Search

Introduction: Attenuated total reflection (ATR)–Fourier transform infrared (FTIR) analysis is a rapid tool and represents a snapshot of all molecules present in a (plant) sample. Most alternative techniques for biochemical analyses of plant biomass require destructive sampling, complex and laborious sample pre-treatment, and precise and costly analysis. These analyses are often limited to soluble compounds instead of all compounds present. Such complicated procedures are not efficient for manipulative studies that involve repeated sampling and rapid nutrient changes over time, such as in agro-industrial cultivation studies.Methods: In our study, the green seaweed species Ulva laetevirens (Chlorophyta) was cultivated under different nutritional regimes in onshore cultivation tanks. The regimes were nitrogen and phosphorus repletion, nitrogen depletion, phosphorus depletion, and light limitation. Samples were taken and tested according to common laborious analysis methods to determine the biochemical composition of polysaccharides, proteins, carbon, and nitrogen. These results were compared with the potential of ATR-FTIR spectroscopy combined with multivariate analysis to allow for prediction of biomass composition.Results: Statistical analysis of the spectra showed that the samples were clustered according to the nutritional regime during the incubation of U. laetevirens. This made it possible to deduce which abiotic factors were replete or deplete during cultivation. Furthermore, partial least square regression analysis proved the most suitable method to predict carbohydrate concentration and nitrogen content present in the biomass.Discussion/conclusion: On the basis of these findings, it is concluded that ATR-FTIR spectroscopy is an efficient and rapid alternative tool for qualitative and quantitative determination of the biochemical composition of U. laetevirens that can be used in industrial cultivation setups.

Rhodomonas salina is a microalgal species, belonging to the cryptophytes, and is widely used as aquaculture feed because of its high nutritional profile and phycoerythrin content. This study investigated the effect of pH on the growth, biochemical composition, and taste of R. salina when cultivated on a semi-large scale under natural light conditions. Two tubular photobioreactors (200 L) were used for the cultivation of R. salina with sunlight as the only illumination source. Two different pH setpoints were applied, 7 and 8.5. Optimal temperature and nutrient conditions were applied, according to previous research findings. The results demonstrated that the productivity of R. salina was higher at pH 7, 0.06–0.14 gdry weight L−1 day−1, compared to pH 8.5, 0.03–0.12 gdry weight L−1 day−1. It was found that protein and total fatty acid concentrations were higher in the biomass that was produced at pH 8.5, 33.7% and 12.3% of dry weight, respectively, while at pH 7, the protein content was 31.9% and the total fatty acids 8.8% of dry weight. The phycoerythrin concentration, like protein, was higher at pH 8.5, 2.7% of dry weight, compared to pH 7, 1% of dry weight. The free amino acid and nucleotide profile of R. salina was affected by the pH, resulting in increased equivalent umami concentration at pH 7. For the sensory evaluation, an expert panel on algae flavors evaluated the effect of pH on the taste of R. salina, reporting that the biomass that was produced at pH 7 had more umami flavor than the biomass that was produced at pH 8.5, which was evaluated as more bitter.

Protein hydrolysates from lesser mealworm (Alphitobius diaperinus, LM) were obtained by enzymatic hydrolysis with protease from Bacillus licheniformis. A preliminary test performed for five hours of hydrolysis generated an insect protein hydrolysate with 15% of degree of hydrolysis (DH), optimum solubility property and oil holding capacity, but emulsifying and foaming ability were completely impaired. In order to investigate the potential implication of DH on techno-functional properties, a set of protein hydrolysates with a different DH was obtained by sub-sampling at different time points during three hours of enzymatic hydrolysis process. An increase in DH% had positive effects on the solubility property and oil holding ability, while a reduced emulsifying ability was observed up to five hours of hydrolysis. These results demonstrated that the enzymatic hydrolysis, if performed under controlled conditions and not for a long period, represents a valid method to extract high quality protein from insects with tailored techno-functionality, in order to produce tailored ingredients for feed and food purpose.

