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Single cell protein production potential of enriched microbial populations from rice paddy soils and roots: Insights into protein yield enhancement by Methylophilaceae
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Methanol, the second most abundant volatile organic compound, primarily released from plants, is a major culprit disturbing atmospheric chemistry. Interestingly, ubiquitously found methanol-utilizing bacteria, play a vital role in mitigating atmospheric methanol effects. Despite being extensively characterized, the effect of nitrogen sources on the richness of methanol-utilizers in the bulk soil and rhizosphere is largely unknown. Therefore, the current study was planned to isolate, characterize and explore the richness of cultivable methylotrophs from the bulk soil and rhizosphere of a paddy field using media with varying nitrogen sources. Our data revealed that more genera of methylotrophs, including Methylobacterium, Ancylobacter, Achromobacter, Xanthobacter, Moraxella, and Klebsiella were enriched with the nitrate-based medium compared to only two genera, Hyphomicrobium and Methylobacterium, enriched with the ammonium-based medium. The richness of methylotrophic bacteria also differed substantially in the bulk soil as compared to the rhizosphere. Growth characterization revealed that majority of the newly isolated methanol-utilizing strains in this study exhibited better growth at 37 °C instead of 30 or 45 °C. Moreover, Hyphomicrobium sp. FSA2 was the only strain capable of utilizing methanol even at elevated temperature 45 °C, showing its adaptability to a wide range of temperatures. Differential carbon substrate utilization profiling revealed the facultative nature of all isolated methanol-utilizer strains with Xanthobacter sp. TS3, being an important methanol-utilizer capable of degrading toxic compounds such as acetone and ethylene glycol. Overall, our study suggests the role of nutrients and plant-microbial interaction in shaping the composition of methanol-utilizers in terrestrial environment.
Global catastrophes such as a supervolcanic eruption, asteroid impact, or nuclear winter could cause global agricultural collapse due to reduced sunlight reaching the Earth’s surface. The human civilization’s food production system is unprepared to respond to such events, but methane single cell protein (SCP) could be a key part of the solution. Current preparedness centers around food stockpiling, an excessively expensive solution given that an abrupt sunlight reduction scenario (ASRS) could hamper conventional agriculture for 5–10 years. Instead, it is more cost-effective to consider resilient food production techniques requiring little to no sunlight. This study analyses the potential of SCP produced from methane (natural gas and biogas) as a resilient food source for global catastrophic food shocks from ASRS. The following are quantified: global production potential of methane SCP, capital costs, material and energy requirements, ramp-up rates, and retail prices. In addition, potential bottlenecks for fast deployment are considered. While providing a more valuable, protein-rich product than its alternatives, the production capacity could be slower to ramp up. Based on 24/7 construction of facilities, 7%–11% of the global protein requirements could be fulfilled at the end of the first year. Despite significant remaining uncertainties, methane SCP shows significant potential to prevent global protein starvation during an ASRS at an affordable price—US$3–5/kg dry.
Methanotrophs are naturally occurring microorganisms capable of oxidizing methane, having an impact on global net methane emissions. Additionally, they have also gained interest for their biotechnological applications in single-cell protein production, biofuels, and bioplastics.
More food needs to be produced for the growing human population, but the possibilities of expanding the area of arable land are limited. Cellular Agriculture is an emerging field of biotechnology, aimed at finding alternatives to agricultural production of various commodities. As a part of Cellular Agriculture, the use of microbes and microalgae as food and feed with high protein content, so-called single cell protein (SCP), is gaining renewed scientific and commercial interest. In this review, we give an introduction to SCP production by heterotrophic microbial species, phototrophs, methanotrophs and autotrophic hydrogen oxidizers, as well as highlight some challenges and the latest developments in the growing SCP industry.
The modern definition of enzymology is synonymous with the Michaelis–Menten equation instituted by Leonor Michaelis and Maud Menten. Most textbooks, or chapters within, discussing enzymology start with the derivation of the equation under the assumption of rapid equilibrium (as done by Michaelis–Menten) or steady state (as modified by Briggs and Haldane) conditions to highlight the importance of this equation as the bedrock on which interpretation of enzyme kinetic results is dependent. However, few textbooks or monographs take the effort of placing the equation within its right historical context and discuss the assumptions that have gone into its institution. This guide will dwell on these in substantial detail. Further, this guide will attempt to instil a sense of appreciation for the mathematical curve rectangular hyperbola, its unique attributes and how ubiquitous the curve is in biological systems. To conclude, this guide will discuss the limitations of the equation, and the method it embodies, and trace the journey of how investigators are attempting to move beyond the steady‐state approach and the Michaelis–Menten equation into full progress curve, pre–steady state and single‐turnover kinetic analysis to obtain greater insights into enzyme kinetics and catalysis.
Significance
The cultivation of microbial biomass, which is rich in proteins as well as other nutrients, can play a vital role in achieving food security while mitigating the negative environmental footprint of agriculture. Here, we analyze the efficiency associated with using solar energy for converting atmospheric CO 2 derived from direct air capture into microbial biomass that can feed humans and animals. We show that the production of microbial foods outperforms agricultural cultivation of staple crops in terms of caloric and protein yields per land area at all relevant solar irradiance levels. These results suggest that microbial foods could substantially contribute to feeding a growing population and can assist in allocating future limited land resources.
