Serge Hiligsmann

Fermentative hydrogen production from glucose and starch using pure strains and artificial co-cultures ofClostridium spp.

BackgroundPure bacterial strains give better yields when producing H2 than mixed, natural communities. However the main drawback with the pure cultures is the need to perform the fermentations under sterile conditions. Therefore, H2 production using artificial co-cultures, composed of well characterized strains, is one of the directions currently undertaken in the field of biohydrogen research.ResultsFour pure Clostridium cultures, including C. butyricum CWBI1009, C. pasteurianum DSM525, C. beijerinckii DSM1820 and C. felsineum DSM749, and three different co-cultures composed of (1) C. pasteurianum and C. felsineum, (2) C. butyricum and C. felsineum, (3) C. butyricum and C. pasteurianum, were grown in 20 L batch bioreactors. In the first part of the study a strategy composed of three-culture sequences was developed to determine the optimal pH for H2 production (sequence 1); and the H2-producing potential of each pure strain and co-culture, during glucose (sequence 2) and starch (sequence 3) fermentations at the optimal pH. The best H2 yields were obtained for starch fermentations, and the highest yield of 2.91 mol H2/ mol hexose was reported for C. butyricum. By contrast, the biogas production rates were higher for glucose fermentations and the highest value of 1.5 L biogas/ h was observed for the co-culture (1). In general co-cultures produced H2 at higher rates than the pure Clostridium cultures, without negatively affecting the H2 yields. Interestingly, all the Clostridium strains and co-cultures were shown to utilize lactate (present in a starch-containing medium), and C. beijerinckii was able to re-consume formate producing additional H2. In the second part of the study the co-culture (3) was used to produce H2 during 13 days of glucose fermentation in a sequencing batch reactor (SBR). In addition, the species dynamics, as monitored by qPCR (quantitative real-time PCR), showed a stable coexistence of C. pasteurianum and C. butyricum during this fermentation.ConclusionsThe four pure Clostridium strains and the artificial co-cultures tested in this study were shown to efficiently produce H2 using glucose and starch as carbon sources. The artificial co-cultures produced H2 at higher rates than the pure strains, while the H2 yields were only slightly affected.

Improving effect of metal and oxide nanoparticles encapsulated in porous silica on fermentative biohydrogen production by Clostridium butyricum.

This paper investigated the enhancement effect of nanometre-sized metallic (Pd, Ag and Cu) or metallic oxide (FexOy) nanoparticles on fermentative hydrogen production from glucose by a Clostridium butyricum strain. These nanoparticles (NP) of about 2-3 nm were encapsulated in porous silica (SiO2) and were added at very low concentration (10(-6) mol L(-1)) in batch hydrogen production test. The cultures containing iron oxide NP produced 38% more hydrogen with a higher maximum H2 production rate (HPR) of 58% than those without NP or with silica particles only. The iron oxide NP were used in a 2.5L sequencing-batch reactor and showed no significant effect on the yields (established at 2.2 mol(hydrogen) mol(glucose)(-1)) but an improvement of the HPR (+113%, reaching a maximum HPR of 86 mL(hydrogen) L(-1) h(-1)). These results suggest an improvement of the electron transfers trough some combinations between enzymatic activity and inorganic materials.

Lack of isocitrate lyase in Chlamydomonas leads to changes in carbon metabolism and in the response to oxidative stress under mixotrophic growth.

Isocitrate lyase is a key enzyme of the glyoxylate cycle. This cycle plays an essential role in cell growth on acetate, and is important for gluconeogenesis as it bypasses the two oxidative steps of the tricarboxylic acid (TCA) cycle in which CO₂ is evolved. In this paper, a null icl mutant of the green microalga Chlamydomonas reinhardtii is described. Our data show that isocitrate lyase is required for growth in darkness on acetate (heterotrophic conditions), as well as for efficient growth in the light when acetate is supplied (mixotrophic conditions). Under these latter conditions, reduced acetate assimilation and concomitant reduced respiration occur, and biomass composition analysis reveals an increase in total fatty acid content, including neutral lipids and free fatty acids. Quantitative proteomic analysis by ¹⁴N/¹⁵N labelling was performed, and more than 1600 proteins were identified. These analyses reveal a strong decrease in the amounts of enzymes of the glyoxylate cycle and gluconeogenesis in parallel with a shift of the TCA cycle towards amino acid synthesis, accompanied by an increase in free amino acids. The decrease of the glyoxylate cycle and gluconeogenesis, as well as the decrease in enzymes involved in β-oxidation of fatty acids in the icl mutant are probably major factors that contribute to remodelling of lipids in the icl mutant. These modifications are probably responsible for the elevation of the response to oxidative stress, with significantly augmented levels and activities of superoxide dismutase and ascorbate peroxidase, and increased resistance to paraquat.

