Methanol,a potential feedstock for biotechnological processes

A wide variety of bacteria and yeasts is able to grow in inexpensive synthetic media with methanol as the sole or major source of carbon and energy. This is due to the presence of a few unique enzymes which enable these organisms to generate metabolic energy and synthesize cell constituents from this one-carbon substrate. In the chemical industry there is currently much interest in the production of fuels and chemicals from methanol. As a feedstock for industrial fermentations methanol is also attractive because of its low cost, ease of handling and abundant availability. In many countries methanol-utilizing microbes are being studied and their potential utility in biotechnological processes is explored. These studies are aimed at making use of their characteristic properties, exploiting known organisms and new strains for improving existing processes and developing novel products.     

Methanol‐driven enhanced biological phosphorus removal with a syntrophic consortium

The presence of suitable carbon sources for enhanced biological phosphorus removal (EBPR) plays a key role in phosphorus removal from wastewater in urban WWTP. For wastewaters with low volatile fatty acids (VFAs) content, an external carbon addition is necessary. As methanol is the most commonly external carbon source used for denitrification it could be a priori a promising alternative, but previous attempts to use it for EBPR have failed. This study is the first successful report of methanol utilization as external carbon source for EBPR. Since a direct replacement strategy (i.e., supply of methanol as a sole carbon source to a propionic‐fed PAO‐enriched sludge) failed, a novel process was designed and implemented successfully: development of a consortium with anaerobic biomass and polyphosphate accumulating organisms (PAOs). Methanol‐degrading acetogens were (i) selected against other anaerobic methanol degraders from an anaerobic sludge; (ii) subjected to conventional EBPR conditions (anaerobic + aerobic); and (iii) bioaugmented with PAOs. EBPR with methanol as a sole carbon source was sustained in a mid‐term basis with this procedure. Biotechnol. Bioeng. 2013; 110: 391–400. © 2012 Wiley Periodicals, Inc.     

Nutrient transport in mycorrhizas: structure,physiology and consequences for efficiency of the symbiosis

Nutrient transport in mycorrhizas occurs across specialized interfaces which are the result of corrdinated development of the organisms. The structural modifications give rise to large areas of either inter- or intra-cellular interface in which wall synthesis is frequently modified and in which altered distribution of membrane bound ATPases is important, particularly with respect to mechanisms that may be involved in bidirectional transfer of nutrients. Except in orchid mycorrhizas, net movement of organic carbon from plant to fungus occurs, complemented by mineral nutrient movement in the opposite direction. The general consensus is that sustained transfer at rates that will maintain the growth and development of the organisms requires increases in the rates at which nutrients are lost from the organisms; possible mechanisms for this are discussed. The transfer processes are essential in determining both plant and fungal productivity and an approach to calculating the efficiency of the symbiosis in terms of the expenditure of carbon (or of phosphorus) is discussed.     

Utilization of methanol by rhodospirillaceae.

Enrichment culture of organisms growing anaerobically in the light in methanol-bicarbonate medium resulted in isolation of strains of Rhodopseudomonas gelatinosa and Rhodopseudomonas acidophila. The pH optimum for growth on methanol for all strains tested was approximately one unit higher than for growth on carbon sources containing more than one carbon atom. At the appropriate pH, 17 strains of Rhodospirillaceae out of 39 in a culture collection grew anaerobically in the light on methanol-bicarbonate. Rhodopseudomonas acidophia strain 10050 showed the most abundant growth and was studied in more detail. Its growth on methanol was stimulated by yeast extract or vitamin-free casamino acids. The organism grew on methanol-bicarbonate, methanol-formate or formate alone as the sole carbon sources. No growth was observed on methylamine or fomaldehyde. In the presence of excess bicarbonate a maximum yield of 98 g cell material from 100 g methanol was obtained. Ribulose diphosphate carboxylase was present in the methanol-bicarbonate-grown organism at six times the specific activity of that in the succinate-grown organism.     

