Microbial community dynamics in mesophilic anaerobic reactors exposed to overfeeding

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Microbial community dynamics in mesophilic anaerobic reactors exposed to overfeeding

Xavier Goux, Magdalena Calusinska, Sébastien Lemaigre, Sébastien Legay, Michael Klocke, Philippe Delfosse

To cite this version:

Xavier Goux, Magdalena Calusinska, Sébastien Lemaigre, Sébastien Legay, Michael Klocke, et al..

Microbial community dynamics in mesophilic anaerobic reactors exposed to overfeeding. 3th World Congress on Anaerobic Digestion, Jun 2013, Santiago de Compostela, Spain. 2013. �hal-01263562�

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13

^th

World Congres s on Anaerobic Diges tion

Santiago de Compostela

W W W .L IP P M A N N .L U

For further information or contact:

Xavier Goux, CRP - Gabriel Lippmann 41, rue du Brill - L-4422 BELVAUX

Tél. (+352) 47 02 61-458 goux@lippmann.lu

Acknowledgements: This work was partly supported by the « Fonds européen de développement régional – INTERREG IV A « Grande Région » 2007-2013 » for the OPTIBIOGAZ project and by the « Fond National de la Recherche » of Luxembourg for the GASPOP Project

MICROBIAL COMMUNITY DYNAMICS IN MESOPHILIC ANAEROBIC REACTORS EXPOSED TO OVERFEEDING

X.Goux

^1,3

, M. Calusinska

¹

, S. Lemaigre

¹

, S. Legay

¹

, M. Klocke

²

, E. Benizri

³

and P. Delfosse

¹

1

Département EVA, Centre de Recherche Public Gabriel Lippmann, 41 rue du Brill, L-4422 Belvaux, LU

2

Abteilung Bioverfahrenstechnik, Leibniz-Institut für Agrartechnik Potsdam-Bornim e.V., Max-Eyth-Allee 100, D-14469 Potsdam-Bornim, DE

3

Laboratoire Sols et Environnement, 2 avenue de la Forêt de Haye, B.P. 172, F-54505 Vandoeuvre lès Nancy cedex, FR

a b

c d

Figure 3. Bacterial community structure dynamic over time for the 4 reactors: (a) R1, (b) R2, (c) R3 and (d) R4.

a b

c d

Figure 4. Archaeal community structure dynamic over time for the 4 reactors: (a) R1, (b) R2, (c) R3 and (d) R4.

Background

Anaerobic digestion (AD) is a complex biological process which involves anaerobic microbial consortia, but little is known about these microbes. Thus, a better knowledge of the Bacteria and Archaea population dynamics and interactions between these two communities are currently considered as a key subjects of research to increase the AD process efficiency. This could provide valuable information regarding unexplained and unpredictable failures or malfuncions of the AD process, including acidosis (pH perturbation).

Aim

Mesophilic CSTRs were monitoring during a gradual overfeeding experiment aiming at causing acidosis. The bacterial and archaeal communitie’s genetic structures were followed using 16S rRNA T-RFLP (Terminal-Restriction Fragment Length Polymorphism).

Material & methods

•Four mesophilic completely/continuously stirred tank reactors (CSTRs) of 100L capacity (named R1, R2, R3, R4)

•PCR products were digested and analysed by T-RFLP using an Applied Biosystems 3130 Genetic Analyser (Stafford, Texas, USA) and the GeneMapper software version 4.0

•To analyse the T-RFLP results, a normalisation was realised as descripted by Dunbar et al. 2001 and the online T-Rex software was used according to Culman et al. 2009. The Richness, Shannon (H), Pielou (J) and Simpson (D) indices were calculated according to Shannon, 1948 ; Simpson 1949 and Pielou 1966 and statistical analysis (one-way ANOVA) were realised using the SPSS software version 19

Res ults

Conclus ion

T-RFLP results showed overt changes in the genetic structure of the microbial community during the organic overloading of mesophilic CSTR reactors. Each reactor was characterized by its own microbial community and its own response to the changing pH. The lower the pH value during the acidosis, the more the composition of the microbial community was altered between the beginning and the end of the experiment. The next step would be to identify TRFs in order to better understand the microbial community structure dynamics during organic overload leading to acidosis.

Overfed reactors (R1, R3, R4)

Date

1 22 38 44 64

kg FM.m-3 .wd-1

0 1 2 3 4 5 6

OLR

Reference reactor (R2)

Date

1 22 38 44 64

kg FM.m-3 .wd-1

0 1 2 3 4 5 6

OLR

D1 D2 D3 D4 D5 D1 D2 D3 D4 D5

Figure 1. Organic Loading Rate (OLR) of the reactors over time in kg.FM.m^-3.wd^-1.

