Diversity of thermophilic populations during thermophilic aerobic digestion of potato peel slurry

ORIGINAL ARTICLE

Diversity of thermophilic populations during thermophilicaerobic digestion of potato peel slurryJ.O. Ugwuanyi, L.M. Harvey and B. McNeil

Strathclyde Fermentation Centre, University of Strathclyde, Glasgow, UK

Introduction

Thermophilic aerobic digestion (TAD) of waste arises as

a result of conservation of the heat generated during

microbial aerobic metabolism, as a result of which the tem-

perature of the waste undergoing digestion rises to thermo-

philic levels (‡45�C) (Surucu et al. 1975; Heinonen-Tanski

et al. 2005). It is a versatile process, which is increasingly

accepted for waste treatment, either in stand-alone reac-

tions, or as part of integrated two stage processes (Messen-

ger and Ekama 1993). Relative to classical waste process

such as activated sludge, TAD is a new process (Surucu

et al. 1975), and its advantages include the capacity for

rapid, cost-effective waste stabilization (Hawash et al.

1994) and pasteurization (Ugwuanyi et al. 1999). It is

encouraged in Europe and North America, where various

legislations seek to limit the levels of human and animal

pathogens in wastes intended for land or sea application

and disposal (EPA, 1992; Council of the European Union.

2001). Of particular acclaim is the potential for use of

TAD in upgrading, by protein enrichment, of a variety of

agricultural wastes for reuse in animal feed (Couillard and

Zhu 1990; Ugwuanyi et al. 2006). This application has con-

siderable implications for global food security; especially

in the tropics where on account of inadequate food supply,

animals and humans often compete directly for the same

sources of calories.

New studies are emerging that seek to properly charac-

terize TAD (Chu et al. 1997; Ugwuanyi et al. 2004,

2005a,b; Cibis et al. 2006). However, in spite of these, lit-

tle has been performed to properly understand the micro-

biology of the process (Cibis et al. 2002). This is due to

both the newness of the process and also because it has

tended to develop mainly along the lines of public health

Keywords

agricultural slurry, microbial diversity,

thermophilic aerobic digestion, thermophilic

Bacillus spp.

Correspondence

J. O. Ugwuanyi, Department of Microbiology,

University of Nigeria, Nsukka, Nigeria.

E-mail: [email protected]

2007 ⁄ 0036: received 10 January 2007,

revised 22 May 2007 and accepted 29 June

2007

doi:10.1111/j.1365-2672.2007.03557.x

Abstract

Aims: To study the diversity of thermophiles during thermophilic aerobic

digestion (TAD) of agro-food waste slurries under conditions similar to full-

scale processes.

Methods and Results: Population diversity and development in TAD were

studied by standard microbiological techniques and the processes monitored by

standard fermentation procedures. Facultative thermophiles were identified as

Bacillus coagulans and B. licheniformis, while obligate thermophiles were identi-

fied as B. stearothermophilus. They developed rapidly to peaks of 107 to 108 in

£48 h. Stability of obligate thermophiles increased with process temperatures.

Thermophiles were unstable at process pH above or below neutral, but devel-

oped rapidly at all aeration rates. Peak populations were higher in the median

than at extremes of aeration rates. Obligate thermophiles were unstable at low

aeration rates. Process self-heating was higher at lower than at higher aeration

rate. Beyond 96 h most thermophiles were present as spores.

Conclusions: Limited range of indigenous thermophiles drives TAD of slurry.

They develop rapidly and are stable at most digestion conditions.

Significance and Impact of the Study: Development and stability of thermo-

philes in TAD suggest that the process may be operated in a wide range of

conditions; and even at short HRT in continuous processes without compro-

mising efficiency.

Journal of Applied Microbiology ISSN 1364-5072

ª 2007 The Authors

Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 79–90 79

engineering (Mohaibes and Heinonen-Tanski 2004). In

addition, outside biogasification and use of caldoactive

species for pure culture high value applications (Krahe

et al. 1996), use of thermophiles in bioprocesses was

essentially unknown before the 1970s (Brock 1986).

Sonnleitner and Fiechter (1983a,b)studied the microbi-

ology of sewage sludge undergoing TAD at 50–67�C and

characterized at least 95% of the isolates as Bacillus spp.

with maximal growth temperatures in excess of 70�C.

They concluded that the active populations responsible

for heat generation during TAD consist entirely of mem-

bers of the extremely thermophilic B. stearothermophilus.

However, Loll (1976) reported that thermophilic and

thermotolerant Bacillus spp. were together responsible

for the stabilization of model wastewater. During TAD

of swine slurry, Beaudet et al. (1990) counted microbial

populations at 55�C, varying from 104 to 107 ml)1 and

identified them as Bacillus spp., including B. lichenifor-

mis. Burt et al. (1990) enumerated 65 and 55�C thermo-

philes in excess of 106 and 108 ml)1, respectively, during

TAD of sewage sludge, while Malladi and Ingham

(1993) reported thermophilic aerobic spore-former

counts of up to 109 ml)1 during TAD of potato process

wastewater. These populations, which develop from the

proliferation of thermophiles indigenous in the waste

(Burt et al. 1990), fluctuate by up to five orders of

magnitude, but are stable enough not to washout in

continuous processes even in short hydraulic retention

time (Sonnleitner and Fiechter 1983a), particularly at

the higher end of the digestion temperatures (Beaudet

et al. 1990).