A comparative study of methanol as a supplementary carbon source for enhancing denitrification in primary and secondary anoxic zones

A comparative study on the use of methanol as a supplementary carbon source to enhance denitrification in primary and secondary anoxic zones is reported. Three lab-scale sequencing batch reactors (SBR) were operated to achieve nitrogen and carbon removal from domestic wastewater. Methanol was added to the primary anoxic period of the first SBR, and to the secondary anoxic period of the second SBR. No methanol was added to the third SBR, which served as a control. The extent of improvement on the denitrification performance was found to be dependent on the reactor configuration. Addition to the secondary anoxic period is more effective when very low effluent nitrate levels are to be achieved and hence requires a relatively large amount of methanol. Adding a small amount of methanol to the secondary anoxic period may cause nitrite accumulation, which does not improve overall nitrogen removal. In the latter case, methanol should be added to the primary anoxic period. The addition of methanol can also improve biological phosphorus removal by creating anaerobic conditions and increasing the availability of organic carbon in wastewater for polyphosphate accumulating organisms. This potentially provides a cost-effective approach to phosphorus removal from wastewater with a low carbon content. New fluorescence in situ hybridisation (FISH) probes targeting methanol-utilising denitrifiers were designed using stable isotope probing. Microbial structure analysis of the sludges using the new and existing FISH probes clearly showed that the addition of methanol stimulated the growth of specific methanol-utilizing denitrifiers, which improved the capability of sludge to use methanol and ethanol for denitrification, but reduced its capability to use wastewater COD for denitrification. Unlike acetate, long-term application of methanol has no negative impact on the settling properties of the sludge.     

Utilization of methanol by rhodospirillaceae

Enrichment culture of organisms growing anaerobically in the light in methanol-bicarbonate medium resulted in isolation of strains of Rhodopseudomonas gelatinosa and Rhodopseudomonas acidophila. The pH optimum for growth on methanol for all strains tested was approximately one unit higher than for growth on carbon sources containing more than one carbon atom. At the appropriate pH, 17 strains of Rhodospirillaceae out of 39 in a culture collection grew anaerobically in the light on methanol-bicarbonate. Rhodopseudomonas acidophila strain 10050 showed the most abundant growth and was studied in more detail. Its growth on methanol was stimulated by yeast extract or vitamin-free casamino acids. The organism grew on methanol-bicarbonate, methanol-formate or formate alone as the sole carbon sources. No growth was observed on methylamine or formaldehyde. In the presence of excess bicarbonate a maximum yield of 98 g cell material from 100 g methanol was obtained. Ribulose diphosphate carboxylase was present in the methanol-bicarbonate-grown organism at six times the specific activity of that in the succinate-grown organism.     

Growth yields of methanotrophs

Summary The growth yield ofMethylococcus capsulatus (Bath) on methane was dependent on the availability of copper in the growth medium. In nitrate mineral salts medium the carbon conversion efficiency increased by 38%, concomitant with the transition from soluble to particulate methane monooxygenase, after transfer from low to high copper medium. An increase in growth efficiency was also observed with ammonia as nitrogen source but not when methanol replaced methane as carbon source. The high growth efficiency is attributed to a reduced NADH requirement for methane oxidation. This could only arise if methanol dehydrogenase was capable of electron transfer, either directly or indirectly to the particulate methane monooxygenase (MMO). The carbon conversion efficiency from methanol with nitrate as nitrogen source was as high as theoretically predicted. It is suggested that the previously low yields of methanotrophs grown on methanol resulted from the use, as nitrogen source, of ammonia which was oxidised by the MMO still present under these growth conditions. The term ‘methanotroph’ is used throughout to distinguish those organisms capable of growth on methane from ‘methylotrophs’ capable of growth on reduced C, compounds other than methane     

Nature, nomenclature and taxonomy of obligate methanol utilizing strains

In a screening program, a number of different bacterial strains with the ability to utilize methanol as a sole carbon and energy source were isolated and described. They are well known methanol utilizing genera Pseudomonas, Klebsiella, Micrococcus, Methylomonas or, on the contrary, the new, unknown genera and species of methylotrophic bacteria. In the last category, Acinetobacter and Alcaligenes are the new reported genera of organisms able to use methanol as a sole carbon and energy source. The present paper reports the very complex physiological and biochemical modifications when very versatile bacteria such as Pseudomonas aeruginosa and Acinetobacter calcoaceticus are cultured on methanol and when the obligate methylotrophic state is compared with the facultative methylotrophic state of the same bacterial strain. Based on experiments and comparisons with literature data, it seems that Methylomonas methanica is the obligate methylotrophic state of Pseudomonas aeruginosa and that Acinetobacter calcoaceticus is the facultative methylotrophic state of Methylococcus capsulatus, an obligate methylotroph. The relationship of the obligate to the facultative and of the facultative to the obligate methylotrophy were established. These new methylotrophic genera and species, the profound physiological and biochemical modifications as well as the new data concerning nature, nomenclature and taxonomy of methanol utilizing bateria were reported for the first time in 1983.     

