Influence of Soil Phosphate on Rhizobacterial Performance in Affecting Wheat Yield.

As a primary nutrient in agricultural soils, phosphorus plays a crucial but growth-limiting role for plants due to its complex interactions with various soil elements. This often results in excessive phosphorus fertilizer application, posing concerns for the environment. Agri-research has therefore shifted focus to increase fertilizer-use efficiency and minimize environmental impact by leveraging plant growth-promoting rhizobacteria. This study aimed to evaluate the in-field incremental effect of inorganic phosphate concentration (up to 50 kg/ha/P) on the ability of two rhizobacterial isolates, Lysinibacillus sphaericus (T19), Paenibacillus alvei (T29), from the previous Breedt et al. (Ann Appl Biol 171:229-236, 2017) study on maize in enhancing the yield of commercially grown Duzi® cultivar wheat. Results obtained from three seasons of field trials revealed a significant relationship between soil phosphate concentration and the isolates' effectiveness in improving wheat yield. Rhizospheric samples collected at flowering during the third season, specifically to assess phosphatase enzyme activity at the different soil phosphate levels, demonstrated a significant decrease in soil phosphatase activity when the phosphorus rate reached 75% for both isolates. Furthermore, in vitro assessments of inorganic phosphate solubilization by both isolates at five increments of tricalcium phosphate-amended Pikovskaya media found that only isolate T19 was capable of solubilizing tricalcium at concentrations exceeding 3 mg/ml. The current study demonstrates the substantial influence of inorganic phosphate on the performance of individual rhizobacterial isolates, highlighting that this is an essential consideration when optimizing these isolates to increase wheat yield in commercial cultivation.

High-throughput screening alternative to crystal violet biofilm assay combining fluorescence quantification and imaging.

The crystal violet assay is widely used for biofilm quantitation despite its toxicity and variability. Here, we instead combine fluorescence labelling with the Cytation 5 multi-mode plate reader, to enable simultaneous acquisition of both quantitative and imaging biofilm data. This high-throughput method produces more robust data and provides information about morphology and spatial species organization within the biofilm.

Simple stain-free screening method for pectinolytic microorganisms under alkalophilic conditions.

OBJECTIVES: To develop a simple pectin-degrading microorganism screening method. RESULTS: We developed a method utilizing the phenomenon whereby cooling an alkaline agar medium containing pectin causes the agar to become cloudy. This highly simplified method involves culturing the microorganisms on pectin-containing agar medium until colony formation is observed, and subsequent overnight cooling of the agar medium to 4 °C. Using this simple procedure, we successfully identified pectin-degrading microorganisms by observing colonies with halos on the clouded agar medium. We used alkaline pectinase and Bacillus halodurans, which is known to secrete alkaline pectinase, to establish the screening method. We demonstrated the screening of pectin-degrading microorganisms using the developed method and successfully isolated pectin-degrading microorganisms (Paenibacillus sp., Bacillus clausii, and Bacillus halodurans) from a soil sample. CONCLUSIONS: The developed method is useful for identifying pectin-degrading microorganisms.

Transcriptional analysis of nitrogen fixation in Paenibacillus durus during growth in nitrogen-enriched medium.

Paenibacillus durus strain ATCC 35681T is a Gram-positive diazotroph that displayed capability of fixing nitrogen even in the presence of nitrate or ammonium. However, the nitrogen fixation activity was detected only at day 1 of growth when cultured in liquid nitrogen-enriched medium. The transcripts of all the nifH homologues were present throughout the 9-day study. When grown in nitrogen-depleted medium, nitrogenase activities occurred from day 1 until day 6 and the nifH transcripts were also present during the course of the study albeit at different levels. In both studies, the absence of nitrogen fixation activity regardless of the presence of the nifH transcripts raised the possibility of a post-transcriptional or post-translational regulation of the system. A putative SigA box sequence was found upstream of the transcription start site of nifB1, the first gene in the major nitrogen fixation cluster. The upstream region of nifB2 showed a promoter recognizable by SigE, a sigma factor normally involved in sporulation.

