Heike Sträuber

Viability states of bacteria - specific mechanisms of selected probes.

Single cell techniques like flow cytometry combined with viability staining can help to obtain information on viability states of bacteria. Many fluorescent dyes are available for this purpose and can be chosen according to the available excitation source, the species used, and the background of scientific questions and relevant specifications. Within this short overview, we focus on two diverse groups of bacteria: the gram− Escherichia coli and representatives of the gram+ Mycobacterium to demonstrate differences and similarities in dye uptake principles, processing and binding. We call for attention to possible diverse responses of different species to various viability assays. The cell surface structure of bacteria and the chemical properties of fluorescent probes considerably determine the success of a certain staining practice. Particular focus was drawn on analysis of membrane integrity, uptake of substrates and transformation of fluorogenic substrates.

Metabolic and microbial community dynamics during the hydrolytic and acidogenic fermentation in a leach-bed process

BackgroundBiogas production from lignocellulosic feedstock not competing with food production can contribute to a sustainable bioenergy system. The hydrolysis is the rate-limiting step in the anaerobic digestion of solid substrates such as straw. Hence, a detailed understanding of the metabolic processes during the steps of hydrolysis and acidogenesis is required to improve process control strategies.MethodsThe fermentation products formed during the acidogenic fermentation of maize silage as a model substrate in a leach-bed process were determined by gas and liquid chromatography. The bacterial community dynamics was monitored by terminal restriction fragment length polymorphism analysis. The community profiles were correlated with the process data using multivariate statistics.ResultsThe batch process comprised three metabolic phases characterized by different fermentation products. The bacterial community dynamics correlated with the production of the respective metabolites. In phase 1, lactic and acetic acid fermentations dominated. Accordingly, bacteria of the genera Lactobacillus and Acetobacter were detected. In phase 2, the metabolic pathways shifted to butyric acid fermentation, accompanied by the production of hydrogen and carbon dioxide and a dominance of the genus Clostridium. In phase 3, phylotypes affiliated with Ruminococcaceae and Lachnospiraceae prevailed, accompanied by the formation of caproic and acetic acids, and a high gas production rate.ConclusionsA clostridial butyric type of fermentation was predominant in the acidogenic fermentation of maize silage, whereas propionic type fermentation was marginal. As the metabolite composition resulting from acidogenesis affects the subsequent methanogenic performance, process control should focus on hydrolysis/acidogenesis when solid substrates are digested.

Evaluation of stable isotope fingerprinting techniques for the assessment of the predominant methanogenic pathways in anaerobic digesters

Laboratory biogas reactors were operated under various conditions using maize silage, chicken manure, or distillers grains as substrate. In addition to the standard process parameters, the hydrogen and carbon stable isotopic composition of biogas was analyzed to estimate the predominant methanogenic pathways as a potential process control tool. The isotopic fingerprinting technique was evaluated by parallel analysis of mcrA genes and their transcripts to study the diversity and activity of methanogens. The dominant hydrogenotrophs were Methanomicrobiales, while aceticlastic methanogens were represented by Methanosaeta and Methanosarcina at low and high organic loading rates, respectively. Major changes in the relative abundance of mcrA transcripts were observed compared to the results obtained from DNA level. In agreement with the molecular results, the isotope data suggested the predominance of the hydrogenotrophic pathway in one reactor fed with chicken manure, while both pathways were important in the other reactors. Short-term changes in the isotopic composition were followed, and a significant change in isotope values was observed after feeding a reactor digesting maize silage. This ability of stable isotope fingerprinting to follow short-term activity changes shows potential for indicating process failures and makes it a promising technology for process control.

Prediction of flocculation ability of brewing yeast inoculates by flow cytometry, proteome analysis, and mRNA profiling.

The ability of brewing yeast to flocculate is an important feature for brewing of qualitatively good beer. Flocculation involves two main cell wall structures, which are the flocculation proteins (flocculins) and mannans, to which these flocculins bind. Unfortunately, in practice, the flocculation ability may get lost after several repitches. Flow cytometry was employed to analyze glucose and mannose structures of the cell surface by application of fluorescent lectins. Validation of the expression of the flocculin genes Lg‐FLO1, FLO1, FLO5, and FLO9 was carried out using microarray techniques. SDS‐PAGE, western blot, and ESI‐MS/MS analyses served to isolate and determine yeast cell flocculins. Mannose and glucose labeling with fluorescent lectins allowed differentiating powdery and flocculent yeast cells under laboratory conditions. Using microarray techniques and proteomics, the four flocculation genes Lg‐FLO1, FLO1, FLO5, FLO9, and the protein Lg‐Flo1p were identified as factors of major importance for flocculation. The expression of the genes was several times higher in flocculent yeast cells than in powdery ones. Flow cytometry is a fast and simple method to quantify the proportions of powdery and flocculent yeast cells in suspensions under defined cultivation conditions. However, differentiation under industrial conditions will require mRNA and protein expression profiling.

