Quantitative comparisons of select cultured and uncultured microbial populations in the rumen of cattle fed different diets

Background： The number and diversity of uncultured ruminal bacterial and archaeal species revealed by 16S rRNA gene firs） sequences greatly exceeds that of cultured bacteria and archaea. However, the significance of uncultured microbes remains undetermined. The objective of this study was to assess the numeric importance of select uncultured bacteria and cultured bacteria and the impact of diets and microenvironments within cow rumen in a comparative manner. Results： Liquid and adherent fractions were obtained from the rumen of Jersey cattle fed hay alone and Holstein cattle fed hay plus grain. The populations of cultured and uncultured bacteria present in each fraction were quantified using specific real-time PCR assays. The population of total bacteria was similar between fractions or diets, while total archaea was numerically higher in the hay-fed Jersey cattle than in the hay-grain-fed Holstein cattle. The population of the genus Prevotello was about one log smaller than that of total bacteria. The populations of Fibrobocter succinogenes, Ruminococcus flovefociens, the genus Butyrivibrio, and R. albus was at least one log smaller than that of genus Prevotello. Four of the six uncultured bacteria quantified were as abundant as F. succinogenes, R. flovefociens and the genus Butyrivibrio. In addition, the populations of several uncultured bacteria were significantly higher in the adherent fractions than in the liquid fractions. These uncultured bacteria may be associated with fiber degradation. Conclusions： Some uncultured bacteria are as abundant as those of major cultured bacteria in the rumen. Uncultured bacteria may have important contribution to ruminal fermentation. Population dynamic studies of uncultured bacteria in a comparative manner can help reveal their ecological features and importance to rumen functions.

Viable but nonculturable bacteria and their resuscitation:implications for cultivating uncultured marine microorganisms

Culturing has been the cornerstone of microbiology since Robert Koch first successfully cultured bacteria in the late nineteenth century. However, even today, the majority of microorganisms in the marine environment remain uncultivated. There are various explanations for the inability to culture bacteria in the laboratory, including lack of essential nutrients, osmotic support or incubation conditions, low growth rate, development of micro-colonies, and the presence of senescent or viable but nonculturable (VBNC) cells. In the marine environment, many bacteria have been associated with dormancy, as typified by the VBNC state. VBNC refers to a state where bacteria are metabolically active, but are no longer culturable on routine growth media. It is apparently a unique survival strategy that has been adopted by many microorganisms in response to harsh environmental conditions and the bacterial cells in the VBNC state may regain culturability under favorable conditions. The resuscitation of VBNC cells may well be an important way to cultivate the otherwise uncultured microorganisms in marine environments. Many resuscitation stimuli that promote the restoration of culturability have so far been identified;these include sodium pyruvate, quorum sensing autoinducers, resuscitation-promoting factors Rpfs and YeaZ, and catalase. In this review, we focus on the issues associated with bacterial culturability, the diversity of bacteria entering the VBNC state, mechanisms of induction into the VBNC state, resuscitation factors of VBNC cells and implications of VBNC resuscitation stimuli for cultivating these otherwise uncultured microorganisms. Bringing important microorganisms into culture is still important in the era of high-throughput sequencing as their ecological functions in the marine environment can often only be known through isolation and cultivation.

From ecophysiology to cultivation methodology:filling the knowledge gap between uncultured and cultured microbes

Earth is dominated by a myriad of microbial communities,but the majority fails to grow under in situ laboratory conditions.The basic cause of unculturability is that bacteria dominantly occur as biofilms in natural environments.Earlier improvements in the culture techniques are mostly done by optimizing media components.However,with technological advancement particularly in the field of genome sequencing and cell imagining techniques,new tools have become available to understand the ecophysiology of microbial communities.Hence,it becomes easier to mimic environmental conditions in the culture plate.Other methods include co-culturing,emendation of growth factors,and cultivation after physical cell sorting.Most recently,techniques have been proposed for bacterial cultivation by employing genomic data to understand either microbial interactions(network-directed targeted bacterial isolation)or ecosystem engineering(reverse genomics).Hopefully,these techniques may be applied to almost all environmental samples,and help fill the gaps between the cultured and uncultured microbial communities.