• Seeding sludge originating from the anaerobic digester of the wastewater treatment plant

• After an acclimation period, the reactors were fed with sugar beet pulp (86.8 % TS, 92.0 % OTS) each working day (wd) as descripted in Figure 1

• Fives date of sampling were analysed (D1, D2, D3, D4 and D5) over time

• DNA extraction were performed with a modified protocol of Klocke et al. 2007 and amplified with FAM labelled primers

Reference

Culman, S.W., Bukowski, R., Gauch, H.G., Cadillo-Quiroz, H., Buckley, D.H., 2009 Software open access T-R EX: software for the processing and analysis of T-RFLP data. BMC Bioinformatics 10, 171.

Dunbar, J ., Ticknor, L.O., Kuske, C.R., 2001 Phylogenetic specificity and reproducibility and new method for analysis of terminal restriction fragment profiles of 16S rRNA genes from bacterial communities. Applie d and Environme ntal Microbiology 67, 190-197.

Klocke, M., Mähnert, P., Mundt, K., Souidi, K., Linke, B., 2007. Microbial community analysis of a biogas-producing completely stirred tank reactor fed continuously with fodder beet silage as mono-substrate. S yste matic and Applie d Microbiology 30, 139-151.

Pielou, E.C., 1966. The measurement of diversity in different types of biological collections. Journal of Theore tical Biology 13, 131-144.

Shannon, C.E., 1948. A mathematical theory of communication. The Bell S yste m Technical Journal 27, 379-423.

Simpson, E.H., 1949. Measurement of diversity. Nature 163, 688.

R1 R2 R3 R4

Date Richness Richness Richness Richness D1 5.00 ± 0.82 a 4.00 ± 0.00 a 4.00 ± 0.00 a 4.00 ± 0.00 a D2 6.67 ± 0.47 ab 4.67 ± 0.47 a 6.67 ± 0.47 ab 3.67 ± 0.47 a D3 9.33 ± 1.25 b 5.67 ± 1.25 a 9.67 ± 2.62 b 7.00 ± 0.00 bc D4 7.67 ± 1.25 ab 4.33 ± 0.47 a 7.67 ± 0.94 a 7.33 ± 0.47 b D5 9.33 ± 0.47 b 5.00 ± 0.00 a 7.33 ± 0.47 a 5.67 ± 0.47 c

R1 R2 R3 R4

Date Richness Richness Richness Richness D1 11.00 ± 0.00 a 10.33 ± 0.47 a 8.33 ± 0.47 a 9.00 ± 0.00 b D2 9.00 ± 0.00 b 9.33 ± 0.47 a 11.00 ± 0.00 b 7.33 ± 0.47 bc D3 10.00 ± 0.00 c 9.33 ± 0.47 a 8.00 ± 0.82 a 11.00 ± 0.00 d D4 9.67 ± 0.47 bc 8.33 ± 1.89 a 10.33 ± 0.47 b 7.33 ± 0.1.25 abc D5 11.00 ± 0.00 a 8.00 ± 0.00 a 7.67 ± 0.47 a 7.00 ± 0.00 c

Table 1. Based on T-RFLP analysis, richness (number of bands) of bacterial (a) and archaeal (b) community of the 4 reactors over time.

Given values are the means of three replicates ± standard deviation. Values of richness for each reactors with the same letter are not significantly different at p<0.05 over time.

a

b

Date H J D H J D H J D H J D

D1 1.17 ± 0.12 a 0.51 ± 0.00 a 0.62 ± 0.02 a 0.83 ± 0.05 a 0.41 ± 0.03 a 0.74 ± 0.02 a 1.00 ± 0.06 a 0.50 ± 0.03 a 0.66 ± 0.02 a 0.84 ± 0.12 a 0.42 ± 0.06 a 0.73 ± 0.05 a D2 1.87 ± 0.24 ab 0.68 ± 0.07 b 0.41 ± 0.07 b 1.10 ± 0.15 ab 0.49 ± 0.04 ab 0.65 ± 0.04 ab 1.80 ± 0.06 ab 0.66 ± 0.03 b 0.43 ± 0.02 b 0.61 ± 0.16 a 0.32 ± 0.06 a 0.81 ± 0.05 a D3 2.30 ± 0.27 bc 0.71 ± 0.04 b 0.33 ± 0.05 bc 1.31 ± 0.27 ab 0.53 ± 0.04 ab 0.59 ± 0.07 ab 2.60 ± 0.53 b 0.80 ± 0.06 d 0.26 ± 0.09 c 1.72 ± 0.05 b 0.61 ± 0.02 b 0.48 ± 0.02 b D4 1.80 ± 0.28 b 0.61 ± 0.05 ab 0.47 ± 0.07 b 0.96 ± 0.09 ab 0.46 ± 0.02 ab 0.69 ± 0.02 ab 2.31 ± 0.13 b 0.79 ± 0.01 cd 0.29 ± 0.02 bc 1.71 ± 0.18 b 0.59 ± 0.05 b 0.48 ± 0.06 b D5 2.80 ± 0.07 c 0.87 ± 0.00 c 0.18 ± 0.01 c 1.38 ± 0.17 b 0.59 ± 0.07 b 0.54 ± 0.07 b 1.94 ± 0.16 b 0.68 ± 0.03 cb 0.39 ± 0.04 bc 1.66 ± 0.12 b 0.67 ± 0.03 b 0.46 ± 0.04 b

