Defined Nutrient Medium Research Articles

Microbial community development during syngas methanation in a trickle bed reactor with various nutrient sources

Microbial community development within an anaerobic trickle bed reactor (TBR) during methanation of syngas (56% H2, 30% CO, 14% CO2) was investigated using three different nutrient media: defined nutrient medium (241 days), diluted digestate from a thermophilic co-digestion plant operating with food waste (200 days) and reject water from dewatered digested sewage sludge at a wastewater treatment plant (220 days). Different TBR operating periods showed slightly different performance that was not clearly linked to the nutrient medium, as all proved suitable for the methanation process. During operation, maximum syngas load was 5.33 L per L packed bed volume (pbv) & day and methane (CH4) production was 1.26 L CH4/Lpbv/d. Microbial community analysis with Illumina Miseq targeting 16S rDNA revealed high relative abundance (20–40%) of several potential syngas and acetate consumers within the genera Sporomusa, Spirochaetaceae, Rikenellaceae and Acetobacterium during the process. These were the dominant taxa except in a period with high flow rate of digestate from the food waste plant. The dominant methanogen in all periods was a member of the genus Methanobacterium, while Methanosarcina was also observed in the carrier community. As in reactor effluent, the dominant bacterial genus in the carrier was Sporomusa. These results show that syngas methanation in TBR can proceed well with different nutrient sources, including undefined medium of different origins. Moreover, the dominant syngas community remained the same over time even when non-sterilised digestates were used as nutrient medium.Key points•Independent of nutrient source, syngas methanation above 1 L/Lpbv/D was achieved.•Methanobacterium and Sporomusa were dominant genera throughout the process.•Acetate conversion proceeded via both methanogenesis and syntrophic acetate oxidation.Graphical abstract

Long-term biological hydrogen production by agar immobilized Rhodobacter capsulatus in a sequential batch photobioreactor.

In this study, agar immobilization technique was employed for biological hydrogen production using Rhodobacter capsulatus DSM 1710 (wild type) and YO3 (hup-mutant) strains in sequential batch process. Different agar and glutamate concentrations were tested with defined nutrient medium. Agar concentration 4% (w/v) and 4mM glutamate were selected for bacterial immobilization in terms of rate and longevity of hydrogen production. Acetate concentration was increased from 40 to 60-100 and 60mM gave best results with both bacterial strains immobilized in 4% (w/v) agar. Cell concentration was increased from 2.5 to 5mg dcwmL-1 agar and it was found that increasing cell concentration of wild-type strain caused decrease in yield and productivity while these parameters improved by increasing cell concentration of mutant strain. Also, the hydrogen production time has extended from 17days up to 60days according to the process conditions and parameters. Hydrogen production by immobilized photosynthetic bacteria is a convenient technology for hydrogen production as it enables to produce hydrogen with high organic acid concentrations comparing to suspended cultures. Besides, immobilization increases the stability of the system and allowed sequential batch operation for long-term application.

Discovery of a Unique Extracellular Polysaccharide in Members of the Pathogenic Bacillus That Can Co-form with Spores

An exopolysaccharide, produced during the late stage of stationary growth phase, was discovered and purified from the culture medium of Bacillus cereus, Bacillus anthracis, and Bacillus thuringiensis when strains were grown in a defined nutrient medium that induces biofilm. Two-dimensional NMR structural characterization of the polysaccharide, named pzX, revealed that it is composed of an unusual three amino-sugar sequence repeat of [-3)XylNAc4OAc(α1-3)GlcNAcA4OAc(α1-3)XylNAc(α1-]n The sugar residue XylNAc had never been described previously in any glycan structure. The XNAC operon that contains the genes for the assembly of pzX is also unique and so far has been identified only in members of the Bacillus cereus sensu lato group. Microscopic and biochemical analyses indicate that pzX co-forms during sporulation, so that upon the release of the spore to the extracellular milieu it becomes surrounded by pzX. The relative amounts of pzX produced can be manipulated by specific nutrients in the medium, but rich medium appears to suppress pzX formation. pzX has the following unique characteristics: a surfactant property that lowers surface tension, a cell/spore antiaggregant, and an adherence property that increases spores binding to surfaces. pzX in Bacillus could represent a trait shared by many spore-producing microorganisms. It suggests pzX is an active player in spore physiology and may provide new insights to the successful survival of the B. cereus species in natural environments or in the hosts.