The paucity of information on the microbiology of

TAD has left room for speculations on the diversity of

micro-organisms in the process. Even the limited infor-

mation that exists has been based on reactions that

employed sewage sludge (Sonnleitner and Fiechter,

1983a,b). This waste type has limitations as the basis for

projection to other more diverse and potentially reusable

(particularly in animal nutrition) wastes. The possibility

of upgrading and recycling of wastes by TAD (Couillard

and Zhu 1990; Ugwuanyi et al. 2006) requires that further

studies on the microbiology of the process be imple-

mented, particularly using such wastes that have potential

for reuse in animal nutrition. The importance of such

studies include the capacity to give a fair picture of the

microbiological safety and limitations of treated waste,

and also for making projections on process capabilities

and reproducibility in the production of materials re-

entering the animal and human food chain. It is impor-

tant for such studies to take account of variables likely to

obtain in full-scale operations. We have reported on the

efficiency of TAD in the treatment of potato peel waste

(Ugwuanyi et al. 2004, 2005a,b). It is the object of this

current work to address some of the microbiological

knowledge gaps in TAD.

Materials and methods

Preparation of model waste for digestion

Peel waste was manually generated in large batches from

the same type of locally procured white potatoes (Pent-

lands variety) for batch digestion. The potatoes were

washed in lukewarm tap water to remove dirt and air-

dried before peeling. Peel was stored in polythene bags in

batches of 2Æ0 kg at )20�C until required. Peel was

defrosted in water bath at 40�C immediately prior to use,

and blended using a commercial blender (Waring Co.,

MA, USA) to a fine paste capable of passing through a

250-lm mesh. The blended peel was used without any

further treatment unless otherwise specified. Minimal

mineral supplement was added to aid stability of thermo-

philic enzymes in the slurry (Tanner 1997). The composi-

tion of the mineral solution used to make up peel waste

volume was (g l)1); CaCl2, 0Æ1; MgSO4, 0Æ1; K2HPO4,

0Æ03; KH2PO4, 0Æ03 (Tanner 1997). The crude (Kjeldahl)

nitrogen content was determined to be approx. 2% (dry

weight basis). The total solids content of waste employed

in a digestion batch was determined by reference to the

dry matter content of peel and quantity of slurry digested

was then fixed at 0Æ2 kg l)1 of waste (wet weight, corre-

sponding to approx. 8Æ0 g l)1 soluble chemical oxygen

demand) (Ugwuanyi et al. 2004, 2005b).

Reactor

All digestion was carried out as batch processes in a fully

instrumented continuously stirred tank reactor CSTR,

IMCS 2000 [Multiple Bioreactor and Sterile Plants (MBR

AG) Zurich, Switzerland] of 15-l total volume (12-l work-

ing volume).

Digestion conditions

Digestion at different temperatures

Digestions were carried out at 45, 50, 55, 60 and 65�C to

study the effect of digestion temperatures on the develop-

ment of thermophilic populations in the slurry. Digestion

temperatures were selected to reflect what occurs in a

full-scale self-heating process (EPA, 1990) and what has

been variously considered optimal process temperatures

(Ginnivan et al. 1981; Messenger and Ekama 1993; LaPara

and Alleman 1999; Ugwuanyi et al. 2005a,b). The lower

temperature, 45�C was selected as the minimum at

which digestion may be considered thermophilic (Surucu

et al. 1975). The higher temperature 65�C was the first

Diversity of thermophiles during thermophilic aerobic digestion J.O. Ugwuanyi et al.

80 Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 79–90

ª 2007 The Authors

https://www.researchgate.net/publication/227884986_Protease_and_xylanase_activities_and_thermophilic_populations_as_potential_process_monitoring_tools_during_thermophilic_aerobic_digestion?el=1_x_8&enrichId=rgreq-e077aa50-3512-49e4-9941-2c00546e4d0d&enrichSource=Y292ZXJQYWdlOzU5MjE5MDM7QVM6MTcxMTUzODgwNTI2ODQ5QDE0MTc4MTc2NzMxMDg=


temperature at which the indigenous populations could

not start the digestion spontaneously. (Although thermo-

philic populations remained viable in low numbers in the

slurry, reaction failed to start as determined by DO and

other process variables). Microbiological data for this

reaction are therefore not presented. Digestions at differ-

ent temperatures were carried out without pH regulation,

while the aeration rate was fixed at 0Æ5-vvm (volume air

per volume slurry per minute). Changes in the pH of

slurry were recorded at 6- or 12-h intervals.

Control reactions in which in addition to the pH the

temperature was also not regulated were conducted at

0Æ5 and 1Æ0 vvm. Changes in temperature and pH of the

control reactions were recorded at 6- or 12-h intervals.

For these reactions, the external wall of the reactor was

insulated with 5-cm thick, dark PU foam to minimize

heat loss and ensure that the temperature of the slurry

rose to thermophilic levels without any heat input other

than from the metabolic heat and bioreactor moving

parts.

Digestion at different pH values

Digestions at the different pH values were conducted at

55�C based on superior performance and earlier studies

(Beaudet et al. 1990; Ugwuanyi et al. 2004, 2005a,b).

These reactions were implemented at 0Æ5 vvm. The pH

was automatically controlled at 6Æ0, 7Æ0, 8Æ0, 9Æ0 and 9Æ5using 2 mol l–1 H2SO4 and 2 mol l–1 NaOH. Above pH

9Æ5, TAD failed to start. Therefore, no digestions were

carried out beyond pH 9Æ5. Control reaction in which the

pH was not regulated was also included at the defined

temperature and aeration rate and the pH was monitored

at 6- and 12-h intervals.