Invited review: Controlling dairy product spoilage to reduce food loss and waste.

Food loss and waste is a major concern in the United States and globally, with dairy foods representing one of the top categories of food lost and wasted. Estimates indicate that in the United States, approximately a quarter of dairy products are lost at the production level or wasted at the retail or consumer level annually. Premature microbial spoilage of dairy products, including fluid milk, cheese, and cultured products, is a primary contributor to dairy food waste. Microbial contamination may occur at various points throughout the production and processing continuum and includes organisms such as gram-negative bacteria (e.g., Pseudomonas), gram-positive bacteria (e.g., Paenibacillus), and a wide range of fungal organisms. These organisms grow at refrigerated storage temperatures, often rapidly, and create various degradative enzymes that result in off-odors, flavors, and body defects (e.g., coagulation), rendering them inedible. Reducing premature dairy food spoilage will in turn reduce waste throughout the dairy continuum. Strategies to reduce premature spoilage include reducing raw material contamination on-farm, physically removing microbial contaminants, employing biocontrol agents to reduce outgrowth of microbial contaminants, tracking and eliminating microbial contaminants using advanced molecular microbiological techniques, and others. This review will address the primary microbial causes of premature dairy product spoilage and methods of controlling this spoilage to reduce loss and waste in dairy products.

Combining co-culturing of Paenibacillus strains and Vitreoscilla hemoglobin expression as a strategy to improve biodesulfurization.

Enhancement of the desulfurization activities of Paenibacillus strains 32O-W and 32O-Y were investigated using dibenzothiophene (DBT) and DBT sulfone (DBTS) as sources of sulphur in growth experiments. Strains 32O-W, 32O-Y and their co-culture (32O-W plus 32O-Y), and Vitreoscilla hemoglobin (VHb) expressing recombinant strain 32O-Yvgb and its co-culture with strain 32O-W were grown at varying concentrations (0·1-2 mmol l-1 ) of DBT or DBTS for 96 h, and desulfurization measured by production of 2-hydroxybiphenyl (2-HBP) and disappearance of DBT or DBTS. Of the four cultures grown with DBT as sulphur source, the best growth occurred for the 32O-Yvgb plus 32O-W co-culture at 0·1 and 0·5 mmol l-1 DBT. Although the presence of vgb provided no consistent advantage regarding growth on DBTS, strain 32O-W, as predicted by previous work, was shown to contain a partial 4S desulfurization pathway allowing it to metabolize this 4S pathway intermediate.

The chemical properties and hygroscopic activity of the exopolysaccharide lubcan from Paenibacillus sp. ZX1905.

This study aims to explore the production and physicochemical properties of an exopolysaccharide (EPS) produced from soil isolate, Paenibacillus sp. ZX1905 in submerged culture. The highest EPS production of 15.67 g/L was achieved in a medium containing soluble starch, peptone and inorganic salts. The purified EPS exhibited excellent skin lubricating properties and was named lubcan. The chemical analysis reviewed that lubcan was an acidic heteropolysaccharide consisted of glucuronic acid, glucose, mannose, galactose, and rhamnose in a molar ratio of 2:3:1:2:2, and the average molecular mass was about 3.27 × 106 Da. NMR and methylation analysis revealed that lubcan backbone was composed of 1,4-α-Man, 1,4,6-α-Glc, 1,3-α-Gal, 1,3-ß-Rha, and 1,3-ß-Gal, together with the branches of 1,3-α-Glc, 1,3-α-Rha, two 1,4-α-GlcA, and terminal-α-Glc(4,6-pyr). The lubcan solution exhibited stability at pH ranging from 5.0 to 7.0, temperature between 5 and 50 °C, and monovalent salts (0.2 M) and divalent salts (0.05 M) addition. The moisture absorption rates of lubcan were 16.98% and 40.41% under the conditions of 43% and 81% relative humidity, which were close to that of hyaluronic acid (17.28% and 41.20%, respectively). These properties make lubcan a good alternative to more expensive hyaluronic acid in the cosmetic industry.

