Metagenome-assembled genomes from microbial communities producing lactic acid from dairy residues

TL;DR

This paper explores microbial communities in bioreactors that convert dairy waste into lactic acid, identifying 42 unique genomes.

Contribution

The study provides new metagenome-assembled genomes from anaerobic bioreactors processing dairy residues.

Findings

Forty-two unique metagenome-assembled genomes were identified from four anaerobic bioreactors.

The genomes represent distinct microbial taxa involved in fermenting dairy residues into lactic acid.

Abstract

To advance the knowledge of microbial communities capable of fermenting agro-industrial residues into value-added products, we report metagenomes of microbial communities from four anaerobic bioreactors fed a mixture of ultra-filtered milk permeate and cottage cheese acid whey. This analysis produced 42 unique metagenome-assembled genomes (MAGs) that represent distinct taxa.

Tables

TABLE 1. Taxonomy, accession numbers, genomic features, and quality statistics of the 42 high- and medium-quality representative MAGs recovered from the bioreactors

MAG ID	Phylum	GTDB classificationa	NCBI classificationb	Reference genomec	ANId	Completeness (%)	Contamination (%)	MAG size (bp)	Contigs	N50 (bp)	%GC	NCBI accession number	NCBI SRA accession numbere
PSEUD1	Pseudomonadota	Pseudomonas_E helleri	Pseudomonas helleri	GCF_001043025.1	97.28	100	0.63	5,950,624	1	5,950,624	58.07	CP181271	SRR31045131
BIF1	Actinomycetota	Bifidobacterium crudilactis	Bifidobacterium crudilactis	GCF_000738005.1	97.36	99.08	2.12	2,454,116	1	2,454,116	57.86	CP181270	SRR31045102
LENTI1	Bacillota	Lentilactobacillus parabuchneri	Lentilactobacillus parabuchneri	GCF_001435315.1	97.43	98.94	0	2,574,352	1	2,574,352	43.68	CP181269	SRR31045178
LACTICAS1	Bacillota	Lacticaseibacillus paracasei	Lacticaseibacillus casei	GCF_000829035.1	98.43	98.64	2.47	2,927,657	29	303,583	46.58	JBKEEX000000000	SRR31045161
LEUC1	Bacillota	Leuconostoc mesenteroides	Leuconostoc mesenteroides	GCF_000014445.1	98.78	91.8	0	1,925,562	8	1,350,794	37.81	JBKEEW000000000	SRR31045102
CLOS1	Bacillota A	Clostridium_H	Clostridium	N/A	N/A	94.71	7.62	2,653,518	18	408,933	31.39	JBKEEV000000000	SRR31045122
CLOS2	Bacillota A	Clostridium_B sp014050525	Clostridium	GCF_014050525.1	99.37	99.59	0	2,617,478	1	2,617,478	30.11	CP181268	SRR31045172
THERMO1	Bacillota A	Thermoanaerobacterium thermosaccharolyticum	Thermoanaerobacterium thermosaccharolyticum	GCF_000145615.1	97.17	99.4	4.11	3,240,700	12	2,846,837	34.01	JBKEEU000000000	SRR31045111
SUTT1	Pseudomonadota	Sutterella	Burkholderia	N/A	N/A	96.89	0	2,299,283	13	427,523	63.29	JBKEET000000000	SRR31045162
ATO1	Actinomycetota	UBA7748 sp900314535	Atopobium	GCA_900314535.1	96.94	99.19	0.81	2,622,559	2	2,593,049	60.25	JBKEES000000000	SRR31045112
LACTIPLAN1	Bacillota	Lactiplantibacillus plantarum	Lactiplantibacillus plantarum	GCF_014131735.1	96.07	99.38	2.78	3,078,116	2	2,813,036	45.26	JBKEER000000000	SRR31045116
PSEUD2	Pseudomonadota	Pseudomonas_E fragi	Pseudomonas fragi	GCF_900105835.1	98.55	86.74	0.27	4,465,640	40	177,538	59.39	JBKEEQ000000000	SRR31045170
LACTO1	Bacillota	Lactobacillus delbrueckii	Lactobacillus delbrueckii	GCF_001433875.1	97.69	99.03	0	2,184,090	1	2,184,090	49.14	CP181267	SRR31045170
LACTO2	Bacillota	Lactobacillus helveticus	Lactobacillus helveticus	GCF_000160855.1	97.68	99.03	0.32	2,234,393	1	2,234,393	36.92	CP181266	SRR31045189
LIMO1	Bacillota	Limosilactobacillus fermentum	Limosilactobacillus fermentum	GCF_013394085.1	98.91	96.86	0	2,025,386	14	154,414	51.82	JBKEEP000000000	SRR31045178
SUCC1	Bacillota C	Succiniclasticum	Succiniclasticum	N/A	N/A	98.58	0	2,160,246	1	2,160,246	55.98	CP181265	SRR31045174