Development of an enzymatic assay for the determination of cellulose bioavailability in municipal solid waste

AbstractAs there is a constant need to assess the biodegradation potential of refuse disposed of in landfills, we have developed a method to evaluate the biodegradability of cellulosic compounds (cellulose and hemicellulose) in municipal solid waste. This test is based on the quantification of monosaccharides released after the hydrolysis of solid waste samples with an optimised enzyme preparation containing commercially available cellulases and hemicellulases. We show that the amounts of monosaccharides could be related to the biodegradability of the cellulosic material contained in the samples. This enzymatic cellulose degradation test was assayed on 37 samples originating from three Belgian landfills and collected at different depths. As results correlated well with those obtained with a classical biochemical methane potential assay, this new and rapid test is sufficiently reliable to evaluate cellulose bioavailability in waste samples.

Comparative study of biological hydrogen production by pure strains and consortia of facultative and strict anaerobic bacteria.

In this paper, a simple and rapid method was developed in order to assess in comparative tests the production of binary biogas mixtures containing CO(2) and another gaseous compound such as hydrogen or methane. This method was validated and experimented for the characterisation of the biochemical hydrogen potential of different pure strains and mixed cultures of hydrogen-producing bacteria (HPB) growing on glucose. The experimental results compared the hydrogen production yield of 19 different pure strains and sludges: facultative and strict anaerobic HPB strains along with anaerobic digester sludges thermally pre-treated or not. Significant yields variations were recorded even between different strains of the same species by i.e. about 20% for three Clostridium butyricum strains. The pure Clostridium butyricum and pasteurianum strains achieved the highest yields i.e. up to 1.36 mol H(2)/mol glucose compared to the yields achieved by the sludges and the tested Escherichia and Citrobacter strains.

Thermophilic and cellulolytic consortium isolated from composting plants improves anaerobic digestion of cellulosic biomass: toward a microbial resource management approach.

A cellulolytic consortium was isolated from a composting plant in order to boost the initial hydrolysis step encountered in anaerobic digestion. Improvement of the cellulose degradation, as well as biogas production, was observed for the cultures inoculated with the exogenous consortium. Metagenomics analyses pointed out a weak richness (related to the number of OTUs) of the exogenous consortium induced by the selective pressure (cellulose as sole carbon source) met during the initial isolation steps. Main microbial strains determined were strictly anaerobic and belong to the Clostridia class. During cellulose anaerobic degradation, pH drop induced a strong modification of the microbial population. Despite the fact that richness and evenness were very weak, the exogenous consortium was able to adapt and to maintain the cellulolytic degradation potential. This important result point out the fact that simplified microbial communities could be used in order to increase the robustness of mixed cultures involved in environmental biotechnology.

Effect of Temperature on Growth of Psychrophilic and Psychrotrophic Members of Rhodotorula aurantiaca

The thermodependence of growth kinetic parameters was investigated for the Antarctic psychrophilic strain Rhodotorula aurantiaca and a psychrotrophic strain of the same species isolated in Belgium (Ardennes area). Cell production, maximum growth rate (μmax), and half-saturation constant for glucose uptake (Ks) of both yeasts were temperature dependent. For the two yeasts, a maximum cell production was observed at about 0°C, and cell production decreased when temperature increased. The μmax values for both strains increased with temperature up to a maximum of 10°C for the psychrophilic strain and 17°C for the psychrotrophic strain. For both yeasts, Ks for glucose was relatively constant at low temperatures. It increased at temperatures above 10°C for the psychrophilic strain and 17°C for the psychrotrophic strain. Although its glucose affinity was lower, the psychrotrophic strain grew more rapidly than the psychrophilicone. The difference in growth rate and substrate affinity was related to the origin of the strain and the adaptation strategy of R. aurantiaca to environmental conditions.