Transcriptome analysis of polysaccharide-based microbial flocculant MBFA9 biosynthesis regulated by nitrogen source.

Microbial flocculant (MBF), an environmentally friendly water treatment agent, can be widely used in various water treatments. However, its use is limited by low yield and high cost. This problem can be solved by clarifying its biosynthesis mechanism and regulating it. Paenibacillus shenyangensis A9, a flocculant-producing bacterium, was used to produce polysaccharide-type MBFA9 by regulating the nitrogen source (nitrogen adequacy/nitrogen deficiency). In this study, RNA-Seq high-throughput sequencing technology and bioinformatic approaches were used to investigate the fermentation and biosynthesis of polysaccharide-type MBFA9 by regulating the nitrogen source (high nitrogen/low nitrogen) in the flocculant-producing bacteria Paenibacillus shenyangensis A9. Differentially expressed genes, functional clustering, and functional annotation of key genes were assessed. Then the MBFA9 biosynthesis and metabolic pathway were reconstructed. Our results showed that when cultured under different nitrogen conditions, bacterial strain A9 had a greater ability to synthesize polysaccharide-type MBFA9 under low nitrogen compared to high nitrogen conditions, with the yield of MBFA9 reaching 4.2 g/L at 36 h of cultivation. The quality of transcriptome sequencing data was reliable, with a matching rate of 85.38% and 85.48% when L36/H36 was mapped to the reference genome. The total expressed genes detected were 4719 and 4730, with 265 differentially expressed genes. The differentially expressed genes were classified into 3 categories: molecular function (MF), cell component (CC), and biological process (BP), and can be further divided into 22 subcategories. There were 192 upregulated genes and 73 downregulated genes, with upregulation being predominant under low nitrogen. UDP-Gal, UDP-Glc, UDP-GlcA, and UDP-GlcNAc, which are in the polysaccharide metabolic pathway, could all be used as precursors for MBFA9 biosynthesis, and murA, wecB, pgm, galU/galF, fcl, gmd, and glgC were the main functional genes capable of affecting the growth of bacteria and the biosynthesis of MBF. Results from this study provide evidence that high-level expression of key genes in MBFA9 biosynthesis, regulation, and control can achieve MBFA9 directional synthesis for large-scale applications.

Bioleaching of silicon in electrolytic manganese residue using single and mixed silicate bacteria.

Electrolytic manganese residue (EMR) is a type of industrial solid waste with a high silicon content. The silicon in EMR can be used as an essential nutrient for plant growth, but most of the silicon is found in silicate minerals with very low water solubility, that is, it is inactive silicon and cannot be absorbed and used by plants directly. Thus, developing a highly effective and environmentally friendly process for the activation of silicon in EMR is important both for reusing solid waste and environmental sustainability. The aim of this study was to investigate the desilication of EMR using cultures of Paenibacillus mucilaginosus (PM) and Bacillus circulans (BC). The results showed that the two types of silicate bacteria and a mixed strain of them were all able to extract silicon from EMR with a high efficiency, but the desilication performance of the mixed PM and BC was the best. Fourier transform infrared spectroscopy indicated that silicate bacteria can induce a suitable micro-environment near the EMR particles and release Si into the solution through their metabolism. X-ray diffraction analysis confirmed that layered crystal minerals, such as muscovite and diopside, were more likely to be destroyed by the bacterial action than quartz, which has a frame structure. Scanning electron microscopy-energy dispersive spectrometry proved that the silicate structures were destroyed and that Si in the residue was decreased, indicating the dissolution of silicon under the action of these microorganisms. This study suggests that bioleaching may be a promising method for the activation of silicon in EMR.

