged soils14) or soils with a pH below tracer studies. FE has the big advantage that the labelled
about 4.539). biomass that develops as substrates decompose can be mea-
Biomass N can also be estimated similarly by measure- sured immediately after substrate addition26). This is impos-
ment and appropriate calibration of the flush of inorganic N sible with FI. In most situations FE has now replaced FI as
which also occurs during FI. the routine method to measure microbial biomass. However,
FI remains the standard method against which all others are
Fumigation-Extraction method calibrated.
Vance et al.40) first showed a close linear relationship be-
tween biomass C measured by FI and Ec, where Ec=[(organ- Adenosine 5’-triphosphate
ic C extracted by 0.5 M K2SO4, from a 24 h fumigated soil) Adenosine 5’-triphosphate (ATP) is only found in living
minus (organic C extracted from a similar, non-fumigated cells and has a very short exocellular existence (a few
soil)]. They proposed that biomass C can be estimated from hours) in soils. It can be quantitatively extracted from the
the relationship: Biomass C=2.64 Ec. biomass by ultrasonification in the presence of a highly
The fumigation-extraction method (FE) does suffer from acidic reagent (e.g. trichloroacetic acid, TCA) to inhibit
the disadvantage that comparatively small amounts of C phosphatase activity. The reagent we use17) is comprised of
have to be measured in 0.5 M K2SO4. Vance et al.40) used a an aqueous solution of paraquat (0.25 M), sodium dihydro-
dichromate digestion method. However, the C can be more gen phosphate (0.5 M) and trichloroacetic acid (0.5 M). Fol-
conveniently determined by an automated system using lowing ultrasonics, the filtered soil extracts can be analysed
persulphate and U.V. oxidation, which gives essentially the immediately or stored frozen (-18°C) for weeks or months.
same results but more rapidly and easily41). A set of extractants ‘spiked’ with a known quantity of ATP
Chloroform fumigation also increases the amount of total (usually 25 pmol 50 ml-1) are extracted simultaneously. The
N extractable with 0.5 M K2SO4. Brookes et al.7) showed partial recoveries of the ‘spike’ are used to correct for
that this extra N also comes from the microbial biomass and incomplete extraction of native microbial ATP. The ATP is
proposed that biomass N could be estimated from the rela- finally assayed by the fire-fly luciferin-luciferase enzyme
tionship: Biomass N=2.22 EN, where EN is analogous to Ec. system using a bioluminometer or scintillation counter set
About 16% of the total N released by CHCl3 after 24 h to count ‘out of coincidence’.
and extracted by K2SO4 is in either ammonium-N or a-
amino N. These forms react with ninhydrin giving a blue/ Characteristics of the soil microbial biomass
purple colour and measurement of ninhydrin-N can be used Biomass size
to estimate the amount of biomass C2). Measurements of soil microbial biomass C usually show
Following CHCl3-fumigation there is also an increase in that it comprises about 1 to 4% of soil organic C, with the
134 BROOKES

largest proportions in grassland or woodland rather than Table 1. Nutrients immobilized in the soil microbial biomass.
arable soils. A more illuminating way of considering the
C N P
biomass size is in terms of its fresh weight. The microbial
biomass in the plough layer of the unfertilized plot of the kg ha-1
Broadbalk Continuous Wheat Experiment at Rothamsted Broadbalka
contains about 500 kg C ha-1. If we assume that living mi- Unfertilized 180 26 7
crobial cells contain 50% C and 90% water then the total C NPK 200 26 6
immobilized in the cells of the biomass converts to 10 t ha-1 Farmyard manure 310 46 27
of living tissue. This is equivalent to approximately 100 Woodland 570 84 54
sheep per hectare which gives some idea of the size of this Highfield Grassland 890 130 65
a
population. All 0–10 cm soil depth
Only a very small proportion of the total species of mi-
cro-organisms in the biomass have been properly identified. Due to its large size, the amounts of nutrients, e.g. N and
It is almost certain that some of them (as yet unidentified) P immobilized in the cells of the microbial biomass are
will have properties which are ultimately very useful (e.g. quite large (Table 1). They are also usually considerably
antibiotic production). For this, if no other reason, it is larger in grassland than arable soils. This reflects the much
clearly in our best interest to conserve soil in a fully func- larger annual inputs of C in grassland than in arable soils. In
tioning state and avoid polluting it with heavy metals, or low-input soils, a large proportion of the plant-available
other toxic materials. While the expression “don’t treat soil nutrients, especially N, P and S, will be derived from the
like dirt” is a much over-used cliché (and lecture title) in mineralization of nutrients immobilized within the cells of
soil science, it does at least carry a useful message. the microbial biomass.