Treatment of gypsum waste in a two stage anaerobic reactor

The reduction of high concentrations of gypsum (up to 110 kg/m 3 ) is investigated in a two stage immobilised cell bioreactor. The first stage is mainly colonised by a consortium of acidogenic bacteria and sulphate reducing bacteria oxidising volatile fatty acids with more than 2 carbons (mainly, butyrate and propionate). The gypsum consumption rate is rather high (11 kg/m 3 .day). Most of acetate remains unconverted in this first stage. It is partially converted in the second stage (residence time : 12 days) which is predominantly colonised by acetate oxidising bacteria. The gypsum consumption rate is much lower than in the first stage: 3 kg/m 3 .day. With both stages, it is possible to reach an almost complete conversion of gypsum with an overall capacity of 6.1 kg gypsum/m 3 .day. We propose also a very simple model to describe the different transformation rates. It allows us to clearly identify the activity levels of the different types of sulphate reducing bacteria in both stages.

Gravimetric water distribution assessment from geoelectrical methods (ERT and EMI) in municipal solid waste landfill

The gravimetric water content of the waste material is a key parameter in waste biodegradation. Previous studies suggest a correlation between changes in water content and modification of electrical resistivity. This study, based on field work in Mont-Saint-Guibert landfill (Belgium), aimed, on one hand, at characterizing the relationship between gravimetric water content and electrical resistivity and on the other hand, at assessing geoelectrical methods as tools to characterize the gravimetric water distribution in a landfill. Using excavated waste samples obtained after drilling, we investigated the influences of the temperature, the liquid phase conductivity, the compaction and the water content on the electrical resistivity. Our results demonstrate that Archies law and Campbells law accurately describe these relationships in municipal solid waste (MSW). Next, we conducted a geophysical survey in situ using two techniques: borehole electromagnetics (EM) and electrical resistivity tomography (ERT). First, in order to validate the use of EM, EM values obtained in situ were compared to electrical resistivity of excavated waste samples from corresponding depths. The petrophysical laws were used to account for the change of environmental parameters (temperature and compaction). A rather good correlation was obtained between direct measurement on waste samples and borehole electromagnetic data. Second, ERT and EM were used to acquire a spatial distribution of the electrical resistivity. Then, using the petrophysical laws, this information was used to estimate the water content distribution. In summary, our results demonstrate that geoelectrical methods represent a pertinent approach to characterize spatial distribution of water content in municipal landfills when properly interpreted using ground truth data. These methods might therefore prove to be valuable tools in waste biodegradation optimization projects.

Determination of Zinc, Cadmium and Lead Bioavailability in Contaminated Soils at the Single-Cell Level by a Combination of Whole-Cell Biosensors and Flow Cytometry

Zinc, lead and cadmium are metallic trace elements (MTEs) that are widespread in the environment and tend to accumulate in soils because of their low mobility and non-degradability. The purpose of this work is to evaluate the applicability of biosensors as tools able to provide data about the bioavailability of such MTEs in contaminated soils. Here, we tested the genetically-engineered strain Escherichia coli pPZntAgfp as a biosensor applicable to the detection of zinc, lead and cadmium by the biosynthesis of green fluorescent protein (GFP) accumulating inside the cells. Flow cytometry was used to investigate the fluorescence induced by the MTEs. A curvilinear response to zinc between 0 and 25 mg/L and another curvilinear response to cadmium between 0 and 1.5 mg/L were highlighted in liquid media, while lead did not produce exploitable results. The response relating to a Zn2+/Cd2+ ratio of 10 was further investigated. In these conditions, E. coli pPZntAgfp responded to cadmium only. Several contaminated soils with a Zn2+/Cd2+ ratio of 10 were analyzed with the biosensor, and the metallic concentrations were also measured by atomic absorption spectroscopy. Our results showed that E. coli pPZntAgfp could be used as a monitoring tool for contaminated soils being processed.