A novel intracellular dextranase derived from Paenibacillus sp. 598K with an ability to degrade cycloisomaltooligosaccharides.

Paenibacillus sp. 598K produces cycloisomaltooligosaccharides (CIs) in culture from dextran and starch. CIs are cyclic oligosaccharides consisting of seven or more α-(1 → 6)-linked-D-glucose residues. The extracellular enzyme CI glucanotransferase (PsCITase), which is the member of glycoside hydrolase family 66, catalyzes the final stage of CI production and produces mainly cycloisomaltoheptaose. We have discovered a novel intracellular CI-degrading dextranase (PsDEX598) from Paenibacillus sp. 598K. The 69.7-kDa recombinant PsDEX598 does not digest isomaltotetraose or shorter isomaltooligosaccharides, but digests longer ones of at least up to isomaltoheptaose. It also digests oligoCIs of cycloisomaltoheptaose, cycloisomaltooctaose, and cycloisomaltononaose better than it does with megaloCIs of cycloisomaltodecaose, cycloisomaltoundecaose, and cycloisomaltododecaose, as well as an α-(1 → 6)-D-glucan of dextran 40. PsDEX598 is produced intracellularly when culture medium is supplemented with cycloisomaltoheptaose or dextran, but not with isomaltooligosaccharides (a mixture of isomaltose, isomaltotriose, and panose), starch, or glucose. The whole genomic DNA sequence of the strain 598K implies that it harbors two genes for enzymes belonging to glycoside hydrolase family 66 (PsCITase and PsDEX598), and PsDEX598 is the only dextranase in the strain. PsDEX598 does not have any carbohydrate-binding modules (CBMs) and has a low similarity (< 30%) with other family 66 dextranases, and the catalytic amino acids of this enzyme are predicted to be Asp191, Asp303, and Glu368. The strain Paenibacillus sp. 598K appears to take up CI-7, so these findings indicate that this bacterium can degrade CIs using a dextranase within the cells and so utilize them as a carbon source for growth.

Isolation and Characterization of Antagonistic Bacteria Paenibacillus jamilae HS-26 and Their Effects on Plant Growth.

Soilborne pathogens affect plant growth and food production worldwide. The application of chemical fertilizers and pesticides to control plant diseases has harmful effects; fortunately, plant growth-promoting rhizobacteria can be used as a potential alternative strategy. Here, Paenibacillus jamilae HS-26 was selected for its highly antagonistic activity against several soilborne pathogens. The bacterium synthesized hydrolytic enzymes and released extracellular antifungal metabolites and volatile organic compounds-primarily, N, N-diethyl-1, 4-phenylenediamine, which was detected by gas chromatography-mass spectrometry and shown to inhibit fungal mycelial growth. Furthermore, HS-26 was useful for nitrogen fixation, phosphate and potassium solubilization, and siderophore and indoleacetic acid production. In vitro tests and pot experiments revealed that HS-26 considerably increased plant biometric parameters. Illumina MiSeq sequencing data showed a significant reduction in soilborne pathogens and increase in beneficial bacteria in the wheat rhizosphere after treatment with strain HS-26.

Evidencia molecular de la ocurrencia de bacterias celulolíticas del género Cohnella en especies neotropicales de Nasutitermitinae colectadas en el NEA / Molecular evidence that cellulolytic bacterial genus Cohnella is widespread among Neotropical Nasutitermitinae from NE Argentina

Cohnella is a highly cellulolytic bacterial genus, which can be found in a variety of habitats. The aim of this study was to assess its presence in the digestive tract of termite species collected in North-eastern Argentina: Nasutitermes aquilinus, N. corniger and Cortaritermes fulviceps. Gut homogenates were incubated with cellulosic substrate for bacterial growth. Bacterial 16S rDNA was partially amplified using new primers for Cohnella spp. and cloned. Sequences obtained showed highest similarity (97.2-99.9%) with those of Cohnella spp. previously reported from diverse environments. Phylogenetic analysis tended to group the clones according to their host species and sampling sites. These results indicate the association of Cohnella-related intestinal symbionts with three common Neotropical termites. Their potential industrial application encourages further research.