The biomass as a sink-source of plant nutrients Soil microbial biomass as an early warning of changing
The soil microbial biomass can be considered as a labile soil conditions
pool of essential plant nutrients such as N, P and S, which There is generally a reasonably close linear relationship
are held in a form largely protected from loss due to leach- between amounts of microbial biomass C and amounts
ing or fixation. Until the development of the fumigation- of soil organic C in arable, grassland and woodland
extraction method it was not possible to quantify the sizes soils4,6,13,16,23,29,31,35). The soil microbial biomass increases or
of the microbial pools of these nutrients as they developed decreases in response to changes in soil management much
during the early decomposition of crop or animal residues. more quickly than soil organic matter as a whole, where
This newer methodology made this possible24). such changes can often take many years before being de-
There is evidence that the biomass may utilize nutrients tectable by classical chemical analysis1,6,16). Ayanaba et al.6)
preferentially from plant residues rather than from the soil and Adams and Laughlin1) reported that changing from
nutrient pool26). Thus, the composition and characteristics of forest or grassland to arable management caused much
plant residues will have a major influence on the availability greater decreases in biomass C than total soil organic C.
of nutrients to crops and upon subsequent recycling. Factors Powlson et al.28) showed that 18 years of straw incorpora-
such as substrate C/N ratio, percentage of readily decom- tion in two Danish field experiments (Studsgaard and Røn-
posable and resistant plant tissue or lignin content will have have) caused 40–50% increases in biomass C and N where-
important effects upon the uptake and subsequent mineral- as total soil organic C and N only increased by 5%, a
ization of nutrients in crop residues3). statistically insignificant increase (Figs. 2–3). Both CO2-C
Therefore the correct management of crop residues and evolution and N mineralization over the 0–60 day period
annual manures, while important enough in the generally were significantly greater in the soils which had received
more productive agriculture in temperate regions, is vital in straw than in control soils where the straw had been burnt.
managing soil fertility in the tropics. It is also clear that the At Rønhave the increase in mineralized N was 38% and at
fertility of both natural and agricultural tropical ecosystems Studsgaard 50% (Fig. 4). This is direct evidence that an in-
depends upon the nutrients being very efficiently cycled creased rate of return of crop residues to soil increases the
within the organic pools of plants, microbes and soil organic labile pool of soil organic matter where mineralization-im-
matter. In this way, losses of nutrients from the ecosystem mobilization occurs, to a much greater extent than the soil
are minimised. organic matter as a whole. Some of this N that is mineral-
Soil Microbial Biomass Dynamics 135

Fig. 2. Percentage soil organic C and N and biomass C and N in Studgaard field soils where straw was burnt or incorporated for 18 years.

ized will be derived from mineral N immobilized during This will often be coupled with a decline in soil organic
straw decomposition and some from N originally present matter with time as the inputs of organic C in the crop resi-
in the straw26,30). The additional mineralization of N in dues or animal manures are seldom equal to the annual loss-
straw-treated soils during the 60 d laboratory incubation es of organic C and N caused by microbial mineralization
was equivalent to more than 20 kg N ha-1 at both sites. and soil erosion. Many farmers are therefore faced with de-
Increases of this magnitude in the field, if they occur, are of clining soil fertility, with a resulting decrease in crop yield.
agronomic significance and would permit fertilizer N applica- It is clear therefore that the correct management of crop
tions to be decreased to some degree. residues and animal manures, while important enough in the
Similar results were also reported by Saffigna et al.32) generally more productive agriculture in temperate regions,
for Australian soils. This, and much other similar work, is an essential part of the agricultural economics in develop-
supports the original idea of Powlson and Jenkinson29) that ing countries, especially in tropical climates. If these organ-
the biomass is a much more sensitive indicator of changing ic inputs could be better managed this would have the direct
soil conditions than is total soil organic matter content so result of improving crop yield, by increasing soil nutrient
that the biomass can serve as an “early warning” of such availability, decreasing erosion, improving soil structure
changes long before they may be determined by classical and increasing soil water holding capacity.
chemical analyses. The rate and efficiency of mineralization of the nutrients
held in crop or animal residues, mediated by the soil micro-
Biomass as a sink or source of plant nutrients in low- bial biomass, is a key factor in determining the availability
input agriculture of nutrients to crops. It is also becoming widely accepted
Soil nutrient availability in low-input farming systems that the fertility of both natural and agricultural tropical eco-
mainly depends upon the mineralization of crop residues, systems depends upon the nutrients being very efficiently
animal manures and of native soil organic matter. Many cycled within the organic pools of plants, microbes and soil
farmers outside of the developed world are too poor to af- organic matter. In this way, losses of nutrients from the eco-
ford much inorganic fertilizer so that there is usually a net system are minimized. For example, many tropical soils
removal of nutrients from the soil in the harvested crop. have exceedingly high P-fixation capacities so that P is
136 BROOKES

