Sven Hoefman

The more, the merrier: heterotroph richness stimulates methanotrophic activity

Although microorganisms coexist in the same environment, it is still unclear how their interaction regulates ecosystem functioning. Using a methanotroph as a model microorganism, we determined how methane oxidation responds to heterotroph diversity. Artificial communities comprising of a methanotroph and increasing heterotroph richness, while holding equal starting cell numbers were assembled. We considered methane oxidation rate as a functional response variable. Our results showed a significant increase of methane oxidation with increasing heterotroph richness, suggesting a complex interaction in the cocultures leading to a stimulation of methanotrophic activity. Therefore, not only is the methanotroph diversity directly correlated to methanotrophic activity for some methanotroph groups as shown before, but also the richness of heterotroph interacting partners is relevant to enhance methane oxidation too. In this unprecedented study, we provide direct evidence showing how heterotroph richness exerts a response in methanotroph–heterotroph interaction, resulting in increased methanotrophic activity. Our study has broad implications in how methanotroph and heterotroph interact to regulate methane oxidation, and is particularly relevant in methane-driven ecosystems.

Optimized Cryopreservation of Mixed Microbial Communities for Conserved Functionality and Diversity

The use of mixed microbial communities (microbiomes) for biotechnological applications has steadily increased over the past decades. However, these microbiomes are not readily available from public culture collections, hampering their potential for widespread use. The main reason for this lack of availability is the lack of an effective cryopreservation protocol. Due to this critical need, we evaluated the functionality as well as the community structure of three different types of microbiomes before and after cryopreservation with two cryoprotective agents (CPA). Microbiomes were selected based upon relevance towards applications: (1) a methanotrophic co-culture (MOB), with potential for mitigation of greenhouse gas emissions, environmental pollutants removal and bioplastics production; (2) an oxygen limited autotrophic nitrification/denitrification (OLAND) biofilm, with enhanced economic and ecological benefits for wastewater treatment, and (3) fecal material from a human donor, with potential applications for fecal transplants and pre/probiotics research. After three months of cryopreservation at −80°C, we found that metabolic activity, in terms of the specific activity recovery of MOB, aerobic ammonium oxidizing bacteria (AerAOB) and anaerobic AOB (AnAOB, anammox) in the OLAND mixed culture, resumes sooner when one of our selected CPA [dimethyl sulfoxide (DMSO) and DMSO plus trehalose and tryptic soy broth (DMSO+TT)] was added. However, the activity of the fecal community was not influenced by the CPA addition, although the preservation of the community structure (as determined by 16S rRNA gene sequencing) was enhanced by addition of CPA. In summary, we have evaluated a cryopreservation protocol that succeeded in preserving both community structure and functionality of value-added microbiomes. This will allow individual laboratories and culture collections to boost the use of microbiomes in biotechnological applications.

Planktonic versus biofilm catabolic communities: importance of the biofilm for species selection and pesticide degradation.

ABSTRACT Chloropropham-degrading cultures were obtained from sludge and soil samples by using two different enrichment techniques: (i) planktonic enrichments in shaken liquid medium and (ii) biofilm enrichments on two types of solid matrixes (plastic chips and gravel). Denaturing gradient gel electrophoresis fingerprinting showed that planktonic and biofilm cultures had a different community composition depending on the presence and type of added solid matrix during enrichment. This was reflected in the unique chloropropham-degrading species that could be isolated from the different cultures. Planktonic and biofilm cultures also differed in chloropropham-degrading activity. With biofilm cultures, slower chloropropham removal was observed, but with less build-up of the toxic intermediate 3-chloroaniline. Disruption of the biofilm architecture resulted in degradation characteristics shifting toward those of the free suspensions, indicating the importance of a well-established biofilm structure for good performance. These results show that biofilm-mediated enrichment techniques can be used to select for pollutant-degrading microorganisms that like to proliferate in a biofilm and that cannot be isolated using conventional shaken-liquid procedures. Furthermore, the influence of the biofilm architecture on the pesticide degradation characteristics suggests that for bioaugmentation the use of biofilm catabolic communities might be a proficient alternative to using planktonic freely suspended cultures.