Patterns in decomposition rates among photosynthetic organisms: the importance of detritus C:N:P content

The strength and generality of the relationship between decomposition rates and detritus carbon, nitrogen, and phosphorus concentrations was assessed by comparing published reports of decomposition rates of detritus of photosynthetic organisms, from unicellular algae to trees. The results obtained demonstrated the existence of a general positive, linear relationship between plant decomposition rates and nitrogen and phosphorus concentrations. Differences in the carbon, nitrogen, and phosphorus concentrations of plant detritus accounted for 89% of the variance in plant decomposition rates of detritus orginating from photosynthetic organisms ranging from unicellular microalgae to trees. The results also demonstrate that moist plant material decomposes substantially faster than dry material with similar nutrient concentrations. Consideration of lignin, instead of carbon, concentrations did not improve the relationships obtained. These results reflect the coupling of phosphorus and nitrogen in the basic biochemical processes of both plants and their microbial decomposers, and stress the importance of this coupling for carbon and nutrient flow in ecosystems. This work was funded through a grant of CICYT (MAR91-0503) to C.M.D.     

Metabolic profiles and genetic diversity of denitrifying communities in activated sludge after addition of methanol or ethanol

External carbon sources can enhance denitrification rates and thus improve nitrogen removal in wastewater treatment plants. The effects of adding methanol and ethanol on the genetic and metabolic diversity of denitrifying communities in activated sludge were compared using a pilot-scale plant with two parallel lines. A full-scale plant receiving the same municipal wastewater, but without external carbon source addition, was the reference. Metabolic profiles obtained from potential denitrification rates with 10 electron donors showed that the denitrifying communities altered their preferences for certain compounds after supplementation with methanol or ethanol and that methanol had the greater impact. Clone libraries of nirK and nirS genes, encoding the two different nitrite reductases in denitrifiers, revealed that methanol also increased the diversity of denitrifiers of the nirS type, which indicates that denitrifiers favored by methanol were on the rise in the community. This suggests that there might be a niche differentiation between nirS and nirK genotypes during activated sludge processes. The composition of nirS genotypes also varied greatly among all samples, whereas the nirK communities were more stable. The latter was confirmed by denaturing gradient gel electrophoresis of nirK communities on all sampling occasions. Our results support earlier hypotheses that the compositions of denitrifier communities change during predenitrification processes when external carbon sources are added, although no severe effect could be observed from an operational point of view.     

Optimal Locations for Methanol and CHP Production in Eastern Finland

Finland considers energy production from woody biomass as an efficient energy planning strategy to increase the domestic renewable energy production in order to substitute fossil fuel consumption and reduce greenhouse gas emissions. Consequently, a number of developmental activities are implemented in the country, and one of them is the installation of second generation liquid biofuel demonstration plants. In this study, two gasification-based biomass conversion technologies, methanol and combined heat and power (CHP) production, are assessed for commercialization. Spatial information on forest resources, sawmill residues, existing biomass-based industries, energy demand regions, possible plant locations, and a transport network of Eastern Finland is fed into a geographically explicit Mixed Integer Programming model to minimize the costs of the entire supply chain which includes the biomass supply, biomass and biofuel transportation, biomass conversion, energy distribution, and emissions. The model generates a solution by determining the optimal number, locations, and technology mix of bioenergy production plants. Scenarios were created with a focus on biomass and energy demand, plant characteristics, and cost variations. The model results state that the biomass supply and high energy demand are found to have a profound influence on the potential bioenergy production plant locations. The results show that methanol can be produced in Eastern Finland under current market conditions at an average cost of 0.22??/l with heat sales (0.34??/l without heat sales). The introduction of energy policy tools, like cost for carbon, showed a significant influence on the choice of technology and CO₂ emission reductions. The results revealed that the methanol technology was preferred over the CHP technology at higher carbon dioxide cost (>145??/t_CO2). The results indicate that two methanol plants (360?MW_biomass) are needed to be built to meet the transport fuel demand of Eastern Finland.     

Microbial carbon limitation: The need for integrating microorganisms into our understanding of ecosystem carbon cycling

