Dynamics and Biodiversity of Bacterial and Yeast Communities during Fermentation of Cocoa Beans

Jatziri Mota-Gutierrez; Cristian Botta; Ilario Ferrocino; Manuela Giordano; Marta Bertolino; Paola Dolci; Marcella Cannoni; Luca Cocolin

doi:10.1128/AEM.01164-18

DOI: 10.1128/AEM.01164-18

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FIG 1
Dynamics of the sugar and organic acid compounds in the cocoa bean pulp inoculated and noninoculated during box and heap fermentations, expressed as milligrams per gram. Data are expressed as mean ± standard deviation (SD) values from triplicate determinations. (A) Sucrose; (B) glucose; (C) fructose; (D) citric acid; (E) succinic acid; (F) malic acid; (G) acetic acid; (H) gluconic acid; and (I) lactic acid.
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FIG 2
Spearman's correlation between the microbial OTUs observed with an incidence >1% in at least 2 samples. The samples are labeled according to the box (A) and heap (B) fermentation methods. Rows and columns are clustered by means of Ward's linkage hierarchical clustering. The intensity of the colors represents the degree of correlation between the fungal and bacterial OTUs, as measured by Spearman's correlation. The intensity of the colors represents the degree of correlation between the yeast and bacteria, where blue represents a negative degree of correlation and red a positive degree of correlation.
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FIG 3
Correlation plot showing Spearman's correlation between the microbial OTUs and metabolites observed with an incidence >1% in at least 2 samples. The samples are labeled according to the box (A) and heap (B) fermentation methods. Only significant associations between the OTUs and metabolites are shown (P < 0.05). The intensity of the colors represents the degree of correlation between the fungal and bacterial OTUs, as measured by Spearman's correlation, where the blue color represents a positive degree of correlation and red a negative correlation between the sugars, organic acids, and OTUs.

TABLE 1

Average changes in the physical and microbiological parameters during the inoculated and noninoculated box and heap fermentations of cocoa bean pulp turned after 48 and 96 h