We report a complete genome sequence of Methylosinus sp. strain C49, a methane-oxidizing bacterium (MOB) in the class Alphaproteobacteria , isolated from MOB-enriched biomass. The genome encodes the functional genes for methane oxidation ( pmoA ) and polyhydroxyalkanoate (PHA) biosynthesis ( phaABC ). Deciphering the genome will help research toward PHA production by MOB.
During late 1960s Green Revolution, researchers utilized semidwarf 1 (sd1) to improve the yield and lodging resistance in rice (Oryza sativa L.). However, sd1 has a negative effect to culm strength and biomass production. To increase yield dramatically in 21th century, development of next generation long-culm rice for non-lodging and high grain yield independent of sd1 has been needed. The present study developed Monster Rice 1, a long-culm and heavy-panicle type of rice line and compared it with Takanari, a high-yielding semidwarf rice variety about yield and lodging resistance associated traits. Brown rice yield and bending moment at breaking of the basal elongated internode were higher in Monster Rice 1 than those in Takanari due to a large number of spikelets per panicle and thicker culm. Furthermore, to identify QTLs with superior alleles for these traits, QTL and haplotype analyses were performed using F2 population and recombinant inbred lines derived from a cross between Monster Rice 1 and Takanari. The results from this study suggest that long-culm and heavy-panicle type of rice with a superior lodging resistance by culm strength can perform its high yield potential by using these identified QTLs contributing yield and lodging resistance.
Citrus is a globally important, perennial fruit crop whose rhizosphere microbiome is thought to play an important role in promoting citrus growth and health. Here, we report a comprehensive analysis of the structural and functional composition of the citrus rhizosphere microbiome. We use both amplicon and deep shotgun metagenomic sequencing of bulk soil and rhizosphere samples collected across distinct biogeographical regions from six continents. Predominant taxa include Proteobacteria, Actinobacteria, Acidobacteria and Bacteroidetes. The core citrus rhizosphere microbiome comprises Pseudomonas, Agrobacterium, Cupriavidus, Bradyrhizobium, Rhizobium, Mesorhizobium, Burkholderia, Cellvibrio, Sphingomonas, Variovorax and Paraburkholderia, some of which are potential plant beneficial microbes. We also identify over-represented microbial functional traits mediating plant-microbe and microbe-microbe interactions, nutrition acquisition and plant growth promotion in citrus rhizosphere. The results provide valuable information to guide microbial isolation and culturing and, potentially, to harness the power of the microbiome to improve plant production and health.
Koshihikari, a Japanese short-grain rice cultivar, was developed in 1956, more than 60 years ago. Despite its age, it has been the most widely grown cultivar in Japan for more than 35 years, making it the most important rice for the Japanese people. In its early days, there was no reason to predict that Koshihikari would become so widely disseminated. However, since the end of the post–World War II food shortage in the 1960s, Japanese preferences changed from high productivity to good eating quality. This triggered wide expansion of Koshihikari cultivation due to the cultivar’s excellent taste and texture. With increasing cultivation of Koshihikari in Japan, several good agronomic characteristics beyond its high eating quality became apparent, such as its good adaptation to different environments, tolerance to pre-harvest sprouting, and cold tolerance during the booting stage. These characteristics outweigh drawbacks such as its low blast resistance and low lodging resistance. The popularity of Koshihikari influenced subsequent rice breeding trends at regional agricultural experimental stations, and the characteristics of newly developed rice cultivars in Japan are usually rated relative to Koshihikari, which is used as the benchmark. Koshihikari was the first japonica rice cultivar whose whole genome has been sequenced by means of next-generation sequencing. Furthermore, comparison of the genomes of Koshihikari and Nipponbare has provided detailed insights into the genetic diversity of Japanese rice cultivars relative to that in rice populations elsewhere in the world. Further progress in rice genomics is gradually unlocking the mechanisms that underlie the agronomic characteristics of Koshihikari. To support both research and the development of novel rice cultivars, a series of isogenic and near-isogenic lines in the Koshihikari genetic background have been continuously developed. These new findings and materials will facilitate genomics-assisted rice breeding, eventually leading to superior cultivars.
Electronic supplementary material
The online version of this article (10.1186/s12284-018-0207-4) contains supplementary material, which is available to authorized users.
In recent years, the role of microorganisms inhabiting rice rhizosphere in promoting arsenic contamination has emerged. However, little is known concerning the species and metabolic properties involved in this phenomenon. In this study, the influence of water management on the rhizosphere microbiota in relation to arsenic dissolution in soil solution was tested. Rice plants were cultivated in macrocosms under different water regimes: continuous flooding, continuous flooding with a 2-week period drainage before flowering, and dry soil watered every 10 days. The active bacterial communities in rhizosphere soil and in rhizoplane were characterized by 16S rRNA pyrosequencing. An in-depth analysis of microbial taxa with direct or indirect effects on arsenic speciation was performed and related contribution was evaluated. Continuous flooding promoted high diversity in the rhizosphere, with the plant strongly determining species richness and evenness. On the contrary, under watering the communities were uniform, with little differences between rhizosphere soil and rhizoplane. Arsenic-releasing and arsenite-methylating bacteria were selected by continuous flooding, where they represented 8% of the total. On the contrary, bacteria decreasing arsenic solubility were more abundant under watering, with relative abundance of 10%. These values reflected arsenic concentrations in soil solution: 135 μg L(-1) and negligible in continuous flooding and under watering, respectively. When short-term drainage was applied before flowering, intermediate conditions were achieved. This evidence strongly indicates an active role of the rhizosphere microbiota in driving arsenic biogeochemistry in rice paddies, influenced by water management, explaining amounts and speciation of arsenic often found in rice grains.