Evolution alters ecological mechanisms of coexistence in experimental microcosms

Summary Species coexistence can be promoted by both equalizing mechanisms that increase fitness equivalence among species and stabilizing mechanisms that decrease niche overlap among species. Strength of the coexistence mechanisms can be altered by evolution. In particular, populations evolved in sympatry may show niche divergence due to competition‐mediated selection, whereas populations evolved in allopatry have a chance to become ecologically equivalent competitors which, on secondary contacts, may form ‘neutral communities’. We addressed how evolution may change ecological mechanisms of coexistence with experimental microcosms of Escherichia coli strains that reproduce asexually and can be considered as analogues of species. We obtained five pairs of bacterial strains, within each of which the two strains could coexist stably. We then allowed the bacterial strains to evolve in a chemically defined nutrient medium for ˜1100 generations, under sympatric (in two‐strain mixtures) or allopatric scenarios (in monocultures). The strength of coexistence mechanisms was assessed based on reciprocal invasion assays and measurements of environmental carrying capacity. Our data confirmed that coexistence could be weaken by either fitness difference or niche overlap among competitors. In allopatrically evolved strain pairs, fitness difference among competitors became larger or unchanged; and the magnitude of niche overlap often showed an increase rather than a decline. Sympatrically evolved competitors showed no consistent changing trend in the strength of the coexistence mechanisms. Overall, sympatrically evolved competitor pairs did not differ from allopatrically evolved ones in the magnitude of fitness difference, but had lower levels of niche overlap. The results are consistent with the ‘character displacement’ view that allopatric populations of competing species occupy more similar niche space compared with sympatric populations. However, the pattern observed here resulted from allopatrically evolved competitors showing niche convergence, but not sympatrically evolved competitors consistently showing further niche divergence. The results also suggest that convergent evolution in allopatry followed by secondary contacts is an unlikely mechanism for the origin of ‘neutral communities’.

Valorization of Hemicelluloses: Production of Bioxylitol from Poplar Wood Prehydrolyzates by Candida guilliermondii FTI 20037

In this study on the valorization of hemicelluloses (a co-product generated during cellulosic bioethanol production), prehydrolyzates obtained from poplar woodchips pretreated in an industrial experimental steam-explosion pilot-plant facility were evaluated for the production of bioxylitol using the yeast, Candida guilliermondii FTI 20037, employing both batch and fed-batch fermentation modes in shake flasks on defined nutrient medium. The prehydrolyzates consisted of monosaccharides (pentose and hexose sugars) as well as xylo-oligosaccharides and undegraded hemicellulose. Xylose (31.6 ± 0.57 g/L) was the major sugar in the prehydrolyzates that also contained acetic acid and degradation products of lignin and sugars (phenolic and furanic compounds). Xylose in the prehydrolyzates could be further increased (106.4 ± 0.02 g/L) through an acid hydrolysis step (0.6 % (w/v) H2SO4). Compounds of a toxic nature in both the acid hydrolyzates and prehydrolyzates were removed by treatment with Amberlite IRA-400 resin (chloride form). Batch fermentation of pure xylose and poplar prehydrolyzate resulted in bioxylitol production of 9.9 ± 0.01 and 4.9 ± 0.17 g/L, respectively, indicating that the poplar prehydrolyzates exhibited an inhibitory effect on fermentation. After detoxification of the poplar prehydrolyzates, bioxylitol production increased to 8.9 ± 0.01 g/L. Fed-batch fermentation of the prehydrolyzate increased the bioxylitol production to 12.39 ± 0.33 g/L, while acid hydrolysis followed by detoxification resulted in a maximum bioxylitol production of 22.0 ± 0.01 g/L, a 348 % increase. The results demonstrated that acid hydrolysis and detoxification followed by fed-batch fermentation was an efficient way to produce bioxylitol from poplar prehydrolyzates.