Digestion at different aeration rates

To study the effect of aeration rates on the development

of thermophilic populations, digestions were carried out

at 0Æ1, 0Æ25, 0Æ5 and 1Æ0 vvm. All aeration rate studies

were carried out at 55�C and pH 7Æ0.

General digestion conditions

Digestions were carried out for 156 h (maximum of

240 h during temperature studies) and terminated when

on-line parameters [dissolved oxygen, oxygen uptake

and carbon dioxide evolution rates and pH (in unregula-

ted reactions)] did not change appreciably over a consec-

utive 24-h period. All digestions were carried out without

any inoculation, by using the indigenous microbial popu-

lations in the waste. Foaming was controlled manually

using sterile polypropylene glycol (BDH) antifoam,

pumped in as necessary. Agitation rate was fixed at

350 rev min–1 throughout all the studies. Any drop in the

volume of waste slurry was noted and the volume was

made up with sterile distilled water at the same tempera-

ture as the digesting slurry, at least 10 min before

sampling.

Sampling

Approximately 50-ml well mixed digestion slurry sample

was collected in a sterile sample bottle at regular 12-h

intervals for the first 36- and 24-h interval thereafter for

the duration of the reaction and processed for microbial

analysis as described below. Samples were analysed imme-

diately on collection (while being maintained in the water

bath at the same temperature as the reactor until inocula-

tion was completed). In all digestions the total volume of

slurry collected as sample for the duration of reaction

remained below 10% of total reaction volume.

On-line determinations

Temperature, pH and dissolved oxygen were measured by

using probes calibrated at the study temperature. Probes

were calibrated at pH 4Æ0, 7Æ0 and 10Æ0 for use in unregu-

lated pH reactions, and at 45�C during unregulated

temperature reaction. Dissolved oxygen and pH probes

were obtained from Ingold Ltd (Switzerland), while

temperature probe was as supplied with the bioreactor.

Oxygen in the exit gas was measured using a paramagnetic

analyser, Servomex, Model 500 (Sybron Taylor Ltd,

Crowborough, UK), while carbon dioxide was measured by

using an infrared analyser ADC, Model 7000 (Analytical

Development Corp., Cambridge, UK).

Enumeration of viable thermophilic populations

Samples were homogenized by mixing briskly in a wrist

action shaker (Gallenkamp, UK). Successive 1 : 10 dilu-

tions of samples were made in sterile peptone water

maintained at 45�C in a water bath and 0Æ1 ml of dilution

was plated by standard spread plate method (Collins et al.

1989) on predried nutrient agar (NA) and plate count

agar (PCA) plates maintained at 45�C before inoculation.

NA and PCA were prepared by fortifying with additional

1Æ5% bacteriological agar (No. 1) and wrapped in plastic

bags to minimize drying before incubation in a static air

incubator at 55 and 65�C for 24–48 h. Count of spores

were carried out after inactivating vegetative cells by heat-

ing samples at 80�C for 10 min in a water bath before

plating and incubating as for total count. All media were

procured from Oxoid Ltd (Basingstoke, UK). Total viable

counts were carried out in triplicate on each of the two

media for each dilution and incubation temperature. By

incubating at two temperatures, the active populations

were separated into obligate thermophiles, which are the

only ones that grow at 65�C, and facultative plus obligate

thermophiles (which together were enumerated at 55�C

J.O. Ugwuanyi et al. Diversity of thermophiles during thermophilic aerobic digestion

ª 2007 The Authors



and constitute the total thermophilic population). The

difference in population obtained at both temperatures

was due to facultative thermophiles that do not grow at

65�C.

Isolation and purification of thermophiles

Isolates which showed distinct morphological features on

NA plates were isolated and purified on further NA

plates. To avoid duplication of populations, as 65�C pop-

ulations are capable of growth at the lower temperature,

isolates obtained at 55�C were tested for their ability to

grow at 65�C, and those that grew at 65�C were included

among the obligate thermophiles for identification.

Biochemical identification of thermophiles

Purified isolates were maintained on NA at 4�C. They

were classified into two groups depending on temperature

of their primary isolation, as well as on the maximum

temperature of growth, as facultative thermophiles enu-

merated at 55�C with a maximum growth temperature

not exceeding 60�C, or obligate thermophiles enumerated

at 65�C with growth temperature extending up to 70�C.

Within each temperature group, populations were

grouped on the basis of their colonial ⁄ cultural character-

istics and microscopic morphology including presence

and nature of endospores. Preliminary tests employed for

the initial characterization of the isolates were based on

the method described by Berkeley et al. (1984) and

Rowan (1996). Sporulation of isolates was determined,

after incubation for a minimum of 96 h in manganous

ion-enriched nutrient broth sporulation medium (Sonn-

leitner and Fiechter 1983a).

Identification was achieved by use of API 50CHB (Bio-

merieux, Basingstoke, UK) for Bacillus spp. supplemented

with the first 12 tests of API 20E (Berkeley et al. 1984;

Rowan 1996). The test isolate was prepared for identifica-

tion by growing it overnight on NA at the appropriate

temperature. A thick suspension of the isolate, prepared

by rubbing off the growth with sterile swab and emulsify-

ing it in sterile normal saline, was used to prepare the

inoculum as described by kit manufacturers, and all test

kits were incubated at 55�C in moist chambers.

The results obtained from the kits were interpreted

using the apilab plus software (Biomerieux, Basingstoke,

UK). In cases where a definitive result could not be

obtained from the APILAB system, because of colour fad-

ing arising from the high temperature of incubation, the

results of the biochemical tests were used to identify iso-

lates by reference to Bergey’s Manual of Systematic Bacte-

riology (Sneath 1984) and the diagnostic profile described

by Berkeley et al. (1984). Identification tests were re-

peated twice in all cases and the results compared with

eliminate ambiguities.