Background
Deposition and secretion from roots influences the composition of the microbial communities surrounding them in the rhizosphere, and microbial activities influence the growth and health of the plant. Different host plant genotypes result in differences in those microbial communities. Crop genomes may have a narrow genetic base because of bottlenecks that occurred when domesticated crops were derived from small populations within the progenitor species. Desirable traits influencing root-associated microbial communities might therefore have been lost in the transition from wild species to modern cultivars. To investigate the diversity of bacterial communities associated with wild and cultivated rice, we surveyed a series of plant species and cultivars spanning the Oryza genus, growing them in the same nutrient-poor soil and assessing the bacterial composition of their rhizospheres and the surrounding soil using 16S rDNA sequencing. ResultsRoot-associated bacterial communities showed small but significant differences dependent on the plant genotype. We found that differences between bacteria associated with differing plant genotypes were only weakly correlated with the phylogenetic distance between the Oryza wild species and cultivars. In ordination plots, domesticated and wild samples could be separated on the basis of their associated bacterial communities. Taxa of the Anaerolineae were overrepresented in wild samples compared to domesticated ones. Certain methanotrophs were overrepresented in the earliest diverged part of the Oryza genus. Conclusions
Bacterial populations associated with the rhizosphere of wild rice species displayed differences with those associated with cultivars, suggesting that root traits selected in domestication could have significant influence on the rhizosphere microbiota composition. Variation within the genus seems to influence the representation of methanotrophs. This suggests that greenhouse emissions from paddy fields could be altered by manipulating plant genotypes through the introgression of wild rice genetic material.
Significance
Land plants continuously contact beneficial, commensal, and pathogenic microbes in soil via their roots. There is limited knowledge as to how the totality of root-associated microbes (i.e., the microbiome) is shaped by various factors or its pattern of acquisition in the root. Using rice as a model, we show that there exist three different root niches hosting different microbial communities of eubacteria and methanogenic archaea. These microbial communities are affected by geographical location, soil source, host genotype, and cultivation practice. Dynamics of the colonization pattern for the root-associated microbiome across the three root niches provide evidence for rapid acquisition of root-associated microbiomes from soil, and support a multistep model wherein each root niche plays a selective role in microbiome assembly.
Purpose
Long-term fertilization can influence soil biological properties and relevant soil ecological processes with implications for sustainable agriculture. This study determined the effects of long-term (>25 years) no fertilizer (CK), chemical fertilizers (NPK) and NPK combined with rice straw residues (NPKS) on soil bacterial and fungal community structures and corresponding changes in soil quality.
Materials and methods
Soil samples were collected from a long-term field site in Wangcheng County established in 1981 in subtropical China between mid summer and early autumn of 2009. Terminal restriction fragment length polymorphism (T-RFLP) and the real-time quantitative polymerase chain reaction (real-time qPCR) of bacterial and fungal community and microbial biomass (MB-C, -N and -P) were analyzed.
Results and discussion
Redundancy analysis of the T-RFLP data indicated that fertilization management modified and selected microbial populations. Of the measured soil physiochemical properties, soil organic carbon was the most dominant factors influencing bacterial and fungal communities. The bacterial and fungal diversity and abundance all showed increasing trends over time (>25 years) coupling with the increasing in SOC, total N, available N, total P, and Olsen P in the fertilized soils. Compared to chemical fertilizer, NPKS resulted in the greater richness and biodiversity of the total microbial community, soil organic C, total N, MB-C, -N and -P. The high biodiversity of microbial populations in NPKS was a clear indication of good soil quality, and also indicated higher substrate use efficiency and better soil nutrient supplementation. Otherwise, unfertilized treatment may have a soil P limitation as indicated by the high soil microbial biomass N: P ratio.
Conclusion
Our results suggest that NPKS could be recommended as a method of increasing the sustainability of paddy soil ecosystems.
