Microbial community analysis with a high PHAstorage capacity

L.S. Serafim*, P.C. Lemos*,**, S. Rossetti***, C. Levantesi***, V. Tandoi*** and M.A.M. Reis*

*Departamento de Quımica, CQFB/REQUIMTE, FCT/Universidade Nova de Lisboa, 2829-516 Caparica,

Portugal (E-mail: amr@dq.fct.unl.pt)

**Instituto de Tecnologia Quımica e Biologica (ITQB), Universidade Nova de Lisboa,

2870-156 Oeiras, Portugal

***CNR, Water Research Institute, Via Reno 1, 00198, Rome, Italy

Abstract Activated sludge was submitted to aerobic dynamic substrate feeding for the production of

biodegradable plastics. Two sequencing batch reactors were operated with acetate or propionate as sole

carbon substrates. With acetate a homopolymer of polyhydroxybutyrate (PHB) was obtained and with

propionate a copolymer of hydroxybutyrate and hydroxyvalerate P(HB/HV) was produced. Three main

morphotypes were identified in both sludges: two belong to the Alphaproteobacteria class and the third to

the Betaproteobacteria class. Bacilli belonging to Betaproteobacteria were shown by FISH analysis, applied

in combination with Nile Blue post-staining, to be the main responsible for PHAs storage. The latter were

affiliated to Azoarcus genus within Betaproteobacteria.

Keywords Acetate; aerobic dynamic substrate feeding; FISH; polyhydroxyalkanoates; propionate; SBR

Introduction

With the increasing necessity to replace synthetic plastics with biodegradable polymers,

the reduction of production costs of the latter materials became vital, since their price is

still very high. Polyhydroxyalkanoates (PHA) are one of the most promising biodegrad-

able plastics due to their large range of applications. Currently, pure cultures or geneti-

cally modified organisms are employed in the industrial production of these bioplastics

(Reddy et al., 2003), with the additional and expensive step of sterilization. In this way

the use of mixed cultures, such as activated sludge, for PHA production becomes a more

cost effective process since sterilization is not required. Selection and enrichment in PHA

accumulating organisms result from the imposed operational conditions. In recent years

many papers on production of PHA by activated sludge have been published. Optimiz-

ation of polymer production was investigated by Serafim et al. (2004) obtaining the high-

est value of PHA content stored by activated sludge, 78.5% of cell dry weight. Modelling

of this type of systems was performed by Beun et al. (2002) and Third et al. (2003).

Other authors studied the effect of different volatile fatty acids in order to obtain a copo-

lymer of hydroxybutyrate and hydroxyvalerate (P(HB/HV)) (Dionisi et al., 2004, Lemos

et al., submitted).

Despite the knowledge of the involved organisms in the feast and famine systems that

is important for a better understanding of the mechanisms of PHA production, little is

known about the populations selected under such conditions. The only reference found in

the literature (Dionisi et al., 2002) reported the presence of filaments like Haliscomeno-

bacter hydrossis and Nostocoida limicola, some tetrad-forming bacteria and floc-formers.

In this work two microbial communities enriched under feast and famine conditions

for PHA production, fed with acetate or propionate as sole carbon source, were regularly

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monitored using conventional and molecular biology techniques. The microbial character-

ization data were compared with the performance of the two systems.

Materials and methods

Two SBRs working under aerobic dynamic substrate feeding (ADF) were operated as

described in Serafim et al. (2004) and Lemos et al. (submitted).

The sludge used as inoculum for both SBRs was obtained from a stable and efficient

P removal sequencing batch reactor (SBR) operated since 7 years with a known microbial

composition as described in Levantesi et al. (2002). SBRs were fed respectively with

acetate (A) and with propionate (P) as a sole carbon source at the concentration of

30Cmmol/l. The mineral medium composition used was the same described in Serafim

et al. (2004) supplemented with allylthiourea (10mg/l) in order to avoid nitrification.

Organic acids, ammonia, volatile suspended solids (VSS) and PHA determinations were

done according to Serafim et al. (2004).

Microscopic analysis on samples collected from both systems was regularly performed

since the beginning of each SBR operation. The biomass was observed with a Zeiss

Axioskop by bright field or epifluorescence microscopy after staining or by phase con-

trast. The Gram and Neisser stainings were performed according to Jenkins et al. (1993).

Intracellular PHA granules were shown by Nile Blue staining (Rees et al., 1992).

Fluorescence in situ hybridisation (FISH) was performed according to Amann et al.

(1995) and the oligonucleotide probes utilized are reported in Table 1.

