Josef Winter

Extraction of chitin from mechanically pre-purified shrimp shells can be achieved by successive NaOH/HCl treatment, protease/HCl treatment or by environmentally friendly fermentation with proteolytic/lactic acid bacteria (LAB). For the last mentioned alternative, scale-up of shrimp shell chitin purification was investigated in 0.25 L (F1), 10 L (F2), and 300 L (F3) fermenters using an anaerobic, chitinase-deficient, proteolytic enrichment culture from ground meat for deproteination and a mixed culture of LAB from bio-yoghurt for decalcification. Protein removal in F1, F2, and F3 proceeded in parallel within 40 h at an efficiency of 89–91 %. Between 85 and 90 % of the calcit was removed from the shells by LAB in another 40 h in F1, F2, and F3. After deproteination of shrimp shells in F3, spent fermentation liquor was re-used for a next batch of 30-kg shrimp shells in F4 (300 L) which eliminated 85.5 % protein. The purity of the resulting chitin was comparable in F1, F2, F3, and F4. Viscosities of chitosan, obtained after chitin deacetylation and of chitin, prepared biologically or chemically in the laboratory, were much higher than those of commercially available chitin and chitosan.

Propionate is the most delicate intermediate during anaerobic digestion as its degradation is thermodynamically unfavorable. To determine its maximum possible degradation rates during anaerobic digestion, a reactor was fed Monday to Friday with an organic loading rate (OLR) of 12/14 kg COD_biowaste·m⁻³·d⁻¹ plus propionate up to a final OLR of 18 kg COD·m⁻³·d⁻¹. No feed was supplied on weekends as it was the case in full-scale. To maintain permanently high propionate oxidizing activity (POA), a basic OLR of 3 kg COD_propionate·m⁻³·d⁻¹ all week + 11 kg COD_biowaste·m⁻³·d⁻¹ from Monday to Friday was supplied. Finally a reactor was operated with an OLR of 12 kg COD_biowaste·m⁻³·d⁻¹ from Monday to Friday and 5 kg COD_propionate·m⁻³·d⁻¹ from Friday night to Monday morning to maintain a constant gas production for permanent operation of a gas engine. The propionate degradation rates (PDRs) were determined for biowaste + propionate feeding. Decreasing PDRs during starvation were analyzed. The POA was higher after propionate supply than after biowaste feeding and decreased faster during starvation of a propionate-fed rather than a biowaste-fed inoculum. Shifts of the propionate-oxidizing and methanogenic community were determined.

Background: After cellulose and starch, chitin is the third-most abundant biopolymer on earth. Chitin or its deacetylated derivative chitosan is a valuable product with a number of applications. It is one of the main components of shrimp shells, a waste product of the fish industry. To obtain chitin from Penaeus monodon, wet and dried shrimp shells were deproteinated with two specifically enriched proteolytic cultures M1 and M2 and decalcified by in-situ lactic acid forming microorganisms. The viscosity of biologically processed chitin was compared with chemically processed chitin. The former was further investigated for purity, structure and elemental composition by several microscopic techniques and ¹³C solid state NMR spectroscopy. Results: About 95% of the protein of wet shrimp shells was removed by proteolytic enrichment culture M2 in 68 h. Subsequent decalcification by lactic acid bacteria (LAB) took 48 h. Deproteination of the same amount of dried shrimps that contained a 3 × higher solid content by the same culture was a little bit faster and was finished after 140 h. The viscosity of chitin was in the order of chemically processed chitin > bioprocessed chitin > commercially available chitin. Results revealed changes in fine structure and chemical composition of the epi-, exo- and endocuticle of chitin from shrimp shells during microbial deproteination and demineralization. From transmission electron microscopy (TEM) overlays and electron energy loss spectroscopy (EELS) analysis, it was found that most protein was present in the exocuticle, whereas most chitin was present in the endocuticle. The calcium content was higher in the endocuticle than in the exocuticle.¹³C solid state NMR spectra of different chitin confirmed < 3% impurities in the final product. Conclusions: Bioprocessing of shrimp shell waste resulted in a chitin with high purity. Its viscosity was higher than that of commercially available chitin but lower than that of chemically prepared chitin in our lab. Nevertheless, the biologically processed chitin is a promising alternative for less viscous commercially available chitin. Highly viscous chitin could be generated by our chemical method. Comprehensive structural analyses revealed the distribution of the protein and Ca matrix within the shrimp shell cuticle which might be helpful in developing shrimp waste processing techniques.