The effects of mineral fertilizer (NPK) and organic manure on phospholipid fatty acid profiles and microbial functional diversity
were investigated in a long-term (21-year) fertilizer experiment. The experiment included nine treatments: organic manure
(OM), organic manure plus fertilizer NPK (OM + NPK), fertilizer NPK (NPK), fertilizer NP (NP), fertilizer NK (NK), fertilizer
N (N), fertilizer P (P), fertilizer K (K), and the control (CK, without fertilization). The original soil was extremely eroded,
characterized by low pH and deficiencies of nutrients, particularly N and P. The application of OM and OM + NPK greatly increased
crop yields, soil pH, organic C, total N, P and K, available N, P and K content. Crop yields, soil pH, organic C, total N
and available N were also clearly increased by the application of mineral NPK fertilizer. The amounts of total PLFAs, bacterial,
Gram-negative and actinobacterial PLFAs were highest in the OM + NPK treatment, followed by the OM treatment, whilst least
in the N treatment. The amounts of Gram-positive and anaerobic PLFAs were highest in the OM treatment whilst least in the
P treatment and the control, respectively. The amounts of aerobic and fungal PLFAs were highest in the NPK treatment whilst
least in the N and P treatment, respectively. The average well color development (AWCD) was significantly increased by the
application of OM and OM + NPK, and the functional diversity indices including Shannon index (H
′
), Simpson index (D) and McIntosh index (U) were also significantly increased by the application of OM and OM + NPK. Principal component analysis (PCA) of PLFA profiles
and C source utilization patterns were used to describe changes in microbial biomass and metabolic fingerprints from nine
fertilizer treatments. The PLFA profiles from OM, OM + NPK, NP and NPK were significantly different from that of CK, N, P,
K and NK, and C source utilization patterns from OM and OM + NPK were clearly different from organic manure deficient treatments
(CK, N, P, K, NP, NK 6 and NPK). Stepwise multiple regression analysis showed that total N, available P and soil pH significantly
affected PLFA profiles and microbial functional diversity. Our results could provide a better understanding of the importance
of organic manure plus balanced fertilization with N, P and K in promoting the soil microbial biomass, activity and diversity
and thus enhancing crop growth and production.
Methanotrophs must become established and active in a landfill biocover for successful methane oxidation. A lab-scale biocover with a soil mixture was operated for removal of methane and nonmethane volatile organic compounds, such as dimethyl sulfide (DMS), benzene (B), and toluene (T). The methane elimination capacity was 211 ± 40 g m(-2) d(-1) at inlet loads of 330-516 g m(-2) d(-1). DMS, B, and T were completely removed at the bottom layer (40-50 cm) with inlet loads of 221.6 ± 92.2, 99.6 ± 19.5, and 23.4 ± 4.9 mg m(-2) d(-1), respectively. The bacterial community was examined based on DNA and RNA using ribosomal tag pyrosequencing. Interestingly, methanotrophs comprised 80 % of the active community (RNA) while 29 % of the counterpart (DNA). Types I and II methanotrophs equally contributed to methane oxidation, and Methylobacter, Methylocaldum, and Methylocystis were dominant in both communities. The DNA vs. RNA comparison suggests that DNA-based analysis alone can lead to a significant underestimation of active members.
Single-cell proteins are the dried cells of microorganism, which are used as protein supplement in human foods or animal feeds. Microorganisms like algae, fungi, yeast and bacteria, utilize inexpensive feedstock and wastes as sources of carbon and energy for growth to produce biomass, protein concentrate or amino acids. Since protein accounts for the quantitatively important part of the microbial cells, these microorganisms, also called single cell protein as natural protein concentrate. With increase in population and worldwide protein shortage the use of microbial biomass as food and feed is more highlighted. Although single cell protein has high nutritive value due to higher protein, vitamin, essential amino acids and lipid content, there is a doubt to be replaced to the conventional protein sources due to their high nucleic acid content and slower in digestibility. They also may be considered as foreign material by body, which may subsequently results in allergic reactions.
Bacterial proteins represent a potential future nutrient source for monogastric animal production because they can be grown rapidly on substrates with minimum dependence on soil, water, and climate conditions. This review summarises the current knowledge on methane-utilising bacteria as feed ingredients for animals. We present results from earlier work and recent findings concerning bacterial protein, including the production process, chemical composition, effects on nutrient digestibility, metabolism, and growth performance in several monogastric species, including pigs, broiler chickens, mink (Mustela vison), fox (Alopex lagopus), Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), and Atlantic halibut (Hippoglossus hippoglossus). It is concluded that bacterial meal (BM) derived from natural gas fermentation, utilising a bacteria culture containing mainly the methanotroph Methylococcus capsulatus (Bath), is a promising source of protein based on criteria such as amino acid composition, digestibility, and animal performance and health. Future research challenges include modified downstream processing to produce value-added products, and improved understanding of factors contributing to nutrient availability and animal performance.
The ongoing revolution in high-throughput sequencing continues to democratize the ability of small groups of investigators to map the microbial component of the biosphere. In particular, the coevolution of new sequencing platforms and new software tools allows data acquisition and analysis on an unprecedented scale. Here we report the next stage in this coevolutionary arms race, using the Illumina GAIIx platform to sequence a diverse array of 25 environmental samples and three known "mock communities" at a depth averaging 3.1 million reads per sample. We demonstrate excellent consistency in taxonomic recovery and recapture diversity patterns that were previously reported on the basis of metaanalysis of many studies from the literature (notably, the saline/nonsaline split in environmental samples and the split between host-associated and free-living communities). We also demonstrate that 2,000 Illumina single-end reads are sufficient to recapture the same relationships among samples that we observe with the full dataset. The results thus open up the possibility of conducting large-scale studies analyzing thousands of samples simultaneously to survey microbial communities at an unprecedented spatial and temporal resolution.
mothur aims to be a comprehensive software package that allows users to use a single piece of software to analyze community
sequence data. It builds upon previous tools to provide a flexible and powerful software package for analyzing sequencing
data. As a case study, we used mothur to trim, screen, and align sequences; calculate distances; assign sequences to operational
taxonomic units; and describe the α and β diversity of eight marine samples previously characterized by pyrosequencing of
16S rRNA gene fragments. This analysis of more than 222,000 sequences was completed in less than 2 h with a laptop computer.