Analysis of results

Results obtained were subjected to anova (single classifi-

cation) using instat� statistical package (Statistical

Services Centre, University of Reading, Reading, UK).

Differences between treatment means were tested using

Tukey–Kramer’s multiple comparison test. Fermenter

experiments were conducted in duplicates. All assays were

carried out in triplicate unless otherwise stated and the

results are reported as the mean of the replicate determi-

nations.

Results

Effect of digestion temperature on development

of thermophiles

Thermophilic populations developed rapidly at all the

digestion temperatures (Fig. 1), reaching peak population

in approx. 48 h. During digestion at 45�C, the total ther-

mophiles (55�C populations) rose rapidly to a peak of

just under 9-log colony forming units (CFU) per millilitre

of slurry in 48 h (Fig. 1A). This population is five orders

of magnitude higher than in the raw waste. Following this

peak, the population decreased gradually for the duration

of reaction. The population of obligate thermophiles rose

from approx. 3-log CFU in the raw waste to peak of

5-log CFU per millilitre in 48 h. The peak count was

maintained for <48 h before dropping dramatically to

population similar to that of the raw waste. At this

temperature, obligate thermophiles accounted for only

0Æ05% of the total population at peak count.

The population of obligate thermophiles increased rap-

idly with temperature of digestion, and at 50�C it

accounted for over 90% of total thermophiles in 48 h.

However, as at 45�C obligate thermophiles declined sig-

nificantly after reaching the peak, and at end of the diges-

tion accounted for only approx. 10% of the total

population. As the digestion temperature increased the

population of obligate thermophiles also increased,

accounting for a higher proportion of the total popula-

tion. During digestion at 55�C, obligate thermophiles

accounted for over 95% of the total population at peak

count (Fig. 1C), and at 60�C for between 99% and 100%,

resulting in an essentially uniform population of obligate

thermophiles (Fig. 1D).

The decline in pH of the digesting slurry appeared to

be temperature-dependent. During digestion at 45 and

50�C, the pH dropped slightly in the first 12 h (remain-

ing above 5Æ0), before rising rapidly (Fig. 1A,B). The



ª 2007 The Authors

decline at 55 and 60�C was greater, resulting in pH

values of <5Æ0 in the first 12 h, and which persisted

for up to 24–36 h before rising to levels similar to

the lower temperatures (Fig. 1C,D). The patterns of

increase in pH were similar at all digestion temperatures,

and the peak values reached were up to pH 8Æ0 except at

50�C.

When the temperature of digestion was not regulated, it

rose rapidly from the starting temperature (25�C) depend-

ing on the aeration rate (Fig. 1E,F). At 0Æ5 vvm, it rose

rapidly to 51Æ5�C in 12 h, then more slowly to a peak of

55Æ5�C in 96 h. Thereafter, it declined slightly and stabi-

lized at approx. 51�C for the duration of the reaction

(Fig. 1E). Both thermophilic populations rose steadily to

1·0 1·0

1·0

1·0

1·0

0

(55°C) (60°C)

(50°C)

(0·5 vvm air) (1·0 vvm air)

(45°C)lo

g C

FU

ml–

1 sl

urry

log.

CF

U m

l–1

slur

ry

log

CF

U m

l–1

slur

ry

log

CF

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l–1

slur

ry

log

CF

U m

l–1

slur

ry

log

CF

U m

l–1

slur

ry

0

0

25·0

35·0

45·0

55·0

30·0

40·0

50·0

60·0

25·0

35·0

45·0

55·0

30·0

40·0

50·0

60·0

0

0

(A)

(C)

(E) (F)

(D)

(B)

Digestion time (days)

Digestion time (days) Digestion time (days)

Digestion time (days)

Digestion time (days)Digestion time (days)0

pH o

f slu

rry

–�–

pH o

f slu

rry

–�–

1 1

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2 2

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3

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6 6

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6

7 7

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7

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pH o

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9

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10 10

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4 5 6 7 8 9 10

5·0 5·0

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4·5 4·5

6·0 6·0

6·0

7·0 7·0

7·0

8·0 8·0

8·0

9·0 9·0

9·0

5·0

4·0

6·0

7·0

8·0

9·09·5

10

10

2·0 2·0

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3·0 3·0

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4·0 4·0

4·0

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5·0 5·0

5·0

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6·0 6·0

6·0

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7·0 7·0

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8·0 8·0

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9·0 9·0

9·0

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2·0

3·0

4·0

5·0

6·0

7·0

8·0

9·0 9·09·0

9·09·0

Figure 1 Development of thermophilic populations during thermophilic aerobic digestion of slurry at different temperatures (0Æ5 vvm aeration

rate). Panels E and F are for digestions without temperature control at 0Æ5 and 1Æ0 vvm aeration rates, respectively. Spore profiles are included for

55 and 60�C processes. pH profiles are shown in figures. , 55�C population; h, 65�C population; s, pH of slurry; , 65�C spore population;

n, 55�C spore population.


ª 2007 The Authors


peaks in 96 h during digestion at 0Æ5 vvm (Fig. 1E). During

digestion at 1Æ0 vvm, the temperature of the slurry rose to

48�C in 12 h and thereafter stabilized at approx. 49�C for

the duration of the digestion (Fig. 1F). At this aeration rate,

increase in thermophilic population was slower and peak

populations for both temperature groups were reached

after 6 days (Fig. 1F). During the unregulated reactions,

the contribution of obligate thermophiles to total popula-

tion was low at temperatures below 50�C (as at 1Æ0 vvm),

but was high when the temperature rose to 55�C (as at

0Æ5 vvm, during the first 96 h).