ACID1	Bacillota C	Acidaminococcus timonensis	Acidaminococcus timonensis	GCF_900106585.1	96.53	99.4	0	2,286,539	1	2,286,539	54.95	CP181264	SRR31045164
CAPRO1	Bacillota A	Caproiciproducens sp012516295	Eubacteriales	GCA_012516295.1	99.7	97.99	0.27	2,530,718	1	2,530,718	48.71	CP181263	SRR31045122
CAPRO2	Bacillota A	Caproicibacter sp024498295	Thermocaproicibacter melissae	GCA_024498295.1	99.27	97.32	0	2,007,289	1	2,007,289	49.47	CP181262	SRR31045122
CAPRO3	Bacillota A	Caproicibacter	Clostridium	N/A	N/A	97.99	0	2,650,324	1	2,650,324	44.45	CP181261	SRR31045102
CAPRO4	Bacillota A	Caproicibacter	Oscillospiraceae	N/A	N/A	97.99	0.67	2,802,872	6	1,505,020	45.14	JBKEEO000000000	SRR31045122
LACTOCO1	Bacillota	Lactococcus lactis	Lactococcus lactic	GCF_900099625.1	98.52	100	0.28	2,595,605	1	2,595,605	35.1	CP181260	SRR31045131
PSEUD3	Pseudomonadota	Pseudomonas_E lundensis	Pseudomonas lundensis	GCF_001042985.1	98.05	94.32	0.11	4,386,151	7	913,462	58.66	JBKEEN000000000	SRR31045166
LACTOCO2	Bacillota	Lactococcus_A	Lactococcus	N/A	N/A	98.11	0.5	2,242,213	1	2,242,213	41.54	CP181259	SRR31045116
LACTOCO3	Bacillota	Lactococcus_A raffinolactis	Lactococcus raffinolactis	GCF_001591765.1	98.66	98.11	0.72	2,239,778	1	2,239,778	39.97	CP181258	SRR31045156
VIRG1	Bacillota	Virgibacillus proomii_A	Virgibacillus proomii	GCF_900162615.1	99.58	100	1.67	4,423,115	10	1,262,884	36.16	JBKEEM000000000	SRR31045166
ENT1	Bacillota	Enterococcus_A gilvus	Enterococcus gilvus	GCF_000407545.1	97.95	98.85	3.4	3,781,005	5	2,660,674	42.13	JBKEEL000000000	SRR31045166
ENT2	Bacillota	Enterococcus_B faecium	Entercococcus faecium	GCF_001544255.1	98.62	99.63	0	2,753,251	1	2,753,251	38.16	CP181257	SRR31045113
DIAL1	Bacillota C	Dialister sp900765565	Dialister	GCA_900765565.1	96.99	99.59	0	2,944,120	1	2,944,120	51.49	CP181256	SRR31045111
MEGA1	Bacillota C	Megasphaera sp000417505	Megasphaera	GCF_000417505.1	98.54	100	0	2,329,985	1	2,329,985	54.02	CP181255	SRR31045111
SELE1	Bacillota C	Selenomonas_C sp934202005	Selenomonas	GCA_934202005.1	95.96	100	0	2,276,171	1	2,276,171	60.17	CP181254	SRR31045116
MITSU1	Bacillota C	Mitsuokella sp900552565	Mitsuokella multacida	GCA_900552565.1	95.27	99.07	0.14	2,353,814	1	2,353,814	53.79	CP181253	SRR31045122
XYLA1	Bacillota A	Xylanivirga thermophila	Xylanivirga thermophila	GCF_004138105.1	98.99	97.58	1.21	2,650,085	14	339,090	36.41	JBKEEK000000000	SRR31045122
SPOR1	Bacillota A	Sporanaerobacter	Sporanaerobacter	N/A	N/A	99.3	0.7	2,267,806	1	2,267,806	31.32	CP181252	SRR31045122
PSEUD4	Pseudomonadota	Pseudomonas_E shahriarae	Pseudomonas shahriarae	GCF_014268455.2	99.27	99.32	1.21	6,278,246	1	6,278,246	60.55	CP181251	SRR31045170
BACI1	Bacillota	Bacillus_H clausii	Shouchella clausii	GCF_002250115.1	97.89	98.67	2	4,672,023	1	4,672,023	44.46	CP181250	SRR31045113
ANA1	Bacillota A	Anaerocolumna	Lachnoclostridium	N/A	N/A	94.62	1.57	3,467,848	49	146,365	37.8	JBKEEJ000000000	SRR31045122
HAF1	Pseudomonadota	Hafnia paralvei	Hafnia paralvei	GCF_001655005.1	99.06	99.86	0.45	4,969,061	1	4,969,061	48.1	CP181249	SRR31045104
SERR1	Pseudomonadota	Serratia_J liquefaciens	Serratia liquefaciens	GCF_000422085.1	98.86	100	0.45	5,585,869	1	5,585,869	55.23	CP181248	SRR31045173
RAHN1	Pseudomonadota	Rahnella inusitata	Rahnella inusitata	GCF_003263515.1	98.97	99.84	0.08	5,186,034	4	3,490,194	53	JBKEEI000000000	SRR31045170
CITRO1	Pseudomonadota	Citrobacter braakii	Citrobacter braakii	GCF_002075345.1	98.53	98.21	0.56	5,116,397	30	324,668	52.12	JBKEEH000000000	SRR31045122
PSEUDOCLA1	Actinomycetota	Pseudoclavibacter_A caeni	Pseudoclavibacter caeni	GCF_008831125.1	99.16	98.07	0	2,066,244	5	507,308	71.16	JBKEEG000000000	SRR31045165