The light-scattering properties of submicroscopic metal particles ranging from 40 to 120 nm in diameter have recently been
investigated. These particles scatter incident white light to generate monochromatic light, which can be seen either by the
naked eye or by dark-field microscopy. The nanoparticles are well suited for detection in microchannel-based immunoassays.
The goal of the present study was to detect Helicobacter pylori- and Escherichia coli O157:H7-specific antigens with biotinylated polyclonal antibodies. Gold particles (diameter, 80 nm) functionalized with a
secondary antibiotin antibody were then used as the readout. A dark-field stereomicroscope was used for particle visualization
in poly(dimethylsiloxane) microchannels. A colorimetric quantification scheme was developed for the detection of the visual
color changes resulting from immune reactions in the microchannels. The microchannel immunoassays reliably detected H. pylori and E. coli O157:H7 antigens in quantities on the order of 10 ng, which provides a sensitivity of detection comparable to those of conventional
dot blot assays. In addition, the nanoparticles within the microchannels can be stored for at least 8 months without a loss
of signal intensity. This strategy provides a means for the detection of nanoparticles in microchannels without the use of
sophisticated equipment. In addition, the approach has the potential for use for further miniaturization of immunoassays and
can be used for long-term archiving of immunoassays.
A total of 180 broiler chickens were fed 1 of 3 diets from day-old to slaughter at 35 d: a control diet with 35% soybean meal (SOY) or diets in which either 6% basic bacterial protein meal (BBP) or 6% autolysed bacterial protein meal (AUT) partially replaced soybean meal protein. Ileal and total tract apparent amino acid digestibility were examined in 5 chickens per diet using TiO(2) as an inert marker. Chickens fed the diets with bacterial protein had higher weight gain and feed consumption than control chicks during the first 3 wk, but there were no differences in growth or feed intake during the last 2 wk or during the total experimental period. The birds fed the BBP diet showed more efficient feed conversion compared with chickens fed the SOY and AUT diets. Litter quality at 5 wk was poorer in pens where the chickens were fed the AUT diet compared with the other 2 treatments. There were no differences among diets in the dressing percentage. Ileal amino acid digestibility at 5 wk of age revealed only minor differences between diets. There was a tendency toward lower ileal digestibility (0.12 > P > 0.07) of Arg, Lys, Met, and Phe in the AUT diet compared with the SOY diet, whereas there were no differences between the SOY and BBP diets. Total tract amino acid digestibilities at 5 wk were similar or slightly lower than the ileal digestibilities within diets. Total tract amino acid digestibility at 2 wk was similar to the total tract amino acid digestibility at 5 wk. The diets containing bacterial protein showed lower total tract digestibility of most amino acids compared with the SOY diet. It was concluded that 6% of either basic or autolysed bacterial protein can replace soybean meal in diets for broiler chickens without impairing growth performance, and the basic bacterial protein seemed to be a slightly better substitute than the autolysed bacterial protein.
The accumulation of a large amount of organic solid waste and the lack of sufficient protein supply worldwide are two major challenges caused by rapid population growth. Anaerobic digestion is the main force of organic waste treatment, and the high-value utilization of its products (biogas and digestate) has been widely concerned. These products can be used as nutrients and energy sources for microorganisms such as microalgae, yeast, methane-oxidizing bacteria(MOB), and hydrogen-oxidizing bacteria(HOB) to produce single cell protein(SCP), which contributes to the achievement of sustainable development goals. This new model of energy conversion can construct a bioeconomic cycle from waste to nutritional products, which treats waste without additional carbon emissions and can harvest high-value biomass. Techno-economic analysis shows that the SCP from biogas and digestate has higher profit than biogas electricity generation, and its production cost is lower than the SCP using special raw materials as the substrate. In this review, the case of SCP-rich microorganisms using anaerobic digestion products for growth was investigated. Some of the challenges faced by the process and the latest developments were analyzed, and their potential economic and environmental value was verified.
Securing a sustainable protein supply at the global level is among the greatest challenges currently faced by humanity. Alternative protein sources, such as second-generation microbial protein (MP), could give rise to innovative circular bioeconomy practices, synthesizing high-value bioproducts through the recovery and upcycling of resources from overabundant biowastes and residues. Within such a multi-feedstock biorefinery scenario, the wide range of microbial pathways and networks that characterize mixed microbial cultures, offers interesting and not yet fully explored advantages over conventional monoculture-based processes. In this review, we combine a comprehensive analysis of waste recovery platforms for second-generation MP production with a critical evaluation of the research gaps and potentials offered by mixed culture-based MP fermentation processes.