Effect of digestion pH on development of thermophiles

Thermophilic populations developed rapidly during diges-

tion at pH 6Æ0–9Æ0 (55�C), and peak populations were

reached in 12 h (earliest sampling time) to 24 h at pH

7Æ0–9Æ0 (Fig. 2B–D). This is significantly faster than at

comparable temperatures when the pH was not regulated

(see Fig. 1). At pH 6Æ0, 9Æ5 and in the unregulated

reaction, thermophilic populations increased more slowly

to reach peaks in 36 h or longer. Patterns of population

increase for both temperature groups were comparable

and the populations were stable in all the reactions. Dur-

ing digestion at pH 7Æ0–9Æ0 and in the unregulated reac-

tion obligate thermophiles accounted for up to 98% or

more of the total count, and in the unregulated reaction

declined only slightly after 60 h of digestion (Fig. 2F).

During digestion at pH 6Æ0, the obligate thermophiles

accounted for 85% of total count after 24 h and remained

so to the end of the reaction. A more pronounced drop

in obligate thermophiles contribution to total count was

obtained during digestion at pH 9Æ5, particularly after the

first 60 h of digestion.

The onset of reaction was followed by decline in the

spore population for both the total and obligate thermo-

philes to under 100 CFU ml)1 of slurry. The spore counts

remained low for up to 84 and 96 h for the total and obli-

gate populations, respectively, before increasing rapidly to

counts only slightly lower than the corresponding total

viable count (Fig. 2B). Endospore count followed the

development of respiration in the slurry. Periods when

spore counts were low corresponded to the periods of

most active metabolism, while increase in the spore count

coincided with rise in the DO (Ugwuanyi et al. 2005a,b).

Significantly, even during period of active metabolism,

considerable spore population remained in slurry.

Effect of aeration rate on the development

of thermophiles

Thermophiles developed rapidly at all the aeration rates

(Fig. 3). However, slightly higher total populations were

obtained at 0Æ25 and 0Æ5 vvm than at 0Æ1 and 1Æ0 vvm

(Fig. 3) and these were not significantly different

(P > 0Æ05). Obligate thermophiles developed significantly

less rapidly at 0Æ1 vvm than at the other aeration rates,

unlike the total populations that developed equally rap-

idly at all aeration rates. They were also less stable at the

lower aeration rates (0Æ1 and 0Æ25 vvm) than the total

thermophiles and declined slightly after peak populations

(Fig. 3A,B). During digestion at the higher aeration rates

(0Æ5 and 1Æ0 vvm), obligate thermophiles accounted for

more than 97% of the total population, but <85% of

total population at 0Æ1 and 0Æ25 vvm.

The patterns of decline and rise in spore population

following onset of digestion were similar at all the aera-

tion rates (Fig. 3). At 1Æ0 vvm, sporulation was rapid in

both populations, starting after 36 and 48 h, respectively,

for the obligate and total populations, and by 120 h

nearly all the thermophiles were present as spores. As the

aeration rate declined to 0Æ5 vvm, the onset of sporulation

was delayed to 84 and 96 h, respectively, for the total and

obligate populations. With further decrease in aeration

rate, the onset of sporulation was delayed further and

during digestion at 0Æ1 vvm only a limited proportion of

the populations had sporulated even at time the digestion

was terminated. As metabolic rate had slowed consider-

ably at this time (Ugwuanyi et al. 2005a), the failure of

thermophiles to sporulate extensively may be related to

oxygen deficiency.

Identity of predominant thermophiles

In the course of the digestion processes, over 220 thermo-

philes, culturally and morphologically distinct on primary

isolation, were selected from both temperature groups.

Colonies were isolated to represent different morphologi-

cal types. Passage through different temperatures was used

to separate the isolates into those capable of growth at

55�C but not 65�C and those capable of growing at 65�C.

All isolates capable of growth at 65�C were included

among the obligate thermophiles. On subculture, most of

the isolates within the 65�C group were found to resemble

each other. Following preliminary tests, 12 isolates,

selected to represent the different cultural and microscopic

forms, were retained for definitive identification. These

were separated into four isolates capable of growth at

65�C and eight isolates that grew at 55�C but not 65�C.

All the isolates obtained at 65�C formed terminal, ellip-

soidal spores some of which distended the sporangium.

Considerable variations were noted in the length of the

vegetative cells and in the staining behaviour of cells par-

ticularly during primary isolation. Some of the 65�C iso-

lates lost the ability to sporulate, or sporulated poorly in

nutrient media and often required extended incubation



ª 2007 The Authors

https://www.researchgate.net/publication/8138787_Effect_of_digestion_temperature_and_pH_on_treatment_efficiency_and_evolution_of_volatile_fatty_acids_during_thermophilic_aerobic_digestion_of_model_high_strength_agricultural_waste?el=1_x_8&enrichId=rgreq-e077aa50-3512-49e4-9941-2c00546e4d0d&enrichSource=Y292ZXJQYWdlOzU5MjE5MDM7QVM6MTcxMTUzODgwNTI2ODQ5QDE0MTc4MTc2NzMxMDg=

https://www.researchgate.net/publication/8138787_Effect_of_digestion_temperature_and_pH_on_treatment_efficiency_and_evolution_of_volatile_fatty_acids_during_thermophilic_aerobic_digestion_of_model_high_strength_agricultural_waste?el=1_x_8&enrichId=rgreq-e077aa50-3512-49e4-9941-2c00546e4d0d&enrichSource=Y292ZXJQYWdlOzU5MjE5MDM7QVM6MTcxMTUzODgwNTI2ODQ5QDE0MTc4MTc2NzMxMDg=