Results and discussion

SBRs performance

The reactor fed with acetate has been working since May 2001 and the reactor fed with

propionate since May 2002. The two systems presented a very stable performance since

the beginning of operation. Figure 1 shows the typical cycles of the reactor fed with acet-

ate (A) and with propionate (P). The kinetic and stoichiometric parameters of the both

SBR biomass were determined by batch experiments. In Table 2 the data obtained for

each biomass fed with acetate or propionate are reported.

Results show that the polymer compositions as well as the kinetic and stoichiometric par-

ameters were different for population A and P. The biomass from reactor A, fed with acetate,

always produced a homopolymer of PHB while the biomass of the reactor P, fed with propio-

nate, produced a copolymer of P(HB/HV). The highest amount of PHA content, higher

Table 1 Oligonucleotide probes used in this study

Probe name Specificity Reference

EUB338 Most Bacteria Amann et al. (1990)EUB338-II Planctomycetales Daims et al. (1999)EUB338-III Verrucomicrobiales Daims et al. (1999)ALF1b Alphaproteobacteria Manz et al. (1992)BET42a Betaproteobacteria Manz et al. (1992)GAM42a Gammaproteobacteria Manz et al. (1992)PAO462 “Candidatus Accumulibacter phosphatis”/PAO Crocetti et al. (2000)PAO651 “Candidatus Accumulibacter phosphatis”/PAO Crocetti et al. (2000)PAO846 “Candidatus Accumulibacter phosphatis”/PAO Crocetti et al. (2000)GAOQ431 “Candidatus Competibacter phosphatis”/GAO Crocetti et al. (2002)ZRA23a Zoogloea ramigera Rosello-Mora et al. (1995)AZA645 Azoarcus spp. Hess et al. (1997)

Probes EUB338, EUB338-II and EUB338-III were applied as a mixture. Probes for detecting PAOs(PAO462, PAO651 and PAO846) were also utilized simultaneously

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specific productivity (qp) and storage yield were obtained by sludge A. Interestingly, a very

high PHB storage (78.5% cell dry weight), the highest amount reported for a mixed culture,

was obtained with population A under particular conditions. More details about SBRs A and

P can be found in Serafim et al. (2004) and in Lemos et al. (submitted).

In order to compare the performance of both populations, batch tests were

also performed by supplying acetate to population P and propionate to the population

A. The population of the system A presented generally a higher PHA storage and carbon

uptake rates than the population P (Table 2). The polymer composition differed

again between the two populations: population A stored a terpolymer of HB, HV and

3-hydroxy-2-methylhydroxyvalerate (HMV) with propionate instead of simple PHB pro-

duced with acetate. Population P produced the same copolymer of P(HB/HV) with both

substrates but an higher HB content was obtained with acetate.

These differences in the metabolism of the two populations were confirmed by in vivo

NMR studies. Using 13C-labelled substrates it was possible to confirm the synthesis of

the different PHA by the two biomass and to verify that the storage and the growth pro-

cesses occurred simultaneously, since labelled glutamate was formed during the feast

phase for the two systems. Further in vivo NMR studies are being performed in order to

clarify the differences verified in the metabolism of both populations.

Biomass composition in SBR A and P

The inoculum of SBR A and P came from a laboratory-scale EBPR reactor previously

described (Levantesi et al., 2002). The latter was mainly composed of three bacterial

populations: the polyphosphate accumulating organisms (PAOs) “Candidatus Accumuli-

bacter phosphatis”, the glycogen accumulating organisms (GAOs) “Candidatus

Competibacter phosphatis” and tetrad forming organisms (TFOs) affiliated to the Alpha-

Figure 1 Feast and famine cycles of reactors fed with acetate (A) and with propionate (P); W – acetate, L

– propionate, A – HB, V – HV, O – ammonia; cycles reduced to the first 4 hours

Table 2 Parameters obtained for culture A and P

SBR A P

Carbon source Acetate Propionate Acetate PropionatePHA PHB P(HB/HV/HMV) P(HB/HV) P(HB/HV)%PHA (cell dry weight) 32.5 15.6 26.2 24.1HB:HV:HMV 100:0:0 31:47:22 68:32:0 28:72:02qS (Cmmol S/Cmmol X.h) 0.80 0.21 0.39 0.202qN (Nmmol/Cmmol X.h) 0.032 0.020 0.030 0.034YX/S (Cmmol X/Cmmol S) 0.20 0.19 0.22 0.25qP (Cmmol HA/Cmmol X.h) 0.47 0.014/0.05/0.04* 0.12/0.014** 0.008/0.031**YP/S (Cmmol HA/Cmmol S) 0.58 0.03/0.22/0.12* 0.49/0.09** 0.05/0.23**

*HB/HV/HMV; **HB/HV

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proteobacteria subphylum. The variation of the biomass composition was monitored

periodically during the SBR A and P operation. The predominant morphotypes were

described by phase contrast observation and after Gram and Neisser staining and their

phylogenetic affiliation was investigated by FISH analysis. Furthermore the role of each

bacterial group, in PHA storage was shown by Nile Blue staining.