Estuary and coastal environments have essential ecosystem functions in greenhouse gas sinks and removal of nitrogen pollution. Methane-oxidizing bacteria (MOB) and ammonia-oxidizing bacteria (AOB) communities play critical functions in the estuary's tidal flat sediments. Therefore, the effects of ammonium on MOB communities and methane on AOB communities need to be further explained. In this study, microcosm incubations with different contents of ammonium or methane were conducted for a relatively short (24 h) or long (28 days) period with tidal flat sediments from the Yangtze River estuary. Subsequently, the tagged highly degenerate primer PCR and DNA-based stable isotope probing method were employed to demonstrate the effects on MOB and AOB populations. The results indicated that the methane consumption was enhanced with ammonium supplements within 24 h of incubation. Supplement of 2 μmol/g d.w.s (μmol per gram dry weight soil) NH4⁺ increased the amount of MOB and its proportion to the total bacteria (p < 0.05) for 28 days incubation. The ammonium supplement increased the proportion of Methylomonas and Methylobacter based on the 16S rRNA gene. According to the functional gene analysis, the MOB primarily engaged in methane oxidation include Methylomonas, Methylobacter, Methylomicrobium, and Methylosarcina, which were associated with Type Ia MOB. It suggested that ammonium supplement may promote methane oxidation by stimulating the Type Ia MOB in tidal flat sediments of the Yangtze River estuary. The current research helps understand the effect of ammonium on methane consumption in the estuary and coastal environments.
Methane-utilizing bacteria (methanotrophs) are a diverse group of gram-negative bacteria that are related to other members of the Proteobacteria. These bacteria are classified into three groups based on the pathways used for assimilation of formaldehyde, the major source of cell carbon, and other physiological and morphological features. The type I and type X methanotrophs are found within the gamma subdivision of the Proteobacteria and employ the ribulose monophosphate pathway for formaldehyde assimilation, whereas type II methanotrophs, which employ the serine pathway for formaldehyde assimilation, form a coherent cluster within the beta subdivision of the Proteobacteria. Methanotrophic bacteria are ubiquitous. The growth of type II bacteria appears to be favored in environments that contain relatively high levels of methane, low levels of dissolved oxygen, and limiting concentrations of combined nitrogen and/or copper. Type I methanotrophs appear to be dominant in environments in which methane is limiting and combined nitrogen and copper levels are relatively high. These bacteria serve as biofilters for the oxidation of methane produced in anaerobic environments, and when oxygen is present in soils, atmospheric methane is oxidized. Their activities in nature are greatly influenced by agricultural practices and other human activities. Recent evidence indicates that naturally occurring, uncultured methanotrophs represent new genera. Methanotrophs that are capable of oxidizing methane at atmospheric levels exhibit methane oxidation kinetics different from those of methanotrophs available in pure cultures. A limited number of methanotrophs have the genetic capacity to synthesize a soluble methane monooxygenase which catalyzes the rapid oxidation of environmental pollutants including trichloroethylene.
Single cell protein (SCP) provides an alternative protein source to partially replace the conventional agricultural resources and support the increased nutritional needs. Inexpensive feeding source is one of the key limiting factors for the expansion of SCP production. The present study examined the valorization of biogas derived from the anaerobic digestion (AD) of sewage sludge and the discarded effluent as nutrients source to produce SCP using methanotrophic bacteria. Results indicated that the mixed methanotrophic culture can grow well on the pasteurized AD supernatant and biogas, succeeding in promising dry weight (DW) yield (0.66±0.01 g-DW/g-CH4 and 11.54±0.12 g-DW/g-NH4+). Methylomonas (56.26%) and Methylophilus (24.60%) spp. were the two main representatives of the mixed culture. The produced dried biomass had a protein content higher than 41% w/w, including essential amino acids like histidine, valine, phenylalanine, isoleucine, leucine, threonine and lysine. The cultivated SCP shows potential utilization as protein source for animal diets.
Methane-oxidizing bacteria (MOB) possess the metabolic potential to assimilate the highly potent greenhouse gas, CH4, and can also synthesize valuable products. Depending on their distinct and fastidious metabolic pathways, MOB are mainly divided into Type I and Type II; the latter are known as producers of polyhydroxyalkanoate (PHA). Despite the metabolic potential of MOB to synthesize PHA, the ecophysiology of MOB, especially under high CH4 flux conditions, is yet to be understood. Therefore, in this study, a rice paddy soil receiving a high CH4 flux from underground was used as an inoculum to enrich MOB using fed-batch operation, then the enriched Type II MOB were characterized. The transitions in the microbial community composition and CH4 oxidation rates were monitored by 16S rRNA gene amplicon sequencing and degree of CH4 consumption. With increasing incubation time, the initially dominant Methylomonas sp., affiliated with Type I MOB, was gradually replaced with Methylocystis sp., Type II MOB, resulting in a maximum CH4 oxidation rate of 1.40 g-CH4/g-biomass/day. The quantification of functional genes encoding methane monooxygenase, pmoA and PHA synthase, phaC, by quantitative PCR revealed concomitant increases in accordance with the Type II MOB enrichment. These increases in the functional genes underscore the significance of Type II MOB to mitigate greenhouse gas emission and produce PHA.
Microbial protein is proposed as an alternative protein source with low environmental impact. Methane oxidizing bacteria are already produced at commercial scale from natural gas. However, their productivity is limited because of the creation of explosive atmospheres in the fermenters during production. This work demonstrates the applicability of bioreactors with a membrane-based gas supply via diffusion. Methanotrophic bacteria were successfully cultivated, with growth yields from 0.26 to 0.43 g-VSS g-CH4⁻¹, slightly below those observed in analogous fermenters relying on bubbling. However, ammonia yields ranged from 5.2 to 6.9 g-VSS g-NH3⁻¹, indicating higher nitrogen assimilation than in conventional fermenters. Indeed, protein content increased during the operational period reaching up to 51% of dry weight. The amino acid profile included the majority of the essential amino acids, demonstrating suitability as feed ingredient. Never during the operational period was an explosive atmosphere established in the reactor. Thus, bubble-free membrane bioreactors are a promising technology for microbial protein production relying on explosive gas mixtures.