(up to 96 h) in sporulation medium to be able to sporu-

late (if they did at all). Within the 55�C populations, var-

iation existed in the location, size and shape of spore,

and size of the vegetative cells both on primary isolation

and on subculture. For both temperature groups, the

microscopic morphology was the more consistent feature

9(A) (B)

8

7

6

5

4

3

log

CF

U m

l–1 s

lurr

y

2

10 12 24 36 48 60 72

Digestion time (h)

(pH 6·0 digestion)(pH 7·0 digestion)

84 96 108 120 132 144 156

9

8

7

6

5

4

3

log

CF

U m

l–1 s

lurr

y

2

1

00 12 24 36 48 60 72

Digestion time (h)

84 96 108 120 132 144 156

9(C) (D)

8

7

6

5

4

3

log

CF

U m

l–1 s

lurr

y

2

10 12 24 36 48 60 72

Digestion time (h)

(pH 8·0 digestion)(pH 9·0 digestion)

84 96 108 120 132 144 156

9

8

7

6

5

4

3

log

CF

U m

l–1 s

lurr

y

2

10 12 24 36 48 60 72

Digestion time (h)

84 96 108 120 132 144 156

9(E) (F)

8

7

6

5

4

3

log

CF

U m

l–1 s

lurr

y

2

10 12 24 36 48 60 72

Digestion time (h)

(pH 9·5 digestion) (Unregulated pH digestion)

84 96 108 120 132 144 156

9

8

10·0

9·0

8·0

7·0

Dig

estio

n pH

6·0

5·0

4·0

7

6

5

4

3log

CF

U m

l–1 s

lurr

y

2

10 12 24 36 48 60 72

Digestion time (h)

84 96 108120132144156

Figure 2 Effect of digestion pH on the development of thermophilic populations during thermophilic aerobic digestion of slurry at 55�C and

0Æ5 vvm aeration rate. Spore counts for both populations were determined at pH 7Æ0 and are shown in (B). Profile of pH in control reaction is

shown in panel F. s, 55�C population; d, 65�C population; h, 55�C spore population; , 65�C spore population.


ª 2007 The Authors


in primary isolation and in subculture. The biochemical

profile based on the use of API 50CHB for members of

the genus Bacillus is shown in Table 1. All the 65�C iso-

lates were identified as B. steraothermophilus while the

55�C isolates were identified as either B. coagulans or B.

licheniformis.

Discussion

The development and diversity of thermophilic popula-

tions during TAD were strongly influenced by the

digestion conditions, particularly the reaction temperature

(see Fig. 1). Burt et al. (1990) reported that obligate

thermophiles do not develop until the temperature of

digestion exceeds 54�C. Similar development is noted in

this study, as the 65�C thermophiles developed poorly

and fluctuated during digestion at 45 and 50�C but

developed rapidly and were stable during digestion at 55

and 60�C. At the higher end, the aeration rate does not

appear to be the principal component in the control of

population relations as counts did not vary significantly

between 0Æ5 and 1Æ0 vvm rates when the temperature was

fixed at 55�C (see Fig. 3C,D). However, it could become

important at the lower aeration rates (0Æ1 and 0Æ25 vvm;

see Fig. 3A,B).

The effect of digestion pH on the development of ther-

mophiles was not as pronounced as that of temperature.

However, obligate thermophiles developed slowly,

declined rapidly, and the peak population obtained was

low at the pH extremes compared with the more neutral

reactions (see Fig. 2). Given this sensitivity of the obligate

thermophiles to extremes of pH, it appears that the drop

in the total population enumerated at pH 9Æ5 was a

reflection of the decline in the number of this subset, and

this may be due to their tendency to fail to sporulate and

so die out after the onset of stationary phase, as reported

by Sonnleitner and Fiechter (1983a). The behaviour of

obligate thermophiles in relation to digestion pH sup-

(0·1 vvm digestion) (0·25 vvm digestion)

(1·0 vvm digestion)(0·5 vvm digestion)

9·0

10·0(A) (B)

(C) (D)

8·0

7·0

6·0

5·0

4·0

3·0

2·0

1·0

00 12

Digestion time (h)

Digestion time (h)

Digestion time (h)

Digestion time (h)

24 36 48 60 72 84 96 108 120 132 144 156 0 12 24 36 48 60 72 84 96 108 120 132 144 156

0 12 24 36 48 60 72 84 96 108 120 132 144 1560 12 24 36 48 60 72 84 96 108 120 132 144 156

log

CF

U m

l–1 s

lurr

y

log

CF

U m

l–1 s

lurr

y

log

CF

U m

l–1 s

lurr

y

log

CF

U m

l–1 s

lurr

y

10·0

9·0

8·0

7·0

6·0

5·0

4·0

3·0

2·0

1·0

0

10·0

9·0

8·0

7·0

6·0

5·0

4·0

3·0

2·0

1·0

0

10·0

9·0

8·0

7·0

6·0

5·0

4·0

3·0

2·0

1·0

0

Figure 3 Effect of aeration rate on the development of thermophilic populations during thermophilic aerobic digestion of slurry at 55�C and pH

7Æ0. Spore profiles are also shown. s, 55�C population; d, 65�C population; h, 55�C spore population; , 65�C spore population.