Cohnella es un género de bacterias celulolíticas que puede ser encontrado en una variedad de hábitats. El propósito de este estudio fue registrar su presencia en el tracto digestivo de termitas (Nasutitermes aquilinus, N. corniger y Cortaritermes fulviceps) colectadas en el noreste argentino (NEA). Se incubaron homogenados de intestinos en sustrato celulósico para multiplicar las bacterias. Utilizando nuevos cebadores para Cohnella spp., se amplificó una porción del ADN ribosomal 16S bacteriano, el cual fue posteriormente clonado. Las secuencias obtenidas mostraron su mayor porcentaje de similitud (97,2-99,9%) con Cohnella spp., previamente reportadas en diversos ambientes. El análisis filogenético tendió a agrupar a los clones de acuerdo a la especie hospedante y al sitio de muestreo. Estos resultados indican que especies de termitas frecuentes en el NEA albergan simbiontes intestinales relacionados con el género Cohnella. Las potenciales aplicaciones industriales de estos microorganismos animan a profundizar los estudios.

Virulence of Melissococcus plutonius and secondary invaders associated with European foulbrood disease of the honey bee.

European foulbrood is a globally distributed brood disease affecting honey bees. It may lead to lethal infections of larvae and, in severe cases, even to colony collapse. Lately, a profound genetic and phenotypic diversity was documented for the causative agent Melissococcus plutonius. However, experimental work on the impact of diverse M. plutonius strains on hosts with different genetic background is completely lacking and the role of secondary invaders is poorly understood. Here, we address these issues and elucidate the impact and interaction of both host and pathogen on one another. Moreover, we try to unravel the role of secondary bacterial invasions in foulbrood-diseased larvae. We employed in vitro infections with honey bee larvae from queens with different genetic background and three different M. plutonius strains. Larvae infection experiments showed host-dependent survival dynamics although M. plutonius strain 49.3 consistently had the highest virulence. This pattern was also reflected in significantly reduced weights of 49.3 strain-infected larvae compared to the other treatments. No difference was found in groups additionally inoculated with a secondary invader (Enterococcus faecalis or Paenibacillus alvei) neither in terms of larval survival nor weight. These results suggest that host background contributes markedly to the course of the disease but virulence is mainly dependent on pathogen genotype. Secondary invaders following a M. plutonius infection do not increase disease lethality and therefore may just be a colonization of weakened and immunodeficient, or dead larvae.

Discovery of a new Pro-Pro endopeptidase, PPEP-2, provides mechanistic insights into the differences in substrate specificity within the PPEP family.

Pro-Pro endopeptidases (PPEPs) belong to a recently discovered family of proteases capable of hydrolyzing a Pro-Pro bond. The first member from the bacterial pathogen Clostridium difficile (PPEP-1) cleaves two C. difficile cell-surface proteins involved in adhesion, one of which is encoded by the gene adjacent to the ppep-1 gene. However, related PPEPs may exist in other bacteria and may shed light on substrate specificity in this enzyme family. Here, we report on the homolog of PPEP-1 in Paenibacillus alvei, which we denoted PPEP-2. We found that PPEP-2 is a secreted metalloprotease, which likewise cleaved a cell-surface protein encoded by an adjacent gene. However, the cleavage motif of PPEP-2, PLP↓PVP, is distinct from that of PPEP-1 (VNP↓PVP). As a result, an optimal substrate peptide for PPEP-2 was not cleaved by PPEP-1 and vice versa. To gain insight into the specificity mechanism of PPEP-2, we determined its crystal structure at 1.75 Å resolution and further confirmed the structure in solution using small-angle X-ray scattering (SAXS). We show that a four-amino-acid loop, which is distinct in PPEP-1 and -2 (GGST in PPEP-1 and SERV in PPEP-2), plays a crucial role in substrate specificity. A PPEP-2 variant, in which the four loop residues had been swapped for those from PPEP-1, displayed a shift in substrate specificity toward PPEP-1 substrates. Our results provide detailed insights into the PPEP-2 structure and the structural determinants of substrate specificity in this new family of PPEP proteases.