Conventional microbial protein production relies on the usage of pure chemicals and gases. Natural gas, which is a fossil resource, is the common input gas for bacterial protein production. Alternative sources for gas feedstock and nutrients can sufficiently decrease the operational cost and environmental impact of microbial protein production processes. In the present study, the effluents streams of municipal biowaste anaerobic digestion, were used to grow methane oxidising bacteria which can be used as protein source. Results demonstrated that a 40:60 CH4:O2 (v/v) gas feeding resulted in microbial biomass production of 0.95 g-DM/L by a Methylophilus dominated community. When raw biogas was used as input for methane corresponding to the same initial methane partial pressure as before, instead of pure methane, the growth was partially hindered (0.61
g-DM/L) due to the presence of H2S (IC50: 1376 ppm). Hence, desulfurization is suggested before using biogas for microbial protein production. At semi-continuous mode, results showed that the produced biomass had relatively high protein content (>40% of dry weight) and the essential amino acids lysine, valine, leucine and histidine were detected at high levels.
Release of abiotic methane from marine seeps into the atmosphere is a major source of this potent greenhouse gas. Methanotrophic microorganisms in methane seeps use methane as carbon and energy source, thus significantly mitigating global methane emissions. Here we investigated microbial methane oxidation at the sediment‐water interface of a shallow marine methane seep. Metagenomics and metaproteomics, combined with 13C‐methane stable isotope probing, demonstrated that various members of the gammaproteobacterial family Methylococcaceae were the key players for methane oxidation, catalyzing the first reaction step to methanol. We observed a transfer of carbon to methanol‐oxidizing methylotrophs of the betaproteobacterial family Methylophilaceae, suggesting an interaction between methanotrophic and methylotrophic microorganisms that allowed for rapid methane oxidation. From our microcosms, we estimated methane oxidation rates of up to 871 nmol of methane per gram sediment and day. This implies that more than 50% of methane at the seep is removed by microbial oxidation at the sediment‐water interface, based on previously reported in situ methane fluxes. The organic carbon produced was further assimilated by different heterotrophic microbes, demonstrating that the methane‐oxidizing community supported a complex trophic network. Our results provide valuable eco‐physiological insights into this specialized microbial community performing an ecosystem function of global relevance. This article is protected by copyright. All rights reserved.
Rice fields are a major source of atmospheric methane (CH4), a greenhouse gas. CH4 emissions from wetland rice fields represents globally 15–20% of the annual anthropogenic CH4 emissions, and about 4% of the global CH4 emissions. Methane emission from rice cultivation may increase from the 1990 level of 97 Tg/year to 145 Tg/year by 2025 due to the increase in acreage and intensification of paddy cultivation. Here we review the role of anaerobic methanogenic bacteria in methane emission. We discuss the factors that influence methane emissions from rice fields, such as water regime, cropping season, soil temperature, fertilizer application, soil physico-chemical properties, crop cultivation, agricultural practices, soil type, soil profile and crop management practices. These practices control soil bacterial communities. Other influencing factors include intercultural operations such as ploughing, puddling and frequent mixing of soil during the paddy field preparation. Methane emission from paddy field follows a seasonal pattern of variation due to influence of climatic factors like temperature, sunlight, and precipitation. Algae, microphytes, macrophytes and anoxygenic photosynthetic bacteria significantly reduce CH4 emissions when they grow actively under illuminated condition. Methane emission is limited by alternate flooding-drying; cultivars with few unproductive tillers, small root system, high oxidative ability, and high harvest index; excessive application of organic amendments; application of potassium, biochar, nitrate, sulfate and ferric iron; and urease and nitrification inhibitors.
The rhizosphere is the most dynamic hotspot of microbial activity in the soil. Despite these dynamics, the spatial pattern of many rhizosphere properties may remain stable because they are continuously reproduced in the changing environment. Low substrate concentration can strongly reduce the rate response of an enzymatic reaction subjected to increased temperature and is recognized as a canceling effect on enzyme temperature sensitivity. Carbon input from rhizodeposits affects C availability in the rhizosphere, and thus the enzyme activities responsible for organic matter decomposition, and their temperature sensitivities, upset the dynamics and stability of biochemical processes in the rhizosphere. However, it is unclear whether a canceling effect occurs in the rhizosphere. We studied temperature effects on chitinase and phosphatase during rice (Oryza sativa L.) growth at 18 and 25 °C. The spatial distribution of enzyme activities was imaged using soil zymography and showed that the overall activities of these enzymes increased with temperature but decreased with rice growth. The temporal dynamics of hotspot areas were enzyme-specific. During growing days 14–30, hotspot areas decreased from 2-2.5% to 0.3–0.5% for chitinase, but increased from 2% to 6–7% for phosphatase. The distribution pattern of both enzymes shifted from being dispersed throughout the soil to being associated with the roots. For the first time, we showed the extent of rhizosphere enzyme activity in paddy soil and demonstrated that it is temporally stationary and independent of temperature. However, the temperature sensitivity of enzyme activities declined radically (Q10∼1.3–1.4) at the root surface compared to that of bulk soil (Q10 ∼1). We conclude that the spatio-temporal pattern of rhizosphere enzymatic hotspots is mainly affected by plant growth. High temperature sensitivity (Q10 > 1) at the root-soil interface for the tested enzymes revealed that warming will lead to faster nutrient mobilization in the rhizosphere than in root-free soil.