Butanol and hexanol production in Clostridium carboxidivorans syngas fermentation: Medium development and culture techniques

Clostridium carboxidivorans was grown on model syngas (CO:H2:CO2 [70:20:10]) in a defined nutrient medium with concentrations of nitrogen, phosphate and trace metals formulated to enhance production of higher alcohols. C. carboxidivorans was successfully grown in a limited defined medium (no yeast extract, no MES buffer and minimal complex chemical inputs) using an improved fermentation protocol. Low partial pressure of CO in the headspace, coupled with restricted mass transfer for CO and H2, was required for successful fermentation. In the absence of substrate inhibition (particularly from CO), growth limitation increased production of alcohols, especially butanol and hexanol. Concentrations of butanol (over 1.0g/L), hexanol (up to 1.0g/L) and ethanol (over 3.0g/L) were achieved in bottle fermentations. Minimal medium and controlled supply of CO and H2 should be used in characterizing candidate butanol and hexanol producing strains to select for commercial potential.

Islet-derived stellate-like cells as a novel source for islet neogenesis in chicks

The therapeutic potential of stem cells has led to renewed interest in regenerative biology. Pancreatic stellate cells have been reported in the mammalian pancreas; however, there are very few reports on stellate cells in the chicken pancreas. The intercalated duct epithelial cells observed in the A and B islets of the chicken pancreas have been claimed to be stellate cells from their morphological appearance. While isolating islets and acinar cells from the chick pancreas, we have found a population of stellate-like cells, which has been successfully propagated in a defined nutrient medium. These cells were immunopositive for vimentin, desmin, and fibronectin and also expressed alkaline phosphatase, indicating their undifferentiated state. On exposure to serum-free medium containing specific nutrients and differentiating agents, these stellate-like-cells gave rise to islet-like cell clusters. Islet-like clusters stained positive for the islet specific stain diphenyl thiocarbazone and were immunopositive for C-peptide indicating de novo insulin synthesis. These clusters secreted insulin in response to glucose challenge, thus suggesting their similarity to islets. Thus stellate cells found in chick pancreatic islets exhibit potential to differentiate into islet-like clusters. Taken together, our study documents for the first time the presence of a stellate-like cell population in chick pancreatic islets providing a source for islet neogenesis.

Nutrient Consumption and Pigmentation of Deep and Surface Colonizing Sapstaining Fungi in Pinus contorta

Summary In this paper, we examined the ability of deep and surface staining fungi to utilize wood tissue nutrients. Fungal isolates were inoculated onto fresh billets and γ-sterilised sawnwood, both from Pinus contorta, and also onto defined nutrient media. The wood samples were assessed for host viability, fungal growth and nutrient status. The results indicated that the most aggressive sapstain species on fresh logs was Ceratocystis coerulescens, followed consecutively by Leptographium spp., Ophiostoma minus, O. piliferum, O. piceae, O. setosum, O. pluriannulatum and Aureobasidium pullulans. HPLC analysis of soluble sugars in fungal-infected wood indicated that mannose was the most depleted sugar, followed by glucose. Lipid analysis of infected wood indicated that Leptographium spp. and C. coerulescens greatly reduced the triglyceride fraction and that there was a wide spectrum of consumption of triglyceridederived fatty acids between the fungi. On defined media, the carbon source mannose led to the darkest pigmentation for all tested fungi. For C. coerulescens, the order of pigmentation intensity for the remaining tested carbon sources was reversed when compared to the other fungal species.