Numerous studies have demonstrated that fertilization with nutrients such as nitrogen, phosphorus, and potassium increases plant productivity in both natural and managed ecosystems, demonstrating that primary productivity is nutrient limited in most terrestrial ecosystems. In contrast, it has been demonstrated that heterotrophic microbial communities in soil are primarily limited by organic carbon or energy. While this concept of contrasting limitations, that is, microbial carbon and plant nutrient limitation, is based on strong evidence that we review in this paper, it is often ignored in discussions of ecosystem response to global environment changes. The plant‐centric perspective has equated plant nutrient limitations with those of whole ecosystems, thereby ignoring the important role of the heterotrophs responsible for soil decomposition in driving ecosystem carbon storage. To truly integrate carbon and nutrient cycles in ecosystem science, we must account for the fact that while plant productivity may be nutrient limited, the secondary productivity by heterotrophic communities is inherently carbon limited. Ecosystem carbon cycling integrates the independent physiological responses of its individual components, as well as tightly coupled exchanges between autotrophs and heterotrophs. To the extent that the interacting autotrophic and heterotrophic processes are controlled by organisms that are limited by nutrient versus carbon accessibility, respectively, we propose that ecosystems by definition cannot be ‘limited’ by nutrients or carbon alone. Here, we outline how models aimed at predicting non‐steady state ecosystem responses over time can benefit from dissecting ecosystems into the organismal components and their inherent limitations to better represent plant–microbe interactions in coupled carbon and nutrient models.     

The dye-linked alcohol dehydrogenase of Rhodopseudomonas acidophila. Comparison with dye-linked methanol dehydrogenases.

1. A dye-linked alcohol dehydrogenase was purified 20-fold from extracts of Rhodopseudomonas acidophila 10050 grown anaerobically in the light on methanol/HCO3-. 2. The enzyme resembled many previously reported methanol dehydrogenases from other methylotrophic organisms in coupling to phenazine methosulphate, requiring ammonia as an activator, possessing a pH optimum of 9 and a mol.wt. of approx. 116000. In many other respects the enzyme showed singular properties. 3. The stability of the enzyme under various conditions of temperature and pH was studied. 4. Primary aliphatic amines containing up to nine carbon atoms (the longest tested) were better activators than ammonia. 5. A wide range of primary alcohols and aldehydes served as substrates, with apparent Km values ranging from 57 mM for methanol to 6 micron for ethanol. 6. O2 was an inhibitor competitive with respect to the alcohol substrate. In the presence of O2, apparent Km values of 145 mM were recorded for methanol. 6. Cyanide and alphaalpha'-bipyridine were inhibitors competitive with respect to the amine activator. 7. The properties of the enzyme from Rhodopseudomonas acidophila are compared with those of similar enzymes from other organisms, and implications of the salient differences are discussed.     

Effects of methanol on the growth of gastrointestinal anaerobes

The effects of methanol on the growth of representative, predominant, anaerobic gut bacteria were studied. Growth yields and rates were determined in a base medium to which methanol was added to produce media with methanol concentrations varying, in twofold steps, over a concentration range of 0.01 to 25%, by volume. The growth of many of the organisms was completely inhibited by a methanol concentration equal to, or less than, 6.2%. Isolates representing cellulolytic species were completely inhibited at a methanol concentration of 3.1%, and inhibitory effects on the yield of some cellulolytic isolates were found at a methanol concentration as small as 0.01%. Although most of the organisms studied were inhibited at relatively small methanol concentrations, isolates of Selenomonas ruminantium, Bacteroides ovatus, and Fusobacterium necrophorum were relatively methanol resistant. A methanol concentration of 12.5% was required to completely inhibit S. ruminantium. Substantial growth of B. ovatus was obtained in media containing 12.5% methanol, and for F. necrophorum, substantial growth occurred in media containing 25% methanol. The yields of F. necrophorum strain B85 and S. ruminantium strain PC18 were enhanced by relatively small methanol concentrations and reduced with further methanol concentration increase Anaerobic, nonsporing gut bacteria exhibit a diversity of responses to methanol.     

果树中糖类代谢和调控研究

糖是生物体内主要的碳源,是光合作用的主要产物,可为生物体提供能量,在植物的生长发育过程中起重要作用。本文综述了近年来关于糖类与果树生长发育及品质形成方面的相关研究进展,重点介绍了糖类运输、积累与基因表达、糖信号传导和糖类调控网络等方面的研究进展,并对今后利用分子生物学手段进行果实品质改良等方面的研究方向进行了展望。     

Metabolic Profiles and Genetic Diversity of Denitrifying Communities in Activated Sludge after Addition of Methanol or Ethanol

External carbon sources can enhance denitrification rates and thus improve nitrogen removal in wastewater treatment plants. The effects of adding methanol and ethanol on the genetic and metabolic diversity of denitrifying communities in activated sludge were compared using a pilot-scale plant with two parallel lines. A full-scale plant receiving the same municipal wastewater, but without external carbon source addition, was the reference. Metabolic profiles obtained from potential denitrification rates with 10 electron donors showed that the denitrifying communities altered their preferences for certain compounds after supplementation with methanol or ethanol and that methanol had the greater impact. Clone libraries of nirK and nirS genes, encoding the two different nitrite reductases in denitrifiers, revealed that methanol also increased the diversity of denitrifiers of the nirS type, which indicates that denitrifiers favored by methanol were on the rise in the community. This suggests that there might be a niche differentiation between nirS and nirK genotypes during activated sludge processes. The composition of nirS genotypes also varied greatly among all samples, whereas the nirK communities were more stable. The latter was confirmed by denaturing gradient gel electrophoresis of nirK communities on all sampling occasions. Our results support earlier hypotheses that the compositions of denitrifier communities change during predenitrification processes when external carbon sources are added, although no severe effect could be observed from an operational point of view.     