Univariate ANOVA is reviewed from a user point-of-view with emphasis on understanding the model building and the assumptions underlying the method. Illustrative examples are taken from organic chemistry and analytical chemistry. The use of graphical techniques to visualize the ANOVA model as well as to analyse residuals is recommended. The main models of ANOVA are developed in some detail including one-factor ANOVA, crossed designs, nested designs, repeated measures ANOVA and variance components estimation. Hypothesis testing by F-tests-and follow up by pairwise comparison methods is shown. The distinction between random effects and fixed effects is explained. Methods to handle non-linearities by transformations or by using response surface methodology are mentioned. Throughout the paper the importance of experimental design is emphasized. References are given to ANOVA methods for more complicated models.
A field experiment was conducted to illustrate the different degree and dynamics of microbial community structure and function in the rhizosphere across four growing stages (before plantation and three growth stages) using a combination of biochemical (enzyme assay and microbial biomass carbon) and molecular approaches of qPCR and PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis). Rice plant cultivation promoted higher enzyme activities (invertase and urease), microbial biomass carbon (Cmic), bacterial (16S rRNA) and fungal (ITS rRNA) genes abundances in the rhizosphere compared to unplanted soil. Principal component analyses of PCR-DGGE profile also revealed that structures of bacterial and fungal communities of rice planted soil were well distinct from unplanted soil. Moreover, enzyme activities showed a significant positive correlation with the total microbial biomass in the rhizosphere throughout growth stages of rice plant. Relative fungal: bacterial ratios were significantly higher in rice planted soil compared to unplanted soil, suggesting rice plantation enhanced the fungal community in the rice rhizosphere environment. These results further suggest a significant linkage between the microbial community dynamics and function in the rhizosphere associated with rice plant over time.
The emission of the greenhouse gas CH4 from ricepaddies is strongly influenced by management practicessuch as the input of ammonium-based fertilisers. Weassessed the impact of different levels (200 and 400kgN.ha–1) of urea and (NH4)2HPO4on the microbial processes involved in production andconsumption of CH4 in rice field soil. We usedcompartmented microcosms which received fertilisertwice weekly. Potential CH4 production rates weresubstantially higher in the rice rhizosphere than inunrooted soil, but were not affected by fertilisation.However, CH4 emission was reduced by the additionof fertiliser and was negatively correlated with porewater NH
4
plus
concentration, probably as theconsequence of elevated CH4 oxidation due tofertilisation. CH4 oxidation as well as numbersof methanotrophs was distinctly stimulated by theaddition of fertiliser and by the presence of the riceplant. Without fertiliser addition,nitrogen-limitation of the methanotrophs will restrictthe consumption of CH4. This may have a majorimpact on the global CH4 budget, asnitrogen-limiting conditions will be the normalsituation in the rice rhizosphere. Elevated potentialnitrifying activities and numbers were only detectedin microcosms fertilised with urea. However, asubstantial part of the nitrification potential in therhizosphere of rice was attributed to the activity ofmethanotrophs, as was demonstrated using theinhibitors CH3F and C2H2.
SUMMARY More than IOO Gram-negative, strictly aerobic, methane-utilizing bacteria were isolated. All used only methane and methanol of the substrates tested for growth. The organisms were classified into five groups on the basis of mor- phology, fine structure, and type of resting stage formed (exospores and different types of cysts) and into subgroups on other properties. Methods of enrichment, isolation and culture are described.
The glutamate dehydrogenase gene of Escherichia coli has been cloned into broad host-range plasmids and can complement glutamate synthase mutants of Methylophilus methylotrophus. Assimilation of ammonia via glutamate dehydrogenase is more energy-efficient than via glutamate synthase, thus the recombinant organism converts more growth substrate, methanol, into cellular carbon.
The methanotrophic bacterium Methylococcus capsulatus (Bath) grows on pure methane. However, in a single cell protein production process using natural gas as methane source, a bacterial consortium is necessary to support growth over longer periods in continuous cultures. In different bioreactors of Norferm Danmark A/S, three bacteria consistently invaded M. capsulatus cultures growing under semi-sterile conditions in continuous culture. These bacteria have now been identified as a not yet described member of the Aneurinibacillus group, a Brevibacillus agri strain, and an acetate-oxidiser of the genus Ralstonia. The physiological roles of these bacteria in the bioreactor culture growing on natural, non-pure methane gas are discussed. The heterotrophic bacteria do not have the genetic capability to produce either the haemolytic enterotoxin complex HBL or non-haemolytic enterotoxin.
The Earth’s energy budget, climate feedbacks, and climate sensitivity
- Forster
Single cell protein production: a review
- Suman