CELL DEATH IN TETRAHYMENA THERMOPHILA: NEW OBSERVATIONS ON CULTURE CONDITIONS

We previously suggested that the cell fate of the protozoan ciliate, Tetrahymena thermophila, effectively relates to a quorum-sensing mechanism where cell-released factors support cell survival and proliferation. The cells have to be present above a critical initial density in a chemically defined nutrient medium in order to release a sufficient level of these factors to allow a new colony to flourish. At a relatively high rate of metabolism and/or macromolecular synthesis and below this critical density, cells began to die abruptly within 30min of inoculation, and this death took the form of an explosive disintegration lasting less than 50 milliseconds. The cells died at any location in the culture, and the frequency of cell death was always lower in well-filled vials than those with medium/air interface. Cell death was inhibited by the addition of Actinomycin D or through modifications of the culture conditions either by reducing the oxygen tension or by decreasing the temperature of the growth medium. In addition, plastic caps in well-filled vials release substances, which promote cell survival. The fate of low-density cultures is related to certain ‘physical’ conditions, in addition to the availability of oxygen within closed culture systems.

Survival and differentiation of purified embryonic chick retinal ganglion cells cultured at low density in a chemically defined medium

Both, a tailored chemically defined nutrient medium (BP5) and a sandwich culture sustain the survival for more than a week and allow the differentiation of embryonic chick retinal ganglion cells (RGCs) seeded at low density. Purification of RGCs from 7–11-day old embryos was accomplished by panning using specific anti-chicken Thy-1 antibodies immobilized in plaques. Yield of RGCs was less than 1% of the calculated number of these cells in the used retinas. This result agrees with the scarce expression of Thy-1 in immature retina; accordingly, the most mature RGCs are those probably selected by the panning. This assumption obtained support on the expression of gangliotetraoxylgangliosides (GTOG), that characterize the differentiated retinal neurons. Thus, the outgrowth of processes observed in cultured cells, might imply axonal regeneration in mature neurons. This manageable RGC culture method approaches a system for studying the in vitro trophic factors and substrata which affect axonal regrowth in central nervous system cells.

Accumulation of sesquiterpenoid cyclohexenone derivatives induced by an arbuscular mycorrhizal fungus in members of the Poaceae

Sixty one members of the Poaceae, including various cereals, were grown in defined nutrient media with and without the arbuscular mycorrhizal (AM) fungus, Glomus intraradices Schenk & Smith. The roots of all species investigated were colonized by the AM fungus, however, to different degrees and independent of their systematic position. High-performance liquid chromatographic analyses of methanolic extracts from the roots of mycorrhizal and nonmycorrhizal species revealed dramatic changes in the patterns of UV-detectable products along with a widespread occurrence of AM-fungus-induced accumulation of sesquiterpenoid cyclohexenone derivatives. The latter occur most often in the tribes Poeae, Triticeae and Aveneae. Some additional control experiments on plant infection with pathogens (Gaeumannomyces graminis) and Drechslera sp.) or an endophyte (Fusarium sp.), as well as application of abiotic stress, proved that the metabolism of these terpenoids is part of a response pattern of many gramineous roots in their specific reaction to AM fungal colonization.

Castanospermine, an HIV inhibitor from tissue cultures of Castanospermum australe

AbstractTree species are important sources of phytotherapeutics. The seeds of the tree Castanospermum australe contain alkaloids effective against HIV. Unorganized callus cultures were established from the leaves of the mature tree on a chemically defined nutrient medium of Murashige and Skoog supplemented with 2,4‐dichlorophenoxyacetic acid and benzyladenine. The alkaloids were separated and identified by gas liquid chromatographic analysis. The cultures synthesized castanospermine (0.004% F.W.) indicating that the biosynthetic ability of the highly differentiated seeds of the tree is expressed in the unorganized cells.