Metabolic Engineering of Corynebacterium glutamicum for Methanol Metabolism

Methanol is already an important carbon feedstock in the chemical industry, but it has found only limited application in biotechnological production processes. This can be mostly attributed to the inability of most microbial platform organisms to utilize methanol as a carbon and energy source. With the aim to turn methanol into a suitable feedstock for microbial production processes, we engineered the industrially important but nonmethylotrophic bacterium Corynebacterium glutamicum toward the utilization of methanol as an auxiliary carbon source in a sugar-based medium. Initial oxidation of methanol to formaldehyde was achieved by heterologous expression of a methanol dehydrogenase from Bacillus methanolicus, whereas assimilation of formaldehyde was realized by implementing the two key enzymes of the ribulose monophosphate pathway of Bacillus subtilis: 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase. The recombinant C. glutamicum strain showed an average methanol consumption rate of 1.7 ± 0.3 mM/h (mean ± standard deviation) in a glucose-methanol medium, and the culture grew to a higher cell density than in medium without methanol. In addition, [¹³C]methanol-labeling experiments revealed labeling fractions of 3 to 10% in the m + 1 mass isotopomers of various intracellular metabolites. In the background of a C. glutamicum Δald ΔadhE mutant being strongly impaired in its ability to oxidize formaldehyde to CO₂, the m + 1 labeling of these intermediates was increased (8 to 25%), pointing toward higher formaldehyde assimilation capabilities of this strain. The engineered C. glutamicum strains represent a promising starting point for the development of sugar-based biotechnological production processes using methanol as an auxiliary substrate.     

Towards a more plant physiological perspective on soil ecology

Soil respiration almost balances carbon fixation by terrestrial photosynthesis and exceeds all anthropogenic carbon emissions by an order of magnitude, yet we lack precise knowledge of the sources of, and controls upon, the release of carbon dioxide from soils. Here, we discuss the increasing evidence that half of this carbon release is from living plant roots, their mycorrhizal fungi and other root-associated microbes, and that this release is driven directly by recent photosynthesis. The new studies challenge the widespread view that soil activity is dominated by decomposer organisms using older detrital material and that root litter inputs equal those of aboveground litter. The new observations emphasize the physiological continuity and dynamic interdependence of the plant-microbe-soil system and highlight the need for closer cooperation between plant and soil scientists.     

Transformation of tetrachloroethylene to trichloroethylene by homoacetogenic bacteria

Abstract Eight homoacetogenic strains of the genera Acetobacterium, Clostridium and Sporomusa were tested for their ability to dechlorinate tetrachloroethylene (perchloroethene, PCE). Of the organisms tested only Sporomusa ovata was able to reductively dechlorinate PCE with methanol as an electron donor. Resting cells of S. ovata reductively dechlorinated PCE at a rate of 9.8 nmol h⁻¹ (mg protein)⁻¹ to trichloroethylene (TCE) as the sole product. The dechlorination activity depended on concomitant acetogenesis from methanol and CO₂. Cell-free extracts of S. ovata, Clostridium formicoaceticum, Acetobacterium woodii , and the methanogenic bacterium Methanolobus tindarius transformed PCE to TCE with Ti(III) or carbon monoxide as electron donors. Corrinoids were shown in S. ovata to be involved in the dechlorination reaction of PCE to TCE as evident from the reversible inhibition with propyl iodide. Rates of dechlorination followed a pseudo-first-order kinetic.     

Growth energetics in the production of bacterial single cell protein from Methanol

Bacteria grown on methanol exhibit a poor efficiency of energy conservation, which is mainly due to the low P/O ratio of 1 associated with methanol oxidation. Thermodynamic considerations indicate that a P/O ratio of at least 2 is possible for this step in substrate oxidation. This low efficiency of energy conservation is reflected in the yield values on methanol, which are very important in the consideration of biomass production from methanol. Unfortunately in continuous culture there is no obvious way to select for organisms with a greater efficiency of energy conservation.