Full text

ANNOUNCEMENT

Microbial communities have the potential to increase the value of agro-industrial residues by fermentation. Metagenomes reported here originate from two sets of anaerobic bioreactors and their inoculant cultures. In each experiment, two chemostats were operated anaerobically, at 50°C, pH 5.5, 6-day solids retention time, and either a 6-, 3-, or 1-day hydraulic retention time. The chemostats were supplied with a 1:1 mixture of ultra-filtered milk permeate and cottage cheese acid whey and inoculated with a culture derived from an acid-phase digester at a wastewater treatment plant (Madison, WI, USA) that was first enriched in a similarly operated bioreactor for 26 days at 50°C.

For each experiment, weekly samples of DNA were extracted from the microbes in each bioreactor with the Qiagen Genomic Tip 20 /G (Qiagen) following the manufacturer’s protocol for “Preparation Gram‐negative and some Gram‐positive Bacterial Samples” with lysostaphin (4000 U mL^−1^, Sigma‐Aldrich) added. A total of 37 samples containing 3,000–10,000 ng DNA were sequenced on a Pacific Biosciences Sequel 2 platform by the Joint Genome Institute (JGI) (Berkeley, USA).

Processing and sequencing of DNA included shearing genomic DNA to either 3 kb or 6–10 kb, depending on DNA quality, and performing ligations using the SMRTbell Express template prep kit (Pacific Biosciences). Size selection was performed with BluePippin (Sage Sciences). Finally, the libraries were sequenced on Sequel II (Pacific Biosciences, Inc. [PacBio], Menlo Park, CA, USA) using Sequel Polymerase Binding Kit and Revio chemistry. Across all 37 samples, there was an average of 513,455 reads with a range from 341,988 reads to 828,421 reads. The average length was 9,120 bp (range of 8,037 bp to 10,220 bp), and the average N50 of the reads was 9,367 bp (range of 8,033 bp to 10,531 bp).

Metagenomic DNA quality checking, assembly, binning, and taxonomy classification were performed as follows, and default parameters were used except where otherwise noted. BBTools (v39.01) (1) was used to remove reads containing inverted repeats. Followed by assembly with MetaMDBG (v0.3) (2) (“metaMDBG asm assembly <fastq.gz> -t 4”), polished with Pbmm2 (v1.13.1) (3) and Racon (v1.5.0) (4) for consensus, aligned with minimap2 (v2.26-r1175) (5). Then, “pileup.sh” from BBTools (1) to calculate coverage and binning with MetaBAT (v2.15) (6). Quality was assessed with checkM (v1.2.2) (7), and MAGs were dereplicated using dRep (v3.4.5) (-N50W 2) (8). GTDB-Tk (v2.3.2, database release 214) (9) was used to assign taxonomy to representative MAGs (“gtdbtk identify”) based on concatenated bacterial marker genes (Bac120). For NCBI submission, some taxonomic changes were required as noted in Table 1. Bakta (v1.9.1) (10) was used for gene annotation of all MAGs.

This announcement reports 42 annotated MAGs that are classified as high- and medium-quality according to Bowers et al. (11), dereplicated and representing distinct taxa (Table 1). These data advance our metagenome-based knowledge of agro-industrial residue and waste bioconverting microbiomes.