Population analysis of a binary bacterial culture by multi-parametric flow cytometry.

To study the degradation of a xenobiotic that requires a mixed culture it is essential to monitor the proportions and to control the population dynamics of the component strains. For these purposes fluorochromising techniques and multi-parametric flow cytometry were used to follow Rhodococcus erythropolis K2-3 and Ochrobactrum anthropi K2-14, both of which are needed to degrade 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB). Although the two strains can grow in constant proportions in mixed cultures on other substrates, 2,4-DB could not be degraded as a sole substrate in a continuous process and R. erythropolis K2-3 was clearly impaired in the binary mixture. Addition of a second, easily assimilable substrate (xylitol) in appropriate concentrations (empirically determined) helped this strain survive, and thus facilitated complete degradation of the xenobiotic. This combination of substrates was found to stabilise the growth of R. erythropolis K2-3 and, consequently promoted the action of O. anthropi K2-14. Thus, the two organisms became established in constant proportions in a continuous process until reaching steady state. Consequently, multiplication and cell division activities of the two components of the binary culture were high and reached similar values to those attained when they are grown in pure culture.

Optimization of hydrolysis and volatile fatty acids production from sugarcane filter cake: Effects of urea supplementation and sodium hydroxide pretreatment.

Different methods for optimization the anaerobic digestion (AD) of sugarcane filter cake (FC) with a special focus on volatile fatty acids (VFA) production were studied. Sodium hydroxide (NaOH) pretreatment at different concentrations was investigated in batch experiments and the cumulative methane yields fitted to a dual-pool two-step model to provide an initial assessment on AD. The effects of nitrogen supplementation in form of urea and NaOH pretreatment for improved VFA production were evaluated in a semi-continuously operated reactor as well. The results indicated that higher NaOH concentrations during pretreatment accelerated the AD process and increased methane production in batch experiments. Nitrogen supplementation resulted in a VFA loss due to methane formation by buffering the pH value at nearly neutral conditions (∼ 6.7). However, the alkaline pretreatment with 6g NaOH/100g FCFM improved both the COD solubilization and the VFA yield by 37%, mainly consisted by n-butyric and acetic acids.

Improved Anaerobic Fermentation of Wheat Straw by Alkaline Pre-Treatment and Addition of Alkali-Tolerant Microorganisms

The potential of two alkali-tolerant, lignocellulolytic environmental enrichment cultures to improve the anaerobic fermentation of Ca(OH)2-pre-treated wheat straw was studied. The biomethane potential of pre-treated straw was 36% higher than that of untreated straw. The bioaugmentation of pre-treated straw with the enrichment cultures did not enhance the methane yield, but accelerated the methane production during the first week. In acidogenic leach-bed fermenters, a 61% higher volatile fatty acid (VFA) production and a 112% higher gas production, mainly CO2, were observed when pre-treated instead of untreated straw was used. With one of the two enrichment cultures as the inoculum, instead of the standard inoculum, the VFA production increased by an additional 36% and the gas production by an additional 110%, again mainly CO2. Analysis of the microbial communities in the leach-bed processes revealed similar bacterial compositions in the fermenters with pre-treated straw, which developed independently of the used inoculum. It was suggested that the positive metabolic effects with the enrichment cultures observed in both systems were due to initial activities of the alkali-tolerant microorganisms tackling the alkaline conditions better than the standard inocula, whereas the latter dominated in the long term.

Trace Elements Induce Predominance among Methanogenic Activity in Anaerobic Digestion