Insect proteins have been proposed as a promising alternative for feed and food formulations. In the present work protease-assisted extraction was studied as a way to separate and extract proteins from two different insect species: Alphitobius diaperinus (AD) and Hermetia illucens (HI). The proteolytic activity of seven enzymes (papain, pancreatin, dispase I, pepsin, protease from Bacillus licheniformis, bromelain and trypsin) was evaluated determining the protein extraction yield, the degree of hydrolysis (DH) and the released free amino acids (FAA). Both insects represent an interesting source of proteins, not only for their amount (more than 40% on dry matter) but also for the nutritional value, with essential amino acid profile exceeding the requirements proposed for human nutrition. Enzyme-assisted protein extraction, performed at laboratory scale, gave for HI an average yield of extraction of 71±8% and for AD 67±6%. Hydrolysates produced from HI gave a DH% ranging between 3 to 18%, whereas hydrolysates produced from AD yielded a DH% between 7 to 23%. The protein hydrolysates were composed by peptides and FAA (which accounted for more than 30% of the extracted protein fraction), which were released according to their abundance in initial protein. A moderate correlation between the DH% and the total amount of FAA was found, except for AD hydrolysed with trypsin and HI with papain. Based on these results, the production of hydrolysates was preliminary scaled up in a proof-of-concept experiment, focusing on the most promising insect-enzyme combination. The final product resulted to be rich in protein (60% on dry matter). This work support enzymatic hydrolysis as an effective method to extract and isolate proteins from insects, with minimal sample preparation, tailoring their composition, preserving the nutritional quality, decreasing the risk of allergic reactions and making them more accessible for their future use as feed/food supplements.

Postprint (published version)

Sphingomonas is an organism of major interest for the degradation of organic contaminants in soils and other environments. A medium based on the aminoglycoside antibiotic streptomycin (Sm) was developed, which, together with the yellow pigmentation of Sphingomonas, facilitated the detection, recovery and quantification of culturable Sphingomonas from soils. All 29 previously described bacterial strains belonging to 17 different Sphingomonas species were able to grow on mineral media containing 200 mug ml(-1) streptomycin, showing that the capacity to resist high concentrations of Sm is a common characteristic within Sphingomonas. Incorporation of Sm into the mineral medium led to a significant reduction in the background microbial population and a concomitant 100 times more sensitive detection of Sphingomonas inoculated in non-sterile soil matrices. The Sm-containing medium was used to examine a variety of hydrocarbon-contaminated soils for the presence and biodiversity of Sphingomonas. Incorporation of Sm in the medium led to a significant increase in the number of yellow-pigmented colonies. Comparison of contaminated and non-contaminated soils derived from the same site revealed colonization by culturable yellow-pigmented Sm-resistant bacteria of the polluted location solely. Both yellow and non-yellow-pigmented colonies were purified from plates containing glucose and Sm, and BOX-polymerase chain reaction (PCR) was used to sort out clonally related strains. Representative strains from the major BOX-PCR clusters were identified using FAME and partial 16S rRNA gene sequencing. Forty-eight of 58 Sm-resistant isolates were identified as Sphingomonas sp. Streptomycin-resistant Sphingomonas isolates generated BOX-PCR diversity patterns that were site dependent and represented different species mainly belonging to Sphingomonas subgroups containing species formerly designated as Sphingopyxis and Sphingobium. The ability to degrade phenanthrene was only found in a minorit

Om bioremediatie ook in bodems met weinig biobeschikbare PAK's, dat wil zeggen, verweerde bodems, efficiënter te laten verlopen, wordt gedacht aan stimulatie van bacteriën die via speciale strategieën de biobeschikbaarheid van PAK's kunnen verhogen. Binnen het Europees project BIOSTIMUL worden dergelijke bacteriën bestudeerd en worden methodes uitgetest, zowel op labo- als pilootschaal, om de bacteriën te detecteren en te stimuleren in bodems

The metabolic pathway of the PAH fluorene and the cometabolic pathway of the PAHs phenanthrene, fluoranthene, anthracene and dibenzothiophene in Sphingomonas sp. LB 126 were examined. To our knowledge this is the first study on the cometabolic degradation of the three-ring PAHs phenanthrene, anthracene and the four-ring PAH fluoranthene by a fluorene-utilizing species. Metabolism of fluorene was shown to proceed via the 9-fluorenone pathway to form o-phthalic acid and protocatechuic acid. The cometabolic mono-hydroxylation found for phenanthrene, fluoranthene and anthracene shows similarity with the hydroxylation of fluorene. Several mono- and dihydroxy products and ring-cleavage products were identified for phenanthrene, fluoranthene and anthracene. It appeared that the cometabolism of those three compounds is a non-specific process, in contrast to the metabolism of fluorene. For dibenzothiophene the metabolites dibenzothiophene-5-oxide and dibenzothiophene-5,5 -dioxide were identified; these compounds appeared to be the products of a dead-end pathway. Since apart from dibenzothiophene no metabolites were found in very high concentrations for any of the other substrates, complete degradation is suggested, even for the cometabolic degradation of phenanthrene, fluoranthene and anthracene. (C) 2003 Editions scientifiques et medicales Elsevier SAS. All rights reserved.