A mixed culture from an anaerobic biowaste digester was enriched on propionate and used to investigate interspecies hydrogen transfer in dependence of spatial distances between propionate degraders and methanogens. From 20.3 mM propionate, 20.8 mM acetate and 15.5 mM methane were formed. Maximum specific propionate oxidation and methane formation rates were 49 and 23 mmol mg⁻¹ day⁻¹, respectively. Propionate oxidation was inhibited by only 20 mM acetate by about 50 %. Intermediate formate formation during inhibited methanogensis was observed. The spatial distribution and the biovolume fraction of propionate degraders and of methanogens in relation to the total population during aggregate formation were determined. Measurements of interbacterial distances were conducted with fluorescence in situ hybridization by application of group-specific 16S rRNA-targeted probes and 3D image analyses. With increasing incubation time, floc formation and growth up to 54 μm were observed. Propionate degraders and methanogens were distributed randomly in the flocs. The methanogenic biovolume fraction was high at the beginning and remained constant over 42 days, whereas the fraction of propionate degraders increased with time during propionate feeding. Interbacterial distances between propionate degraders and methanogens decreased with time from 5.30 to 0.29 μm, causing an increase of the maximum possible hydrogen flux from 1.1 to 10.3 nmol ml⁻¹ min⁻¹. The maximum possible hydrogen flux was always higher than the hydrogen formation and consumption rate, indicating that reducing the interspecies distance by aggregation is advantageous in complex ecosystems.

Background: Selenium (Se) is an essential trace element, but is toxic at high concentrations. Depending upon the geological background, the land use or on anthropogenic pollution, different amounts of Se may be present in soil. Its toxicity is related to the oxyanions selenate and selenite as they are water soluble and bioavailable. Microorganisms play an important role in Se transformations in soil and its cycling in the environment by transforming water-soluble oxyanions into water insoluble, non-toxic elemental Se (0). For this study, soil samples were collected from selenium-contaminated agricultural soils of Punjab/India to enrich and isolate microbes that interacted with the Se cycle. Results: A mixed microbial culture enriched from the arable soil of Punjab could reduce 230 mg/l of water soluble selenite to spherical Se (0) nanoparticles during aerobic growth as confirmed by SEM-EDX. Four pure cultures (C 1, C 4, C 6, C 7) of Gram negative, oxidase and catalase positive, aerobic bacteria were isolated from this mixed microbial consortium and identified by 16 S rDNA gene sequence alignment as two strains of Duganella sp. (C 1, C 4) and two strains of Agrobacterium sp.(C 6, C 7). SEM/TEM-EDX analyses of the culture broth of the four strains revealed excretion of uniformly round sharply contoured Se (0) nanoparticles by all cultures. Their size ranged from 140–200 nm in cultures of strains C 1 and C 4, and from 185–190 nm in cultures of strains C 6 and C 7. Both Duganella sp. revealed better selenite reduction efficiencies than the two Agrobacterium sp. Conclusions: This is the first study reporting the capability of newly isolated, aerobically growing Duganella sp. and Agrobacterium sp. from soils of Punjab/India to form spherical, regularly formed Se (0) nanoparticles from water soluble selenite. Among others, the four strains may significantly contribute to the biogeochemical cycling of Se in soil. Bioconversion of toxic selenite to non-toxic Se (0) nanoparticles under aerobic conditions in general may be useful for detoxification of agricultural soil, since elemental Se may not be taken up by the roots of plants and thus allow non-dangerous fodder and food production on Se-containing soil.