Consistent associations with beneficial bacteria in the seed endosphere of barley (Hordeum vulgare L.).

The importance of the plant microbiome for host fitness has led to the concept of the "plant holobiont". Seeds are reservoirs and vectors for beneficial microbes, which are very intimate partners of higher plants with the potential to connect plant generations. In this study, the endophytic seed microbiota of numerous barley samples, representing different cultivars, geographical sites and harvest years, was investigated. Cultivation-dependent and -independent analyses, microscopy, functional plate assays, greenhouse assays and functional prediction were used, with the aim of assessing the composition, stability and function of the barley seed endophytic bacterial microbiota. Associations were consistently detected in the seed endosphere with Paenibacillus, Pantoea and Pseudomonas spp., which were able to colonize the root with a notable rhizocompetence after seed germination. In greenhouse assays, enrichment with these bacteria promoted barley growth, improved mineral nutrition and induced resistance against the fungal pathogen Blumeria graminis. We demonstrated here that barley, an important crop plant, was consistently associated with beneficial bacteria inside the seeds. The results have relevant implications for plant microbiome ecology and for the holobiont concept, as well as opening up new possibilities for research and application of seed endophytes as bioinoculants in sustainable agriculture.

Aerobic and anaerobic cellulose utilization by Paenibacillus sp. CAA11 and enhancement of its cellulolytic ability by expressing a heterologous endoglucanase.

For cost-effective lignocellulosic biofuel/chemical production, consolidated bioprocessing (CBP)-enabling microorganisms utilizing cellulose as well as producing biofuel/chemical are required. A novel strain Paenibacillus sp. CAA11 isolated from sediment was found to be not only as a cellulose degrader under both aerobic and strict anaerobic conditions but also as a producer of cellulosic biofuel/chemicals. Paenibacillus sp. CAA11 secreted cellulolytic enzymes by its own secretion system and produced ethanol as well as short-chain organic acids (formic acid, acetic acid, lactic acid) from cellulose. Cellulolytic activity of the strain was significantly enhanced by expressing a heterologous endoglucanase 168Cel5 from Bacillus subtilis under both aerobic and anaerobic conditions. The strain harboring the 168cel5 gene revealed 2-fold bigger halo zone on Congo-red plate and 1.75-fold more aerobic cellulose utilization in liquid medium compared with the negative control. Notably, under anaerobic conditions, the recombinant strain expressing 168Cel5 consumed 1.83-fold more cellulose (5.10 g/L) and produced 5-fold more ethanol (0.65 g/L) along with 5-fold more total acids (1.6 g/L) compared with the control, resulting 2.73-fold higher yields. This result demonstrates the potential of Paenibacillus sp. CAA11 as a suitable aerobic and anaerobic CBP-enabling microbe with cellulolytic production of ethanol and short-chain organic acids.

Regulated ploidy of Bacillus subtilis and three new isolates of Bacillus and Paenibacillus.