Parameter by fermentation	Inoculation^a	Data by fermentation time (h)^b
Parameter by fermentation	Inoculation^a	0	48	96	120
Box
°C	S	26.73 ± 0.60	36.20 ± 1.53	41.88 ± 1.78	43.70 ± 2.94
	ST	26.48 ± 0.34	35.10 ± 2.46	41.73 ± 2.06	42.78 ± 3.68
	C	26.73 ± 0.62	36.20 ± 1.53	41.88 ± 1.78	43.70 ± 2.94
	Avg	26.58 ± 0.08 C	35.80 ± 1.22 B	42.09 ± 0.50 A	43.33 ± 0.70 A
pH	S	3.55 ± 0.03	3.88 ± 0.16	4.15 ± 0.11	3.96 ± 0.18
	ST	3.54 ± 0.01	4.00 ± 0.16	4.20 ± 0.15	4.18 ± 0.26
	C	3.55 ± 0.03	3.88 ± 0.17	4.27 ± 0.11	3.96 ± 0.18
	Avg	3.57 ± 0.03 C	4.08 ± 0.11 B	4.31 ± 0.09 A	4.15 ± 0.17 AB
Log CFU
Yeast	S	7.08 ± 0.05	7.19 ± 0.15	NC	NC
	ST	7.14 ± 0.01	7.05 ± 0.06	NC	NC
	C	7.19 ± 0.17	7.55 ± 0.27	NC	NC
	Avg	7.14 ± 0.11 A	7.26 ± 0.29 A	NC	NC
LAB	S	5.38 ± 0.30	7.18 ± 0.02	5.11 ± 1.28	6.75 ± 0.17
	ST	6.13 ± 0.22	6.85 ± 0.27	4.33 ± 0.38	7.35 ± 0.25
	C	6.21 ± 0.74	6.88 ± 0.40	4.00 ± 0.00	5.49 ± 0.59
	Avg	5.91 ± 0.61 B	6.97 ± 0.31 A	5.44 ± 2.85 C	6.55 ± 0.94 A
AAB	S	6.41 ± 0.11	7.01 ± 0.12	5.54 ± 1.78	5.63 ± 1.88
	ST	6.28 ± 0.21	6.96 ± 0.06	5.60 ± 1.84	7.34 ± 0.09
	C	6.28 ± 0.25	7.31 ± 0.26	4.00 ± 0.00	5.76 ± 0.49
	Avg	6.32 ± 0.20 A	7.09 ± 0.23 A	5.05 ± 2.01 B	6.69 ± 2.85 A
Heap
°C	S	28.20 ± 1.15	38.17 ± 0.75	36.57 ± 1.80	40.07 ± 0.12
	ST	27.37 ± 0.32	39.00 ± 2.21	36.57 ± 0.90	39.38 ± 0.32
	C	26.27 ± 0.06	38.97 ± 0.32	39.37 ± 2.57	40.30 ± 0.56
	Avg	27.28 ± 0.97 C	38.71 ± 0.47 B	38.40 ± 1.59 B	40.07 ± 0.23 A
pH	S	3.55 ± 0.01	4.24 ± 0.17	4.48 ± 0.79	4.90 ± 0.97
	ST	3.53 ± 0.01	4.32 ± 0.23	4.05 ± 0.40	4.52 ± 0.74
	C	3.50 ± 0.05	3.87 ± 0.08	4.24 ± 0.29	3.99 ± 0.33
	Avg	3.54 ± 0.02 B	4.28 ± 0.24 A	4.26 ± 0.21 A	4.71 ± 0.90 A
Log CFU
Yeast	S	7.16 ± 0.92	7.80 ± 0.15	7.13 ± 0.16	8.03 ± 0.29
	ST	6.76 ± 0.85	7.72 ± 0.15	7.24 ± 1.41	7.43 ± 0.07
	C	7.02 ± 0.71	6.62 ± 0.02	6.34 ± 0.04	7.24 ± 0.28
	Avg	6.98 ± 0.20 C	7.38 ± 0.66 B	6.90 ± 0.48 D	7.57 ± 0.41 A
LAB	S	5.67 ± 0.25	7.28 ± 0.19	7.36 ± 0.04	7.69 ± 0.28
	ST	5.95 ± 0.29	7.07 ± 0.09	7.00 ± 0.01	7.50 ± 0.04
	C	5.72 ± 0.03	6.17 ± 0.21	7.40 ± 0.04	8.10 ± 0.13
	Avg	5.78 ± 0.15 D	6.84 ± 0.59 C	7.25 ± 0.22 B	7.76 ± 0.30 A
AAB	S	6.20 ± 0.23	6.81 ± 0.14	8.33 ± 0.02	7.80 ± 0.18
	ST	5.70 ± 0.04	7.15 ± 0.28	8.08 ± 0.11	7.66 ± 0.01
	C	6.60 ± 0.04	5.65 ± 0.15	8.42 ± 0.02	8.56 ± 0.04
	Avg	6.17 ± 0.45 D	6.54 ± 0.79 C	8.28 ± 0.18 A	8.00 ± 0.49 B

↵a S, S. cerevisiae; ST, S. cerevisiae and T. delbrueckii; C, noninoculated.
↵b Values are expressed as the mean ± SD from triplicate determinations. Different letters indicate statistical differences related to the fermentation period using the least significant difference test (P < 0.05). P values were adjusted using Bonferroni's method. NC, below the detection limit.

TABLE 2

Incidence of the fungal taxonomic groups, achieved by means of amplicon sequencing, expressed as relative abundances^a