Fig. 3. Percentage soil organic C and N and biomass C and N in Rønhave field soils where straw was burnt or incorporated for 18 years.

rapidly and irreversibly fixed and becomes unavailable forms. Recent breakthroughs in methodology now make
to plants. However, if cycled within the organic pools, as this possible24,26,43), using both unlabelled and isotopically-
described above, such losses from plant-available forms labelled plant material and other substrates.
can be minimised34).
There is evidence that the microbial biomass constitutes Improving phosphorus fertilizer use efficiency in
an organic matter pool of potentially available, but protect- P-fixing soils in Africa
ed, plant nutrients in tropical ecosystems. Thus, Singh et We are testing the concept of the microbial biomass as a
al.37) reported that the microbial biomass is an important pool of potentially available plant nutrients experimentally
source of plant-available N in tropical soils. The biomass in Africa5). Phosphorus is the limiting nutrient in many Afri-
declined in size as N mineralization increased following the can soils. This is partly because more is removed in the crop
rewetting of such soils, precisely during the period when than is replaced by additions of manures or (even more rare-
plant growth was most rapid. They therefore considered that ly) inorganic fertilizer. Another reason is that many soils
the microbial biomass acted both as a sink and a source of chemically fix P on soil surfaces, where it is then removed
nutrients in these nutrient-poor systems. It thus functioned from the plant-available pool. One way of possibly over-
by accumulating and conserving nutrients in a biologically coming this problem is to apply inorganic fertilizer P to-
active form during the dry period (large biomass—slow gether with an organic fertilizer, e.g. farmyard manure. The
turnover) when the ability of plants to extract nutrients from organic matter may decrease P fixation by masking sites
soil was low. It then released nutrients rapidly once the soils which would otherwise fix P. The microbial biomass which
became wet, so stimulating plant productivity (small bio- decomposes the manure will also have a large demand for P
mass—fast turnover). Until recently it was not possible to as it grows. Thus P will thus be immobilized within the
quantify the sizes of the microbial pools of plant nutrients, microbial cells and so protected from fixation by the soil
e.g. N, P, S as they formed during the early decomposition colloids. As the biomass declines, following exhaustion of
of crop or animal residues. Neither was it possible to moni- the manure, the microbial P will be mineralized to inorganic
tor the fluxes of nutrients under these conditions as they P, which plants can use again.
passed through the biomass, and thence into mineralizable Preliminary results were most encouraging. In both 1997
Soil Microbial Biomass Dynamics 137

and 1998 the (Farmyard Manure+P) treatment gave signifi-

cantly larger yields than when FYM or P were applied sin-
gly to a strongly P-fixing soil at Malava (Fig. 6). Differenc-
es were much less when the treatments were applied to a
non-P fixing soil at Mau Summit. The improvements in
yield were certainly not due to the additional inorganic nu-
trients in the manures. Less than 2 kg P ha-1 was supplied in
this way.

Turnover of the soil microbial biomass

The methods available to measure the ‘standing crop’ of
soil microbial biomass, while having their limitations, have
given estimates of pool sizes of C, N and P in the biomass
which generally fit with perceived reality. They certainly al-
low us to work towards an understanding of soil nutrient dy-
namics which would be impossible if we could only study
micro-organisms as single species or families in soil27).
Linked with this is the concept of biomass turnover, leading
to estimates of the flux of nutrients through the biomass. It
is really by these processes that soil nutrients are made
available to plants by microbial activity. Estimates of bio-
mass turnover times, defined for example for P as: [(Bio-
mass P content, kg P ha-1)/(Annual input of P into the bio-
mass, kg P ha-1 yr-1)] and flux of P through the biomass, as
Fig. 4. N mineralised during 60 days in the laboratory from Danish
field soils where straw was burnt or incorporated for 18 years. [(Biomass P content, kg P ha-1)/(Biomass P turnover time,
yr-1)] can provide estimates of soil nutrient fluxes in

Fig. 5. Possible strategies to overcome the effects of P fixation in soils.