After 49 days of reactor operation under feast and famine aerobic conditions, the

populations dominating in the inoculum were mainly replaced by other bacteria. As

shown by FISH analysis with the specific probes (Table 1), the PAOs and GAOs disap-

peared confirming the importance of alternate anaerobic/aerobic conditions for the selec-

tion of these bacteria. On the contrary, the alphaproteobacterial TFOs were retained in

the system. Furthermore, while the inoculum contained some filamentous bacteria, such

as Haliscomenobacter hydrossis and the type 021N (Jenkins et al., 1993), the latter were

almost completely washed out in the feast and famine reactors.

The microbial communities developed in the A and P SBR systems under steady state

were very similar to each others and were mainly composed of the three different mor-

photypes described in Table 3 and shown in Figure 2.

By FISH analysis morphotype I was affiliated to the Betaproteobacteria while morpho-

types II and III were Alphaproteobacteria. As reported in Table 3 morphotype I recalled in

appearance the Zoogloea sp., usually observed in activated sludge systems. However, no

positive hybridisation was observed on this morphotype with the oligonucleotide probe

specific for Zoogloea ramigera. By molecular approach a sequence belonging to Azoarcus

genus was retrieved from the SBR biomass (data not shown). FISH analysis with a probe

targeting this genus (probe AZA645, Hess et al., 1997) was performed on the original

sludge. All the cells with morphotype I hybridised with AZA645 probe indicating the

importance of Azoarcus species in the PHA accumulation in this system.

Although usually organised in pairs, the cells of morphotypes II appeared also as

loosely aggregated tetrads being hardly differentiated from morphotypes III.

Nile Blue staining elucidated the storage capabilities of the described bacterial groups.

In the SBR A and P all the biomass was involved in storage phenomena although the

higher fluorescence intensity, corresponding to higher PHA content was observed in the

morphotype I (Figure 2). The presence of intracellularly stored lipids was followed during

complete cycles in both reactors and it was possible to observe their increase during the

feast phase and their degradation during the next famine period.

The composition of the biomass in terms of morphotypes present remained constant in the

two reactors at steady state while their relative abundance changed during the SBRs oper-

ation. The biomass variation seemed, however, not to affect the SBRs performance

(i.e. storage capacity and type of polymer produced) being constant throughout the operation

period.

Table 3 Morphotypes dominating in the SBR A and P

Morphotypes Description FISH result

Morphotype I Bacilli (1 £ 1.2–1.5mm) organised in loosely aggregated(surrounded by EPS) or compactclusters recalling Zoogloea. Gram and Neisser negative

Betaproteobacteria

Morphotype II Coccobacilli (1 £ 2mm) usually in pairs, orloosely aggregated tetrads. Gram negative,stain violet with Neisser

Alphaproteobacteria

Morphotype III Large cocci (2 £ 2mm) organised in tightly packedtetrads. Gram negative, stain violetwith Neisser

Alphaproteobacteria

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Conclusions

An enrichment (fed with acetate as carbon source), characterized by high storage capability

has been obtained and the microscopic characterization showed the presence of mainly

three distinctive morphotypes. The same morphotypes were enriched in another system fed

with a different carbon source (propionate). FISH applied in combination with Nile Blue

staining showed that the microbial group able to store the higher amount of PHA belonged

to the Azoarcus genus within Betaproteobacteria. Despite both systems presenting a simi-

lar microbial composition, the selected populations in the systems fed with acetate and

with propionate presented different metabolisms that resulted in diverse polymer compo-

sitions, kinetic and stoichiometric parameters. Additional NMR studies and comprehensive

biomass characterization are required to clarify this different behaviour.

Acknowledgements

This research was developed within the agreement for Scientific and Technological

Cooperation between the Gabinete de Relacoes Internacionais da Ciencia e do Ensino

Superior (Portugal) and National Research Council (Italy). The authors acknowledge the

financial support of the Fundacao para a Ciencia e Tecnologia (FCT) through the project

POCTI/35675/Bio/2000. Paulo C. Lemos and Luisa S. Serafim acknowledge Fundacao para

a Ciencia e Tecnologia for grants SFRH/BPD/14662/2003 and SFRH/BPD/14663/2003.

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