ª 2007 The Authors

Table 1 Result of biochemical test for the identification of members of the genus Bacillus based on API 50CH B

Isolate Glycerol Erythritol D-Arabinose L-Arabinose Ribose D-Xylose L-Xylose Adonitol B-Methyl xyl Galactose D-Glucose D-Fructose

1V7 + ) ) + + + ) + ) + + +

1V8 + ) ) + + + ) + ) + + +

5V6 + ) ) + + + ) + ) + + +

P70 + ) ) + + + ) + ) + + +

1V3 + ) ) + + + ) ) ) + + +

2V4 + ) ) + + + ) ) ) + + +

2V5 + ) ) + + + ) ) ) + + +

5V2 + ) ) + + + - ) ) + + +

R5A + ) + + + + ) + ) + + +

R5E + ) + + + + ) + ) + + +

P63 + ) + + + + ) + ) + + +

P91 + ) + + + + ) + ) + + +

Isolate D-Mannose L-Sorbose Rhamnose Dulcitol Inositol Mannitol Sorbitol

a-methyl-

D-man

a-methyl-

D-gluco

Nacetyl

glucose Amygdalin Arbutin Aesculin

1V7 + + + ) + + + ) + + + + +

1V8 + + + ) + + + ) + + + + +

5V6 + + + ) + + + ) + + + + +

P70 + + + ) + + + ) + + + + +

1V3 + + + ) + + + ) + + + + +

2V4 + + + ) + + + ) + + + + +

2V5 + + + ) + + + ) + + + + +

5V2 + + + ) + + + ) + + + + +

R5A + + + ) + + + ) + + + + +

R5E + + + ) + + + ) + + + + +

P63 + + + ) + + + ) + + + + +

P91 + + + ) + + + ) + + + + +

Isolate Salicin Cellobiose Maltose Lactose Melibiose Sucrose Trehalose Inulin Melezitose D-Raffinose Starch Glycogen Xylitol

1V7 + + + + ) + + ) + + + + +

1V8 + + + + ) + + ) + + + + +

5V6 + + + + ) + + ) + + + + +

P70 + + + + ) + + ) + + + + +

1V3 + + + + + + + + ) + + + )2V4 + + + + + + + + ) + + + )2V5 + + + + + + + + ) + + + )5V2 + + + + + + + + ) + + + )R5A + + + + + + + ) ) + + + )R5E + + + + + + + ) ) + + + )P63 + + + + + + + ) ) + + + )P91 + + + + + + + ) ) + + + )

Isolate B-Gentiobiose D-Turanose D-Lyxose D-Tagatose D-fucose L-Fucose D-Arabitol L-Arabitol Gluconate 2Ketogluconate 5Ketogluconate

Identity of

Isolate

1V7 + + ) + ) + + ) ) ) + B.s

1V8 + + ) + ) + + ) ) ) + B.s

5V6 + + ) + ) + + ) ) ) + B.s

P70 + + ) + ) + + ) ) ) + B.s

1V3 + + ) + ) ) ) ) + ) ) B.l

2V4 + + ) + ) ) ) ) + ) ) B.l

2V5 + + ) + ) ) ) ) + ) ) B.l

5V2 + + ) + ) ) ) ) + ) ) B.l

R5A + + ) + ) ) + ) + + + B.c

R5E + + ) + ) ) + ) + + + B.c

P63 + + ) + ) ) + ) + + + B.c

P91 + + ) + ) ) + ) + + + B.c

Note: B. s is Bacillus stearothermophilus; B. l is B. licheniformis; B. c is Bacillus coagulans. Results were interpreted by reference to the table of Berkeley et al 31

and the APILAB software.


ª 2007 The Authors


ports earlier reports that highly thermophilic digestions

employing these organisms progress efficiently only at

neutral to slightly alkaline pH (Kosseva et al. (2001);

Ugwuanyi et al. 2005a). Although there is as yet no pub-

lished report of controlled pH digestion, Sonnleitner and

Fiechter (1983a) reported that TAD thermophiles are

neutrophilic in growth characteristics. Also Beaudet et al.

(1990) and Kosseva et al. (2001) reported that waste deg-

radation and population stability declined when the pH

of digesting slurry increased beyond neutral in a continu-

ous process, even though individual isolates were able to

grow up to pH 9Æ5. The enhanced stability of obligate

thermophiles obtained during the unregulated pH reac-

tion may be due to gradual adaptation of the populations

to the gradually increasing pH.

Obligate thermophiles developed poorly during diges-

tion under oxygen limitations such as obtained at

0Æ1 vvm in this study. It may be assumed that, during

digestion under conditions that select for obligate ther-

mophiles, a low aeration rate may result in poor process

performance (particularly in a batch process). This has

indeed been shown to be the case (Ugwuanyi et al.

2005b). As B. stearothermophilus (which predominated at

high temperatures in this study) does not grow in the

absence of oxygen (Sneath 1984), it is possible that they

were metabolically disadvantaged by the reduced avail-

ability of oxygen at 0Æ1 vvm. In summary, unless other

process requirements demand it, the high cost of strong

aeration may be avoided without compromising thermo-

philic activity by operating the process in median of air

supply. In fact, whereas comparable xylanase activities are

maintained at 0Æ5 and 1Æ0 vvm, protease activity at

0Æ5 vvm was over 20 times higher than at 1Æ0 vvm during

TAD of similar slurries (Ugwuanyi et al. 2004).