一株耐热纤维素酶产生菌的筛选及酶学特性

从辽宁鞍山汤岗子温泉附近土样中分离得到能产生纤维素酶的真菌,通过形态观察和18S rRNA序列分析,该菌株为蒙昧的散囊菌纲(Uncultured Eurotiomycetes)。实验中对酶学性质进行了检验,测定得出该菌株产生的纤维素酶最适温度为65℃,在温度高达75℃仍能保持70%的酶活力,它的最适pH值为6.5,pH在5～8的范围内酶活力保持稳定。实验表明该菌株所产纤维素酶具有较高的pH稳定性和温度稳定性,值得对该酶进行进一步的研究。

Cultivating the uncultured:Harnessing the“sandwich agar plate”approach to isolate heme-dependent bacteria from marine sediment

In the classical microbial isolation technique,the isolation process inevitably destroys all microbial interactions and thus makes it difficult to culture the many microorganisms that rely on these interactions for survival.In this study,we designed a simple coculture technique named the“sandwich agar plate method,”which maintains microbial interactions throughout the isolation and pure culture processes.The total yield of uncultured species in sandwich agar plates based on eight helper strains was almost 10-fold that of the control group.Many uncultured species displayed commensal lifestyles.Further study found that heme was the growth-promoting factor of some marine commensal bacteria.Subsequent genomic analysis revealed that heme auxotrophies were common in various biotopes and prevalent in many uncultured microbial taxa.Moreover,our study supported that the survival strategies of heme auxotrophy in different habitats varied considerably.These findings highlight that cocultivation based on the“sandwich agar plate method”could be developed and used to isolate more uncultured bacteria.

Current Research and Strategy on the Biosurfactants Used in the Remediation of Petrochemical Polluted Environment

Biosurfactants are biologically active metabolites, and the efficiency of direct screening of new biosurfactants from nature using traditional methods is low, which should be enhanced in the following studies by adopting advanced biotechnologies. Rapid development and wide application of microbial culture independent methods, such as metagenomics, metatranscriptomics, metaproteomics and metabonomics, etc., contributes to quickly and precisely screening of novel biological surfactants. We mainly represented the current status of research and applications of biosurfactants in the remediation of petrochemical polluted environment, and also prospected avenues for future research.

Significance of dormant forms of Helicobacter pylori in ulcerogenesis

Nearly half of the global population are carriers of Helicobacter pylori(H. pylori),a Gram-negative bacterium that persists in the healthy human stomach. H. pylori can be a pathogen and causes development of peptic ulcer disease in a certain state of the macroorganism. It is well established that H. pylori infection is the main cause of chronic gastritis and peptic ulcer disease(PUD). Decontamination of the gastric mucosa with various antibiotics leads to H. pylori elimination and longer remission in this disease. However,the reasons for repeated detection of H. pylori in recurrent PUD after its successful eradication remain unclear. The reason for the redetection of H. pylori in recurrent PUD can be either reinfection or ineffective anti-Helicobacter therapy. The administration of antibacterial drugs can lead not only to the emergence of resistant strains of microorganisms,but also contribute to the conversion of H. pylori into the resting(dormant) state. The dormant forms of H. pylori have been shown to play a potential role in the development of relapses of PUD. The paper discusses morphological H. pylori forms,such as S-shaped,C-shaped,U-shaped,and coccoid ones. The authors proposes the classification of H. pylori according to its morphological forms and viability.