Niche differentiation in nitrogen metabolism among methanotrophs within an operational taxonomic unit

BackgroundThe currently accepted thesis on nitrogenous fertilizer additions on methane oxidation activity assumes niche partitioning among methanotrophic species, with activity responses to changes in nitrogen content being dependent on the in situ methanotrophic community structure Unfortunately, widely applied tools for microbial community assessment only have a limited phylogenetic resolution mostly restricted to genus level diversity, and not to species level as often mistakenly assumed. As a consequence, intragenus or intraspecies metabolic versatility in nitrogen metabolism was never evaluated nor considered among methanotrophic bacteria as a source of differential responses of methane oxidation to nitrogen amendments.ResultsWe demonstrated that fourteen genotypically different Methylomonas strains, thus distinct below the level at which most techniques assign operational taxonomic units (OTU), show a versatile physiology in their nitrogen metabolism. Differential responses, even among strains with identical 16S rRNA or pmoA gene sequences, were observed for production of nitrite and nitrous oxide from nitrate or ammonium, nitrogen fixation and tolerance to high levels of ammonium, nitrate, and hydroxylamine. Overall, reduction of nitrate to nitrite, nitrogen fixation, higher tolerance to ammonium than nitrate and tolerance and assimilation of nitrite were general features.ConclusionsDifferential responses among closely related methanotrophic strains to overcome inhibition and toxicity from high nitrogen loads and assimilation of various nitrogen sources yield competitive fitness advantages to individual methane-oxidizing bacteria. Our observations proved that community structure at the deepest phylogenetic resolution potentially influences in situ functioning.

Exploration and prediction of interactions between methanotrophs and heterotrophs

Methanotrophs can form the basis of a methane-driven food web on which heterotrophic microorganisms can feed. In return, these heterotrophs can stimulate growth of methanotrophs in co-culture by providing growth additives. However, only a few specific interactions are currently known. We incubated nine methanotrophs with 25 heterotrophic strains in a pairwise miniaturized co-cultivation setup. Through principal component analysis and k-means clustering, methanotrophs and heterotrophs could be grouped according to their interaction behaviour, suggesting strain-dependent methanotroph-heterotroph complementarity. Co-cultivation significantly enhanced the growth parameters of three methanotrophs. This was most pronounced for Methylomonas sp. M5, with a threefold increase in maximum density and a fourfold increase in maximum increase in density in co-culture with Cupriavidus taiwanensis LMG 19424. In contrast, co-cultivation with Methylobacterium radiotolerans LMG 2269 and Pseudomonas aeruginosa LMG 12228 inhibited growth of most methanotrophs. Functional genomic analysis suggested the importance of vitamin metabolism for co-cultivation success. The generated data set was then successfully exploited as a proof-of-principle for predictive modelling of co-culture responses based on other interactions of the same heterotrophs and methanotrophs, yielding values of the area under the receiver operating characteristic curve of 0.73 upon 50% missing values for the maximum increase in density parameter. As such, these modelling-based tools were shown to hold great promise in reducing the amount of data that needs to be generated when conducting large co-cultivation studies.

Methyloparacoccus murrellii gen. nov., sp. nov., a methanotroph isolated from pond water.

Two novel methanotrophic strains, R-49797(T) and OS501, were isolated from pond water in South Africa and Japan, respectively. Strains R-49797(T) and OS501 shared 99.7% 16S rRNA gene sequence similarity. Cells were Gram-stain-negative, non-motile cocci with a diplococcoid tendency and contained type I methanotroph intracytoplasmic membranes. The pmoA gene encoding particulate methane monooxygenase was present. Soluble methane monoooxygenase (sMMO) activity, the mmoX gene encoding sMMO and the nifH gene encoding nitrogenase were not detected. Methane and methanol were utilized as sole carbon source. The strains grew optimally at 25-33 °C (range 20-37 °C) and at pH 6.3-6.8 (range 5.8-9.0). The strains did not support growth in media supplemented with 1% (w/v) NaCl. For both strains, the two major fatty acids were C(16 : 1)ω7c and C(16 : 0) and the DNA G+C content was 65.6 mol%. The isolates belong to the family Methylococcaceae of the class Gammaproteobacteria and cluster most closely among the genera Methylocaldum, Methylococcus and Methylogaea, with a 16S rRNA gene sequence similarity of 94.2% between strain R-49797(T) and its closest related type strain (Methylocaldum gracile VKM 14L(T)). Based on the low 16S rRNA gene sequence similarities with its nearest phylogenetic neighbouring genera, the formation of a separate lineage based on 16S rRNA and pmoA gene phylogenetic analysis, and the unique combination of phenotypic characteristics of the two isolated strains compared with the genera Methylocaldum, Methylococcus and Methylogaea, we propose to classify these strains as representing a novel species of a new genus, Methyloparacoccus murrellii gen. nov., sp. nov., within the family Methylococcaceae. The type strain of Methyloparacoccus murrellii is R-49797(T) ( = LMG 27482(T) = JCM 19379(T)).