Sphingomonas sp. LB126 is able to utilize fluorene as sole source of carbon and energy. In the present study, a mutagenic vector was constructed and a "plasmid rescue" strategy was set up to isolate a 16.5-kb DNA fragment containing genes required for fluorene degradation. A 14.5-kb portion of the cloned DNA was sequenced revealing thirteen open reading frames. Two encoded hypothetical proteins (FldE and FldY) similar to transcriptional regulators and one (ORF360) located on an IS-like element (ISSsp126) encoded a putative transposase. Three other putative proteins (FldB, FldU and FldV) displayed strong similarity with enzymes of the protocatechuate 4,5-degradation pathway utilized by Sphingomonaspaucimobilis SYK-6 for the degradation of lignin breakdown products. The remaining hypothetical proteins displayed only limited similarity with enzyme sequences available from databases. Suicide plasmid-directed mutagenesis and genetic complementations showed that integrity of the protocatechuate catabolic pathway was an absolute requirement for fluorene degradation to proceed. These findings were further supported by the analysis of metabolites in bacterial culture supernatants obtained from appropriate mutants. The results presented here demonstrated the suitability of the genetic tool constructed and supplied the first genetic evidence for the participation of a protocatechuate 4,5-degradation pathway in a bacterial fluorene degradation pathway.

The promoter probe mini-Tn5-luxAB-tet was used to create a luxAB transcriptional fusion responding to fluorene in the fluorene utilising bacterium Sphingomonas sp. LB126. The mutant strain, named L-132, was impaired in fluorene utilisation and strongly emitted light upon addition of fluorene to the growth medium. L-132 was initially characterised and examined for its potential use as a whole-cell biosensor in the perspective of quantifying fluorene in environmental samples. Activity of the reporter gene as a response to fluorene was detectable after 30 rain and was optimal after 4 h. A linear response to fluorene concentrations within the water solubility range was achieved, with a detection limit of 200 mug per litre. Besides fluorene, L-132 weakly responded to the polycyclic aromatic hydrocarbons phenanthrene and dibenzothiophene, whereas strong responses were obtained with 9-fluorenone, 9-hydroxyfluorene, phthalic acid and protocatechuic acid. The latter four compounds are metabolites formed in course of fluorene degradation, which suggested that a fluorene metabolite rather than fluorene itself was the true inducer of the luxAB fusion in L-132. (C) 2001 Editions scientifiques et medicales Elsevier SAS.

Two different procedures were compared to isolate polycyclic aromatic hydrocarbon (PAH)-utilizing bacteria from PAH-contaminated soil and sludge samples, i.e., (i) shaken enrichment cultures in liquid mineral medium in which PAHs were supplied as crystals and (ii) a new method in which PAH degraders were enriched on and recovered from hydrophobic membranes containing sorbed PAHs. Both techniques were successful, but selected from the same source different bacterial strains able to grow on PAHs as the sole source of carbon and energy. The liquid enrichment mainly selected for Sphingomonas spp,, whereas the membrane method exclusively led to the selection of Mycobacterium spp. Furthermore, in separate membrane enrichment set-ups with different membrane types, three repetitive extragenic palindromic PCR-related Mycobacterial strains were recovered. The new Mycobacterium isolates were strongly hydrophobic and displayed the capacity to adhere strongly to different surfaces. One strain, Mycobacterium sp. LB501T, displayed an unusual combination of high adhesion efficiency and an extremely high negative charge. This strain may represent a new bacterial species as suggested by 16S rRNA gene sequence analysis. These results indicate that the provision of hydrophobic sorbents containing sorbed PAHs in the enrichment procedure discriminated in favor of certain bacterial characteristics. The new isolation method is appropriate to select for adherent PAM-degrading bacteria, which might be useful to biodegrade sorbed PAHs in soils and sludge.