Trace elements (TE) play an essential role in all organisms due to their functions in enzyme complexes. In anaerobic digesters, control, and supplementation of TEs lead to stable and more efficient methane production processes while TE deficits cause process imbalances. However, the underlying metabolic mechanisms and the adaptation of the affected microbial communities to such deficits are not yet fully understood. Here, we investigated the microbial community dynamics and resulting process changes induced by TE deprivation. Two identical lab-scale continuous stirred tank reactors fed with distiller’s grains and supplemented with TEs (cobalt, molybdenum, nickel, tungsten) and a commercial iron additive were operated in parallel. After 72 weeks of identical operation, the feeding regime of one reactor was changed by omitting TE supplements and reducing the amount of iron additive. Both reactors were operated for further 21 weeks. Various process parameters (biogas production and composition, total solids and volatile solids, TE concentration, volatile fatty acids, total ammonium nitrogen, total organic acids/alkalinity ratio, and pH) and the composition and activity of the microbial communities were monitored over the total experimental time. While the methane yield remained stable, the concentrations of hydrogen sulfide, total ammonia nitrogen, and acetate increased in the TE-depleted reactor compared to the well-supplied control reactor. Methanosarcina and Methanoculleus dominated the methanogenic communities in both reactors. However, the activity ratio of these two genera was shown to depend on TE supplementation explainable by different TE requirements of their energy conservation systems. Methanosarcina dominated the well-supplied anaerobic digester, pointing to acetoclastic methanogenesis as the dominant methanogenic pathway. Under TE deprivation, Methanoculleus and thus hydrogenotrophic methanogenesis was favored although Methanosarcina was not overgrown by Methanoculleus. Multivariate statistics revealed that the decline of nickel, cobalt, molybdenum, tungsten, and manganese most strongly influenced the balance of mcrA transcripts from both genera. Hydrogenotrophic methanogens seem to be favored under nickel- and cobalt-deficient conditions as their metabolism requires less nickel-dependent enzymes and corrinoid cofactors than the acetoclastic and methylotrophic pathways. Thus, TE supply is critical to sustain the activity of the versatile high-performance methanogen Methanosarcina.

Evidence of Cytochrome P450-Catalyzed Cleavage of the Ether Bond of Phenoxybutyrate Herbicides in Rhodococcus Erythropolis K2-3

Bacterial strain Rhodococcus erythropolis K2-3 can cleave theether bond of the phenoxybutyrate herbicides, i.e., 4-(2,4-dichlorophenoxy)butyrate(2,4-DB) and 4-(4-chloro-2-methylphenoxy)butyrate (MCPB), by anenzyme system that is constitutively expressed. The enzyme(s) involved were investigated in this study. The rate ofdisappearance of 2,4-DB determined in a whole cell assay amounted to0.6 mmol/h ¶ gdry mass.Carbon monoxide difference spectra of dithionite-reduced wholecells and crude cell extracts suggested that strain K2-3 contains a soluble cytochrome P450(P450), named P450PB-1. The addition of various phenoxybutyrate substrates to crude cell extracts resulted in typical difference spectra following the type I pattern ofsubstrate binding with P450. The rate of 2,4-DB cleavage was reduced by inhibitors of P450: 5 mM metyrapone and carbon monoxide at a CO/O2 ratio of 10 reduced the activity by about 20%, and 70%, respectively. The ether cleaving activity completely disappearedafter disruption of the cells and could not be detected in crude extracts. To elucidate theenzymatic basis of this reaction, P450 was partially purified. With the resulting enzyme preparation,2,4-DB cleavage activity was re-established, becoming measurable after the addition of eitherphenazine methosulfate or ferredoxin and ferredoxin/NADP oxidoreductase from spinach. We detected no activities attributable to α-ketoglutarate-dependent dioxygenase orNAD(P)H-dependent monooxygenase. These results collectively indicatethat cleavage of the ether bond of phenoxybutyrate herbicides is catalyzed by P450-mediated activityin this strain. One of the products derived from this reaction is dichlorophenol, and comparativechromatographic analyses suggest that the other product is a C4-carbonicacid, most likely succinic semialdehyde/succinate.

Biogas production from coumarin-rich plants--inhibition by coumarin and recovery by adaptation of the bacterial community.

Plants like sweet clover (Melilotus spp.) are not suitable as fodder for cattle because of harmful effects of the plant secondary metabolite coumarin. As an alternative usage, the applicability of coumarin-rich plants as substrates for biogas production was investigated. When coumarin was added to continuous fermentation processes codigesting grass silage and cow manure, it caused a strong inhibition noticeable as decrease of biogas production by 19% and increase of metabolite concentrations to an organic acids/alkalinity ratio higher than 0.3(gorganic acids) gCaCO3 (-1). Microbial communities of methanogenic archaea were dominated by the genera Methanosarcina (77%) and Methanoculleus (11%). This community composition was not influenced by coumarin addition. The bacterial community analysis unraveled a divergence caused by coumarin addition correlating with the anaerobic degradation of coumarin and the recovery of the biogas process. As a consequence, biogas production resumed similar to the coumarin-free control with a biogas yield of 0.34 LN g(volatile solids) (-1) and at initial metabolite concentrations (∼ 0.2 g(organic acids) gCaCO3 (-1)). Coumarin acts as inhibitor and as substrate during anaerobic digestion. Hence, coumarin-rich plants might be suitable for biogas production, but should only be used after adaptation of the microbial community to coumarin.