Evaluation of solid, slurry, and submerged fermentations for the production of cyclodextrin glycosyl transferase by Bacillus cereus

Cyclodextrin glycosyl transferase, a multifunctional extracellular enzyme used for the preparation of cyclodextrins, was produced by Bacillus cereus, an indigenous strain. Solid substrate fermentations with wheat bran and slurry fermentations consisting of 10% wheat bran were compared with submerged fermentations using defined nutrient medium. Solid state fermentation displayed the maximum activity of 59 units ml −1 in 24 h when compared to slurry fermentation (58 units ml −1) and submerged fermentation (40 units ml −1). The use of wheat bran enhanced the activity by 50%. The promoting action of wheat bran was evident, with the supplementation of wheat bran extract in submerged fermentation resulting in 2.7-fold (110 units ml −1) higher activity in comparison to submerged fermentation.

Insulin rescues the unicellular eukaryote Tetrahymena from dying in a complete, synthetic nutrient medium

The presence of 10-6 M bovine insulin in chemically defined nutrient medium prevented cell death and improved cell proliferation of the ciliate Tetrahymena pyriformis when inoculated at low initial cell density. The action of insulin was found to be restricted to the 22-30 fragment of the B-chain. These results suggest that small peptides are involved in regulation of cell survival and cell proliferation in Tetrahymena pyriformis.

In vitro sporulation of Frankia strain HFPCcI3 from Casuarina cunninghamiana

Optimization of the in vitro sporulation of Frankia is a prerequisite for the development of spore preparations as a practical inoculum for actinorhizal plants. The in vitro sporulation of Frankia strain HFPCcI3 previously isolated from Casuarina cunninghamiana was increased from 0 to 12 million spores per millilitre of culture medium when nitrogen and phosphorus were excluded from a defined nutrient medium. The same medium did not increase sporulation of Frankia isolates HFPAr13 from Alnus rubra and HFPCpI1 from Comptonia peregrina. The addition of 10 mM aliphatic L amino acids increased the in vitro sporulation of HFPCcI3 by as much as 39% when grown in a defined nutrient medium with 1 mM potassium phosphate lacking ammonium chloride, whereas the addition of 10 mM acidic, basic, aromatic, and sulfur amino acids decreased sporulation. The in vitro sporulation of HFPCcI3 was inhibited by culture at 33 °C relative to culture at 23 and 28 °C. Rotary shaking at 100 rpm increased sporulation at the low culture temperatures. Key words: actinorhizal, Casuarina, Frankia, HFPCcI3, sporulation.

Rhythm generation in brainstem cultures grown in a serum-free medium

Electrophysiological studies were carried out on long term cultured brainstem tissue taken from neonatal rats with the object of investigating mechanisms underlying respiratory rhythm generation. The preparations were derived from 360 microns thick horizontal medullary slices which were explanted into a chemically defined nutrient medium and which remained organotypically intact for ca. 1 month. In 44 of the 50 explants examined both periodic and aperiodic bioelectric activity was detected, the cycle length of the former ranging from 0.5 to 10 s (mean, 2.7 s) at a pH of 7.4 and bath temperature of 32 degrees C. Periodic activity could take several forms, but commonly consisted of regularly repeated, 100-300 ms long, depolarizing (D-) waves or sequences of inhibitory and/or excitatory postsynaptic potentials. Lowering the pH of the superfusate by lowering the bicarbonate concentration, increasing the pCO2 or adding H+ shortened the interval between periodic events, and increased both the amplitude and duration of the D-waves. The interval was also shortened when the bath temperature was increased (Q10: ca.2.5). The mean resting membrane potential of neurons exhibiting periodic activity was -49 mV (n = 62) and not significantly different from that of aperiodically discharging neurons either in the same preparations or in cultured explants from the neocortex. These observations suggest that brainstem cultures constitute a useful 'model' system for studying pH-dependent rhythm generation in small neuronal networks of the medulla.