From the result of this study, microbial succession will

take place in this process as digestion temperature

increases, with obligate thermophiles replacing the facul-

tative ones. As nonmetabolizing populations may be pres-

ent as viable spores, the total viable count may not be

relied upon as an index of active population unless steps

are taken, as in this study, to distinguish between active

vegetative and spore forms. Most of the thermophiles

enumerated in the last few hours of the reactions were

present as spores (see Fig. 3). This may be a stress

response to exhaustion of nutrients. Simplicity of assay

and the clear profile obtained with the spore count sug-

gest that it may be a revealing parameter for monitoring

the development of TAD.

To date, there are few studies on the microbiology of

TAD, because early and continuing research on the pro-

cess has been dominated by the engineering aspects of

process optimization (Vismara 1985). However, Sonnleit-

ner and Fiechter (1983a,b, 1985) reported that over 95%

of the populations associated with the TAD of sludge

were B. stearothermophilus, the balance not being so des-

ignated only because they could not sporulate. Malladi

and Ingham (1993) also identified thermophilic Bacillus

spp. as the predominant populations during the TAD of

potato slop. Although all the thermophiles identified in

the present study belong to the genus Bacillus, they were

not all extremely thermophilic B. stearothermophilus. On

the contrary when the digestion temperature declined to

50�C and below the combination of B. coagulans and

B. licheniformis predominated, comparable to the work of

Burt et al. (1990) with swine slurry, while B. stearother-

mophilus predominated only above 55�C. The use of wide

temperature ranges in the digestion process would have

contributed significantly to the variety of thermophiles

identified in this study. It is clear that when TAD is oper-

ated up to 60�C the majority of the active populations

are members of the B. stearothermophilus group, although

a variety of other species may be isolated, that only sur-

vive in the process by virtue of their ability to sporulate

(Grueninger et al. 1984; Bomio et al. 1989).

A major difficulty in comparing data from microbio-

logical studies of TAD arises from differences in waste

types and operational conditions used. However, it is seen

that a mixture of thermotolerant and thermophilic organ-

isms carry out TAD, with selection and succession

responding to local environments. The principal selection

pressure is the reaction temperature, rather than pH or

aeration rate although these also play a role in affecting

the biochemical potential of populations favoured from

among a variety capable of growing at a given tempera-

ture.

The isolation and identification protocol employed in

this study may have reduced the variety of species

encountered in this study with the possibility of omitting

some populations from particular treatment process.

However, such participating organisms need to be consis-

tent in the processes to be of significance in TAD, as

distinct from participation in a single reaction. It is con-

sidered that the omission of such organisms, if such

occurred, does not imperil the definition of the popula-

tions participating in TAD. (In any case, it is unlikely that

other than routine media and methods will be of any real

use in full scale waste treatment processes). Difficulties

associated with inconsistencies in colonial morphology of

isolates on primary isolation, and rapid changes in cul-

tural morphology of specific temperature groups within

the course of a single digestion constrained attempts to

count the different biochemical groups in the process

hence the recourse to temperature groups. It was also dif-

ficult, for the same reason to construct a profile of suc-

cession on the basis of specific species, particularly

between B. coagulans and B. licheniformis, while only one



ª 2007 The Authors

https://www.researchgate.net/publication/238370444_A_model_for_autothermic_aerobic_digestionEffects_of_scale_depending_on_aeration_efficiency_and_sludge_concentration?el=1_x_8&enrichId=rgreq-e077aa50-3512-49e4-9941-2c00546e4d0d&enrichSource=Y292ZXJQYWdlOzU5MjE5MDM7QVM6MTcxMTUzODgwNTI2ODQ5QDE0MTc4MTc2NzMxMDg=


https://www.researchgate.net/publication/227234459_Growth_and_biocatalytic_activities_of_aerobic_thermophilic_populations_in_sewage_sludge?el=1_x_8&enrichId=rgreq-e077aa50-3512-49e4-9941-2c00546e4d0d&enrichSource=Y292ZXJQYWdlOzU5MjE5MDM7QVM6MTcxMTUzODgwNTI2ODQ5QDE0MTc4MTc2NzMxMDg=

species, B. stearothermophilus, was responsible for activity

because of obligate thermophiles. Participation is likely to

remain most relevant at the level of individual tempera-

ture groups adapted to particular waste type.

In conclusion, a mixed population of facultative and

obligately thermophilic Bacillus spp. drive TAD of potato

slurry. Temperature of digestion is a principal selection

factor, and the starting indigenous mixed populations are

gradually replaced by a near uniform obligately thermo-

philic B. stearothermophilus as the temperature of diges-

tion increases to 55�C and above. The roles of digestion

pH and aeration rate are less pronounced in population

selection. Digesting populations are neutrophilic to

slightly alkaline in their pH preference, and developed

better at the median aeration rates of 0Æ25 and 0Æ5 vvm

than at the extremes of 0Æ1 or 1Æ0 vvm. It is unlikely that

in any waste process a homogeneous population will

carry out waste stabilization, and the variety may also be

influenced by the nature of the waste. However, if the

aim of the process is to use treated waste in animal feed,

there may be need for microbiological control on the

basis of temperature-dependent selection as obtained at

60�C, (besides the need for pasteurization and protein

enrichment, both of which benefit from temperature ele-

vation (Ugwuanyi et al. 1999, 2006).

Acknowledgement

The authors wish to thank the British Commonwealth

Commission for support in carrying out this work.

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ª 2007 The Authors

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Diversity of thermophilic populations during thermophilic aerobic digestion of potato peel slurry