138 BROOKES

Fig. 6. Effect of applied fertilizer P with or without FYM on maize grain yield at Malava.

agricultural or natural ecosystems. soil containing KH232PO4 which has been allowed to equili-
Jenkinson and Rayner21) proposed a turnover time of 2.5 brate for 5 d with unlabelled native soil inorganic P prior to
yr for biomass C measured in the Broadbalk Continuous glucose addition. The apparent turnover times of biomass C
Wheat Experiment under UK field conditions and a turn- and P were estimated by applying first-order kinetics rate
over time for N of 1.52 y was proposed for the biomass in equations to the declines in 32P- and 14C-labelled biomasses
soils of the same experiment, again measured under field at 25°C and 40% water holding capacity (WHC). Assuming
conditions18). The measurements of turnover of biomass C that turnover times of biomass under field conditions in a
were based upon measurements of inputs and declines of temperate climate are about 4 times slower than under the
14C in soil as a result of the atomic bomb tests in the 1960s. above laboratory conditions, Chaussod et al.11), this gives
At this time a pulse of 14C-labelled C entered the global soil mean field turnover rates for both native and recently syn-
organic matter pool giving, hopefully, a unique chance to thesised biomass P of about 0.4 yr and for biomass C of
undertake these measurements. The turnover time for bio- about 1.0 yr, measured in a Rothamsted soil of about 23%
mass N of 1.52 yr18), also obtained under field conditions, clay. Using these values, the mean biomass P flux through 6
was done by adding 15N-labelled inorganic N fertilizer to the UK arable soils was about 40 kg P ha-1 yr-1 and about 140
soil in both cases to obtain these values. Full experimental kg P ha-1 yr-1 for UK grasslands.
and theoretical details of these and other field measure- The faster turnover time for P than C seems reasonable as
ments of biomass turnover are given by others11,18,21). While the P will be almost entirely within the cell membranes and
it is clear that these and similar measurements are best made cytoplasm of the micro-organisms, while the C will also be
under field conditions, the cost, expertise and time required an important constituent of the cell wall. Microbial cell
(often several years) often makes this an impossibility. The walls are known to be much more stable in soil than the in-
need to use radioactive isotopes in many cases causes tracellular components16). The results strongly indicate that
further restrictions, although the increasing use of the non- the microbial biomass is far from being a static component
radioactive isotope 13C may accelerate research into C of the total soil organic matter pool and that the flux of
dynamics under field conditions. Certainly, any proposal to nutrients through it can be surprisingly large. Phosphorus
measure biomass P dynamics, using 32P or 33P in the field coming from biomass turnover will help replenish soil
would now face a plethora of restrictions and regulations inorganic P pools.
which would daunt all but the strongest hearted. It must be emphasised that our ideas about the measure-
Recently we developed a procedure (based on that pro- ment of biomass turnover times and the quantification of
posed by Wu42)) to measure the turnover times of biomass P fluxes of nutrients through the biomass are still evolving. In
and biomass C simultaneously in the same soil, under the particular the fluxes of P through the biomass as measured
same conditions in the laboratory22). The method involves by 32P seem large, although there are few, if any, similar
addition of 14C-labelled substrates (in this case glucose) to measurements to serve as comparisons. Calculated fluxes of
Soil Microbial Biomass Dynamics 139

Table 2. Biomass turnover times and C and P fluxes.

Acknowledgements
Soils Turnover times Flux through biomass
IACR receives grant-aided support from the Biotechnolo-
Biomass C Biomass P C P gy and Biological Sciences Research Council of the United
Years kg ha-1 y-1 Kingdom. I also especially thank Profs. D.S. Jenkinson and
Arable 0.94 0.39 300 44
D.S. Powlson for their contributions to this work, in addi-
Grassland 0.94 0.39 927 146 tion to the many visiting scientists who contributed much
also.

P through the biomass, usually based on ‘standing crop’

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140 BROOKES

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