Strategies for culturing active/dormant marine microbes

Microorganisms are ubiquitous in the ocean environment and they play key roles in marine ecosystem function and service.However,many of their functions and phenotypes remain unknown because indigenous marine bacteria are mostly difficult to culture.Although many novel techniques have brought previously uncultured microbes into laboratory culture,there are still many most-wanted or key players that need to be cultured from marine environments.This review discusses possible reasons for‘unculturable microbes’and categorizes uncultured bacteria into three groups:dominant active bacteria,rare active bacteria,and dormant bacteria.This review also summarizes advances in cultivation techniques for culturing each group of unculturable bacteria.Simulating the natural environment is an effective strategy for isolating dominant active bacteria,whereas culturomics and enrichment culture methods are proposed for isolating rare active bacteria.For dormant bacteria,resuscitation culture is an appropriate strategy.Furthermore,the review provides a list of the most-wanted bacteria and proposes potential strategies for culturing these bacteria in marine environments.The review provides new insight into the development of strategies for the cultivation of specific groups of uncultured bacteria and therefore paves the way for the detection of novel microbes and their functions in marine ecosystems.

One cell at a time:droplet-based microbial cultivation,screening and sequencing

Microbes thrive and,in turn,influence the earth's environment,but most are poorly understood because of our limited capacity to reveal their natural diversity and function.Developing novel tools and effective strategies are critical to ease this dilemma and will help to understand their roles in ecology and human health.Recently,droplet microfluidics is emerging as a promising technology for microbial studies with value in microbial cultivating,screening,and sequencing.This review aims to provide an overview of droplet micro flu idics techniques for microbial research.First,some critical points or steps in the microfluidic system are introduced,such as droplet stabilization,manipulation,and detection.We then highlight the recent progress of droplet-based methods for microbiological applications,from high-throughput single-cell cultivation,screening to the targeted or whole-genome sequencing of single cells.

Application of in situ cultivation in marine microbial resource mining

Microbial communities in marine habitats are regarded as underexplored reservoirs for discovering new natural products with potential application.However,only a few microbes in nature can be cultivated in the laboratory.This has led to the development of a variety of isolation and cultivation methods,and in situ cultivation is one of the most popular.Diverse in situ cultivation methods,with the same basic principle,have been applied to a variety of environmental samples.Compared with conventional approaches,these new methods are able to cultivate previously uncultured and phylogenetically novel microbes,many with biotechnological potential.This review introduces the various in situ cultivation methods for the isolation of previously uncultured microbial species and their potential for marine microbial resource mining.Furthermore,studies that investigated the key and previously unidentified mechanisms of growing uncultivated microorganisms by in situ cultivation,which will shed light on the understanding of microbial uncultivability,were also reviewed.

New approaches for archaeal genome-guided cultivation

Archaea,one of the three domains of life along with Bacteria and Eukarya,contains ancient life forms such as methanogen that are observed today on Earth,and one lineage Asgard archaea is also considered the closest ancestor of Eukarya.Recently,with the development of interdisciplinary studies from Earth and Life sciences,archaeal organisms are considered to play pivotal roles in geochemical cycling in nature.However,our understanding of the attributes,origin and evolution,geochemical and ecological functions of Archaea is hampered by the scarcity of archaeal isolates,which has represented a challenge to researchers for the last 40 years.Cultivation-independent sequencing and phylogenomic analyses have demonstrated a considerable diversity of Archaea with more than 20 novel phyla.However,only four archaeal phyla have cultured representatives,leaving large gaps in our knowledge of the metabolic capabilities and ecological functions of the majority of archaeal strains identified exclusively by DNA sequencing.In this review,we summarize the discovery and development of archaeal research,highlight the knowledge gap between uncultured and cultured archaeal microbes,and call on the importance of devoting greater research efforts to archaeal cultivation.Finally,we outlined new ideas and strategic approaches,namely,(1)genome-based methods,(2)microbial network information-based methods,(3)genome-scale model-guided methods,and(4)machine learning methods,to enable the cultivation of uncultivated archaeal microbes using accumulated high-throughput sequencing data.