Taxonomic group	Relative abundance
	S. cerevisiae								S. cerevisiae + T. delbrueckii							Noninoculated
	Box				Heap				Box				Heap			Box				Heap
	T0	T48	T96	T120	T0	T48	T96	T120	T0	T48	T96	T120	T0	T48	T120	T0	T48	T96	T120	T0	T48	T96	T120
Botryosphaeria	1.33	0.43	0.46	0.21	0.59	1.55	0.13	0.13	0.53	0.44	0.91	3.51	0.56	0.32	0.59	0.66	0.20	1.08	0.16	11.07	0.00	0.47	0.12
Candida	3.33	1.35	1.21	1.27	1.03	0.46	0.31	0.74	1.89	0.97	0.77	5.56	0.53	0.34	0.72	2.83	1.48	2.59	0.49	0.89	0.24	1.39	1.73
Candida butyri	2.23	0.27	0.42	0.50	0.16	0.03	0.07	0.09	0.77	0.69	0.34	0.40	0.15	0.04	0.07	0.70	0.21	1.16	0.09	0.07	0.00	0.07	0.03
Candida diversa	0.00	0.00	0.00	0.00	0.01	0.00	0.00	0.00	0.00	0.00	0.00	5.50	0.00	0.00	0.00	0.00	1.00	0.96	0.00	0.00	0.00	0.00	0.00
Candida inconspicua	0.00	0.58	2.63	7.82	0.06	0.03	0.25	0.32	0.00	0.74	1.51	12.97	0.00	0.04	0.94	0.03	2.61	0.96	13.47	0.00	0.87	1.62	1.43
Candida jaroonii	13.07	4.40	3.63	1.24	4.01	1.65	0.59	1.00	8.06	3.80	2.29	1.24	3.41	1.55	1.96	4.34	2.18	3.04	0.86	1.89	0.24	1.27	0.80
Candida quercitrusa	2.27	1.03	0.78	0.32	1.31	0.94	0.32	0.81	1.40	0.72	1.17	0.49	1.27	0.99	1.34	2.43	1.08	1.04	0.16	0.47	0.09	1.46	0.89
Ceratocystis	3.08	5.98	2.93	1.42	1.43	0.99	0.50	0.50	2.46	2.91	2.91	3.41	1.27	1.56	2.32	4.19	2.45	9.32	4.81	2.07	0.87	1.53	0.80
Hanseniaspora opuntiae	38.33	40.98	39.72	28.21	49.76	39.10	48.67	64.37	36.78	37.25	15.62	0.38	43.73	29.51	11.70	54.21	48.11	29.13	21.78	69.22	72.35	35.05	44.81
Issatchenkia	0.09	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	1.27	0.25	0.00	0.37	0.00	0.41	0.37	0.00	0.00	0.00	2.46	1.18
Kluyveromyces marxianus	0.00	0.28	0.18	8.91	0.06	0.13	11.18	1.79	0.00	0.42	0.38	15.76	0.00	0.13	2.80	0.01	4.61	1.03	43.54	0.09	7.76	7.23	1.98
Lasiodiplodia theobromae	0.83	0.61	2.07	1.33	0.49	0.49	0.09	0.07	3.30	0.91	1.22	0.90	0.80	0.09	0.25	4.94	0.61	0.92	0.27	0.10	0.16	0.07	0.16
Penicillium	0.62	0.39	1.09	0.16	0.19	0.13	0.00	0.06	0.12	0.16	0.31	0.25	0.10	0.19	0.27	0.55	0.12	0.24	0.10	0.15	0.02	0.52	0.21
Pichia	0.56	0.09	0.05	0.16	0.12	0.12	0.01	0.78	0.06	0.09	0.15	0.60	0.07	0.01	0.49	0.37	0.41	0.55	0.19	0.19	0.00	1.21	1.28
Pichia pijperi	10.90	10.23	12.75	15.36	10.21	14.55	6.79	8.42	7.67	9.57	9.05	18.01	13.62	18.35	17.13	11.32	10.91	14.24	8.38	10.37	6.29	29.42	24.14
Saccharomyces cerevisiae	12.95	28.62	28.30	30.22	26.08	37.67	30.29	19.11	7.73	21.50	31.97	15.98	7.67	21.57	33.96	0.28	19.95	27.12	3.35	0.37	10.28	11.92	17.96
Saccharomycopsis	0.72	0.35	0.32	0.15	0.13	0.15	0.07	0.13	1.12	0.30	0.32	0.03	0.27	0.10	0.32	0.82	0.49	1.21	0.22	0.52	0.13	0.32	0.34
Torulaspora delbrueckii	0.06	0.01	0.04	0.01	0.03	0.00	0.01	0.04	25.35	16.32	28.18	8.48	23.02	20.45	22.50	0.02	0.07	0.27	0.03	0.12	0.09	0.13	0.09

↵a Only OTUs with an incidence above 1% in at least 2 samples are shown. Values are expressed as the mean of duplicate determinations. The abundances of OTUs in the 2 biological replicates of each sampling time were averaged. Samples are labeled according to the fermentation period (0 h [T0], 48 h [T48], 96 h [T96], and 120 h [T120]), fermentation method (box and heap), and condition (inoculated with S, inoculated with ST, and noninoculated).

TABLE 3

Classification at the family/genus level of the occurrence of the bacterial taxonomic groups, achieved by means of amplicon sequencing, expressed as relative abundances^a