R1 R2 R3 R4

Date H J D H J D H J D H J D

D1 3.04 ± 0.02 a 0.88 ± 0.01 a 0.15 ± 0.00 a 2.74 ± 0.04 a 0.81 ± 0.00 ab 0.20 ± 0.00 a 2.27 ± 0.05 a 0.74 ± 0.01 b 0.28 ± 0.01 a 2.62 ± 0.02 a 0.83 ± 0.01 b 0.21 ± 0.00 a D2 2.69 ± 0.01 b 0.85 ± 0.00 b 0.21 ± 0.00 b 2.62 ± 0.07 a 0.81 ± 0.00 ab 0.21 ± 0.01 a 2.83 ± 0.01 b 0.82 ± 0.00 a 0.19 ± 0.00 b 2.48 ± 0.06 a 0.86 ± 0.01 cd 0.22 ± 0.01 a D3 3.01 ± 0.01 a 0.91 ± 0.00 c 0.15 ± 0.00 a 2.73 ± 0.04 a 0.85 ± 0.01 a 0.19 ± 0.00 a 2.47 ± 0.21 a 0.83 ± 0.03 a 0.23 ± 0.04 a 3.08 ± 0.00 bc 0.89 ± 0.00 d 0.15 ± 0.00 b D4 2.65 ± 0.04 b 0.81 ± 0.01 d 0.22 ± 0.00 c 2.45 ± 0.34 a 0.81 ± 0.03 ab 0.25 ± 0.05 a 2.78 ± 0.06 b 0.82 ± 0.00 a 0.20 ± 0.01 b 2.16 ± 0.21 b 0.76 ± 0.02 a 0.31 ± 0.03 c D5 3.21 ± 0.00 c 0.93 ± 0.00 e 0.12 ± 0.00 d 2.32 ± 0.01 a 0.77 ± 0.00 b 0.27 ± 0.00 a 2.50 ± 0.04 a 0.85 ± 0.01 a 0.22 ± 0.00 b 2.37 ± 0.01 ab 0.84 ± 0.00 bc 0.24 ± 0.00 a

R3 R4

R1 R2

Table 2 . Based on T-RFLP analysis, Shannon (H), Pielou (J) and Simpson (D) indices of bacterial (a) and archaeal (b) community of the 4 reactors over time.

Given values are the means of three replicates ± standard deviation. Values for each index with the same letter are not significantly different at p<0.05 over time.

a

b

Date

1 22 38 44 64

pH

4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0

R1 R2 R3 R4

D1 D2 D3 D4 D5

Figure 2. pH evolution of the reactors over time.

• Concerning the reference reactor (R2), where the pH remained between 7.1 and 7.4 (Figure 2), several prevailing TRF were detected all along the different date of sampling (Figure 3 and 4)

• No significant differences were found for the Richness, H and D indices over time for the bacterial community of R2 (Table 1a and 2a)

• Regarding the archaeal community structure of the reference reactor, no significant differences were found for the Richness (Table 1b) and significant differences were found only between the D1 and D5 for the H, J and D indices (Table 2b)

• Changes over time in the microbial community of the overfed reactors (R1, R3 and R4) were more visible (Figure 3 and 4)

• The Richness and the three indices were significantly different over time for the three overfed reactors (Table 1 and 2)

• When the overfed reactors reached the lower pH (D4) during the experiment, the H index was significantly different compared with D1 for the bacterial and archaeal community (Table 2)

• The increase of the Richness, the H and J index, and in the same time the decrease of the D index reflected for the Archaeal community a gain of diversity between D1 and D5 (Table 1b and 2b).