Bacteria were long assumed to be monoploid, maintaining one copy of a circular chromosome. In recent years it became obvious that the majority of species in several phylogenetic groups of prokaryotes are oligoploid or polyploid. The present study aimed at investigating the ploidy in Gram-positive aerobic endospore-forming bacteria. First, the numbers of origins and termini of the widely used laboratory strain Bacillus subtilis 168 were quantified. The strain was found to be mero-oligoploid in exponential phase (5.9 origins, 1.2 termini) and to down-regulate the number of origins in stationary phase. After inoculation of fresh medium with stationary-phase cells the onset of replication preceded the onset of mass increase. For the analysis of the ploidy in fresh isolates, three strains were isolated from soil, which were found to belong to the genera of Bacillus and Paenibacillus. All three strains were found to be mero-oligoploid in exponential phase and exhibit a growth phase-dependent down-regulation of the ploidy level in stationary phase. Taken together, these results indicate that mero-oligoploidy as well as growth phase-dependent copy number regulation might be widespread in and typical for Bacillus and related genera.

Facilitated enumeration of the silicate bacterium Paenibacillus mucilaginosus comb. nov. (formerly Bacillus mucilaginosus) via tetrazolium chloride incorporation into a double agar-based solid growth medium.

Accurate enumeration of Paenibacillus mucilaginosus (formerly Bacillus mucilaginosus) bacterium from environmental samples on solid medium is challenging owing to its extensive extracellular polysaccharides (EPS) excretion. In the present study, P. mucilaginosus enumeration has been facilitated by a simple modification: addition of triphenyl tetrazolium chloride (TTC) to growth medium and application of a second soft agar layer. Results show distinctively better and accurate colonies' count. This method can be applied to all bacterial species that produce excessive EPS that may interfere with direct count.

Low-abundant species facilitates specific spatial organization that promotes multispecies biofilm formation.

Microorganisms frequently co-exist in matrix-embedded multispecies biofilms. Within biofilms, interspecies interactions influence the spatial organization of member species, which likely play an important role in shaping the development, structure and function of these communities. Here, a reproducible four-species biofilm, composed of Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus, was established to study the importance of individual species spatial organization during multispecies biofilm development. We found that the growth of species that are poor biofilm formers, M. oxydans and P. amylolyticus, were highly enhanced when residing in the four-species biofilm. Interestingly, the presence of the low-abundant M. oxydans (0.5% of biomass volume) was observed to trigger changes in the composition of the four-species community. The other three species were crucially needed for the successful inclusion of M. oxydans in the four-species biofilm, where X. retroflexus was consistently positioned in the top layer of the mature four-species biofilm. These findings suggest that low abundance key species can significantly impact the spatial organization and hereby stabilize the function and composition of complex microbiomes.

Characterization of four Paenibacillus species isolated from pasteurized, chilled ready-to-eat meals.

Food spoilage is often caused by microorganisms. The predominant spoilage microorganisms of pasteurized, chilled ready-to-eat (RTE) mixed rice-vegetable meals stored at 7°C were isolated and determined as Paenibacillus species. These sporeforming psychrotrophic bacteria are well adapted to grow in the starch-rich environment of pasteurized and chilled meals. Growth of the Paenibacillus isolates appeared to be delayed by decreased (<7°C) temperature or chilled temperature (7°C) combined with decreased pH (<5), increased sodium chloride (>5.5%, corresponding with an aw<0.934), or decreased aw (<0.931; using sucrose). To gain insight in the effect of the pasteurization processing of the meal on spore inactivation, heat-inactivation kinetics were determined and D-values were calculated. According to these kinetics, pasteurization up to 90°C, necessary for inactivation of vegetative spoilage microorganisms and pathogens, does not significantly contribute to the inactivation of Paenibacillus spores in the meals. Furthermore, outgrowth of pasteurized spores was determined in the mixed rice-vegetable meal at several temperatures; P. terrae FBR-61 and P. pabuli FBR-75 isolates did not substantially increase in numbers during storage at 2°C, but had a significant increase within a month of storage at 4°C or within several days at 22°C. Overall, this work shows the importance of Paenibacillus species as spoilage microorganisms of pasteurized, chilled RTE meals and that the meals' matrix, processing conditions, and storage temperature are important hurdles to control microbial meal spoilage.