Taxonomic group	Relative abundance
	S. cerevisiae								S. cerevisiae + T. delbrueckii								Noninoculated
	Box				Heap				Box				Heap				Box				Heap
	T0	T48	T96	T120	T0	T48	T96	T120	T0	T48	T96	T120	T0	T48	T96	T120	T0	T48	T96	T120	T0	T48	T96	T120
Acetobacter pasteurianus	3.16	13.81	62.90	52.83	1.05	40.27	76.99	69.22	3.02	42.67	63.42	86.96	0.96	57.24	79.19	24.74	2.40	18.22	42.67	64.24	4.70	28.52	36.67	45.93
Acetobacteraceae	0.19	1.73	1.01	1.29	0.00	16.87	6.23	5.08	0.29	4.70	0.86	1.05	0.10	11.79	4.41	0.86	0.10	0.58	3.45	2.40	0.38	5.42	1.44	0.10
Acinetobacter	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.29	0.00	0.00	0.05	0.00	0.00	0.00	0.00	4.99	0.00	0.00	0.00	0.00	0.00	0.00	0.05	0.00
Acinetobacter guillouiae	0.24	0.00	0.00	0.00	0.00	0.00	0.00	0.86	0.10	0.00	0.10	0.00	0.00	0.00	0.00	1.25	0.00	0.00	0.00	0.00	0.05	0.00	0.00	0.14
Acinetobacter rhizosphaerae	0.00	0.00	0.00	0.00	0.00	0.00	0.19	1.44	0.00	0.00	0.00	0.00	0.00	0.00	0.00	1.25	0.00	0.00	0.00	0.00	0.05	0.00	0.19	0.24
Bacillus	0.00	0.00	0.00	16.35	0.00	0.00	0.00	0.77	0.00	0.00	11.94	0.29	0.00	0.00	0.00	3.36	0.00	0.10	0.00	4.41	0.05	0.00	0.96	8.96
Dyella	1.68	0.58	0.10	0.14	0.77	0.10	0.00	0.00	0.43	0.38	0.10	0.00	0.19	0.19	0.00	0.00	1.25	0.29	0.58	0.19	0.96	1.10	0.53	0.14
Enterobacteriaceae	3.26	1.34	1.20	0.29	0.00	0.00	0.00	0.10	0.96	0.58	0.10	0.00	0.19	0.00	0.00	0.10	1.82	1.53	0.58	0.19	2.21	1.53	0.62	0.34
Erwinia	4.94	1.68	1.10	0.53	0.19	0.19	0.00	0.00	1.63	0.72	0.19	0.00	0.29	0.10	0.00	0.00	3.45	1.53	0.77	0.58	2.30	1.82	1.05	0.34
Gluconobacter	3.36	1.25	0.38	0.05	1.63	1.34	0.38	0.19	4.07	1.15	0.05	0.10	2.97	1.25	0.38	0.10	2.40	0.67	0.58	0.00	2.88	1.34	0.10	0.10
Klebsiella	0.34	0.43	0.19	0.24	0.00	0.00	0.19	0.19	1.10	0.19	0.10	0.00	0.00	0.00	0.10	0.48	1.25	1.15	0.48	0.10	0.38	0.34	0.34	3.93
Lactobacillaceae	0.05	2.06	3.88	1.20	0.00	0.38	0.00	0.10	0.05	1.68	0.96	0.00	0.00	0.10	0.19	0.10	0.10	5.37	2.21	1.25	0.19	0.62	1.10	0.34
Lactobacillus plantarum group	1.44	15.58	12.27	4.31	0.00	2.11	0.38	0.19	0.91	10.74	1.92	0.67	0.00	1.15	0.19	1.63	0.58	28.57	14.09	5.47	0.29	3.74	6.62	1.49
Lactobacillus fermentum	0.19	31.59	3.40	8.63	0.00	10.07	7.19	5.75	0.14	6.14	10.12	1.44	0.00	9.20	6.90	12.18	0.10	3.55	0.58	7.00	0.05	30.87	13.71	16.73
Lysinibacillus	0.00	0.00	0.00	0.58	0.00	0.00	0.00	0.67	0.00	0.00	1.63	0.00	0.00	0.00	0.00	0.96	0.00	0.00	0.00	0.67	0.00	0.00	0.19	0.05
Trabulsiella	5.13	3.12	2.49	1.53	0.38	0.29	0.00	0.00	2.64	0.81	0.53	0.19	0.58	0.10	0.00	0.10	5.75	1.73	1.53	0.86	2.97	4.12	0.96	0.38

↵a Only OTUs with an incidence above 1% in at least 2 samples are shown. Values are expressed as the mean of duplicate determinations. The abundances of OTUs in the 2 biological replicates of each sampling time were averaged. Samples are labeled according to the fermentation period (0 h [T0], 48 h [T48], 96 h [T96], and 120 h [T120]), fermentation method (box and heap), and condition (inoculated with S, inoculated with ST, and noninoculated).

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Supplemental file 1 -
Relative percentages of volatile compounds (Table S1); yeast community of fermented cocoa beans (Table S2); bacterial community of fermented cocoa beans (Table S3).

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