Miniaturized extinction culturing is the preferred strategy for rapid isolation of fast‐growing methane‐oxidizing bacteria

Methane‐oxidizing bacteria (MOB) have a large potential as a microbial sink for the greenhouse gas methane as well as for biotechnological purposes. However, their application in biotechnology has so far been hampered, in part due to the relative slow growth rate of the available strains. To enable the availability of novel strains, this study compares the isolation of MOB by conventional dilution plating with miniaturized extinction culturing, both performed after an initial enrichment step. The extinction approach rendered 22 MOB isolates from four environmental samples, while no MOB could be isolated by plating. In most cases, extinction culturing immediately yielded MOB monocultures making laborious purification redundant. Both type I (Methylomonas spp.) and type II (Methylosinus sp.) MOB were isolated. The isolated methanotrophic diversity represented at least 11 different strains and several novel species based on 16S rRNA gene sequence dissimilarity. These strains possessed the particulate (100%) and soluble (64%) methane monooxygenase gene. Also, 73% of the strains could be linked to a highly active fast‐growing mixed MOB community. In conclusion, miniaturized extinction culturing was more efficient in rapidly isolating numerous MOB requiring little effort and fewer materials, compared with the more widely applied plating procedure. This miniaturized approach allowed straightforward isolation and could be very useful for subsequent screening of desired characteristics, in view of their future biotechnological potential.

Survival or revival: long-term preservation induces a reversible viable but non-culturable state in methane-oxidizing bacteria.

Knowledge on long-term preservation of micro-organisms is limited and research in the field is scarce despite its importance for microbial biodiversity and biotechnological innovation. Preservation of fastidious organisms such as methane-oxidizing bacteria (MOB) has proven difficult. Most MOB do not survive lyophilization and only some can be cryopreserved successfully for short periods. A large-scale study was designed for a diverse set of MOB applying fifteen cryopreservation or lyophilization conditions. After three, six and twelve months of preservation, the viability (via live-dead flow cytometry) and culturability (via most-probable number analysis and plating) of the cells were assessed. All strains could be cryopreserved without a significant loss in culturability using 1% trehalose in 10-fold diluted TSB (TT) as preservation medium and 5% DMSO as cryoprotectant. Several other cryopreservation and lyophilization conditions, all of which involved the use of TT medium, also allowed successful preservation but showed a considerable loss in culturability. We demonstrate here that most of these non-culturables survived preservation according to viability assessment indicating that preservation induces a viable but non-culturable (VBNC) state in a significant fraction of cells. Since this state is reversible, these findings have major implications shifting the emphasis from survival to revival of cells in a preservation protocol. We showed that MOB cells could be significantly resuscitated from the VBNC state using the TT preservation medium.

Safeguarding bacterial resources promotes biotechnological innovation

Environmental research delivers valuable bacterial resources for biotechnology. We believe that systematic long-term preservation of bacteria will promote future biotechnological innovations, by safeguarding the accessibility of bacteria already recognized to have interesting features and providing a “pool” of bacterial resources for novel applied research. To this end, we want to advocate the incorporation of preservation tests in environmental or applied microbiological research. This paper introduces non-specialists to different preservation methods for bacteria. Several parameters that influence long-term storage of bacterial resources are explained and practical tips and guidelines are formulated. Also, the vital role of public culture collections is highlighted and the state-of-the-art of preservation of non-pure cultures is described.

Methylomonas lenta sp. nov., a methanotroph isolated from manure and a denitrification tank.

Two methanotrophic bacteria, strains R-45377(T) and R-45370, were respectively isolated from a slurry pit of a cow stable and from a denitrification tank of a wastewater treatment plant in Belgium. The strains showed 99.9 % 16S rRNA gene sequence similarity. Cells were Gram-negative, motile rods containing type I methanotroph intracytoplasmic membranes. Colonies and liquid cultures appeared white to pale pink. The pmoA gene encoding particulate methane monooxygenase (pMMO) and the nifH gene encoding nitrogenase were present. Soluble methane monooxygenase (sMMO) activity, the presence of the mmoX gene encoding sMMO and the presence of the pxmA gene encoding a sequence-divergent pMMO were not detected. Methane and methanol were utilized as sole carbon sources. The strains grew optimally at 20 °C (range 15-28 °C) and at pH 6.8-7.3 (range pH 6.3-7.8). The strains grew in media supplemented with up to 1.2 % NaCl. The major cellular fatty acids were C16 : 1ω8c, C16 : 1ω5c, C16 : 1ω7c, C14 : 0, C15 : 0 and C16 : 0 and the DNA G+C content was 47 mol%. 16S rRNA gene- and pmoA-based phylogenetic analyses showed that the isolates cluster among members of the genus Methylomonas within the class Gammaproteobacteria, with pairwise 16S rRNA gene sequence similarities of 97.5 and 97.2 % between R-45377(T) and the closest related type strains, Methylomonas scandinavica SR5(T) and Methylomonas paludis MG30(T), respectively. Based on phenotypic characterization of strains R-45377(T) and R-45370, their low 16S rRNA gene sequence similarities and the formation of a separate phylogenetic lineage compared with existing species of the genus Methylomonas, we propose to classify these strains in a novel species, Methylomonas lenta sp. nov., with R-45377(T) ( = LMG 26260(T) = JCM 19378(T)) as the type strain.