Physiological Characteristics of Fe Accumulation in the `Bronze' Mutant of Pisum sativum L., cv `Sparkle' E107 (brz brz)

The pea (Pisum sativum L.) mutant, E107 (brz, brz) accumulated extremely high concentrations of Fe in its older leaves when grown in light rooms in either defined nutrient media or potting mix, or outdoors in soil. Leaf symptoms (bronze color and necrosis) were correlated with very high Fe concentrations. When E107 plants were grown in nutrient solutions supplied 10 mum Fe, as the Fe(III)-N,N'-ethylenebis[2-(2-hydroxyphenyl)glycine] chelate, their roots released higher concentrations of Fe(III) reducing substances to the nutrient media than did roots of the normal parent cv, ;Sparkle.' Reciprocal grafting experiments demonstrated that the high concentrations of Fe in the shoot was controlled by the genotype of the root. In short-term (59)Fe uptake studies, 15-day-old E107 seedlings exhibited higher rates of Fe absorption than did ;Sparkle' seedlings under Fe-adequate growth conditions. Iron deficiency induced accelerated short-term Fe absorption rates in both mutant and normal genotypes. Iron-treated E107 roots also released larger amounts of both protons and Fe(III) reductants into their nutrient media than did iron-treated ;Sparkle' roots. Furthermore, the mutant translocated proportionately more Fe to its shoot than did the parent regardless of Fe status.

The cell cycle and its relationship to development in Acanthamoeba Castellanii

ABSTRACT The cell cycle and the relationship between particular cell cycle phases and the differentiation of trophozoites into cysts were reinvestigated in Acanthamoeba castellanii using flow fluorometric measurements of nuclear DNA content and synthesis, and synchronization of cells by release from the stationary phase. The investigation was performed with cultures growing in non-defined medium (ND cells) showing a high degree of encystation in response to starvation, and with subcultures growing in chemically defined nutrient medium (D cells) exhibiting a very low encystation competence. In both cultures the cell cycle starts with a short S phase taking place simultaneously with cytokinesis, followed by a long G2 phase. A G1 phase seems to be either absent or very short. Synchronization experiments reveal that in ND cells encystation is initiated from a particular position of late G2. The high encystation competence of stationary phase ND cells seems to be due to arrest of cells at this particular cell cycle position. The lack of encystation competence of stationary phase D cells correlates with the loss of accumulation of cells at this particular stage of the cell cycle. This change of the property of cells is related to the growth condition and not to an irreversible loss of encystation competence of D cells.

Effect of essential amino acids on the phosphorylation of a 40S ribosomal protein and protein synthesis in Acanthamoeba castellanii.

Reversible and multiple phosphorylation of a 40S ribosomal protein is observed in a variety of eukaryotic cells. In the primitive eukaryote Acanthamoeba, one or three phosphorylated S3 derivatives are observed during growth phase in nondefined nutrient medium (ND cells) or in chemically defined nutrient medium (D cells), respectively. In both cases, stationary phase cells exhibit nonphosphorylated S3; however, transfer of these cells into the respective fresh nutrient media results in a transient accumulation of four phosphorylated S3 derivatives. Transfer of D cells into nutrient medium, deficient in all or any single essential amino acids, leads to reversible inhibition of S3 phosphorylation and growth arrest. The low level of phosphorylated S3 is not simply the consequence of growth arrest, since in cells where growth is arrested differently, the level of phosphorylated S3 can be high. In response to amino acid deficiency, a number of other changes can be observed. These include a 2-3-fold decrease of total protein synthesis, 13 changes in the cellular protein pattern, and specific alterations in the ribosome absorbance profiles and in the distribution of poly-A+ RNA within subribosomal and ribosomal fractions. While the rate of total protein synthesis seems to be associated with the level of phosphorylated S3, the level of the synthesis of at least 10 of the particular proteins can be dissociated from the level of S3 phosphorylation.

Fertilization and seed production with pollen from in-vitro-cultured maize tassels

Immature tassel meristems (1-1.5 cm) of maize (Zea mays L.) explanted to a defined nutrient medium underwent further growth and floral development. Microsporogenesis, gametogenesis and pollen maturation were completed within 25 d in vitro. The pollen, recovered from the cultured tassel, germinated on nutrient agar and also on receptive silks. Viable seed produced from controlled pollinations germinated and grew into mature, normal plants. Thus, a significant component of the life cycle of maize can be completed in vitro where analyses and manipulations are possible for both basic and applied research.