Replacing soybean meal with microalgae biomass in diets with contrasting carbohydrate profile can reduce in vitro methane production and improve short-chain fatty acids production.

The objective of this study was to evaluate the interaction of dietary carbohydrate profile and soybean meal (SBM) replacement with either Chlorella pyrenoidosa (CHL) or Spirulina platensis (SPI) on in vitro fermentation. This experiment was conducted as a randomized complete block design, with fermentation run (3 runs) considered as blocks. The treatments were arranged in a 2 × 5 factorial design, where the first factor was the carbohydrate profile, which was composed of diets containing 42.5% NDF and 26.8% starch (HF-LS) or 26.8% NDF and 40.6% starch (LF-HS) and the second factor was the protein source, in which a control diet (100% SBM), partial replacement of SBM with CHL (1/2 CHL) or SPI (1/2 SPI), or total replacement of SBM with CHL or SPI were used. All experimental diets were formulated to have 17% crude protein. The ruminal fluid was collected from 2 lactating Holstein cows, buffered with Van Soest medium at a ratio of 1:2 and added to serum bottles containing 0.50 g of the experimental diets. Bottles were incubated at 39°C for 24 and 48 h in triplicate; headspace pressure was measured, along with gas collection for methane (CH4) quantification at 0, 2, 4, 8, 16, 24, 36, and 48 h after incubation. The final medium was used to measure pH, ammonia, and volatile-fatty acid (VFA). After incubation, feed bags were recovered and used for estimation of degradability of DMD, NDF, and OMD. Statistical analysis was carried out using the MIXED procedure of SAS, with carbohydrate profile, protein source, assay, and its interactions as fixed effects, with run and bottle as random effects. Orthogonal contrasts were used to compare carbohydrate profile, algae species, carbohydrate profile × algae interaction, and linear and quadratic effects of SBM replacement with CHL or SPI. There was no interaction effect between carbohydrate profile and algae source. LF-HS improved gas production, degradability of nutrients, and VFA, mainly increasing the production of butyrate and propionate. When compared with CHL, SPI had a greater degradability of nutrients and branched VFA, along with reduction in total gas production and tended to reduce total CH4 yield. The replacement of SBM with algae linearly reduced the degradability of nutrients, along with a linear reduction in gas production. When replacement of SBM with only SPI was evaluated, SPI slightly reduced the degradability of nutrients; however, it promoted a linear reduction in CH4 yield, as well as reduction in CH4 yield by unit of degraded DM, NDF, and OM. In summary, there was no interaction of carbohydrate profile and protein source, which means that SBM replacement had a similar effect, regardless of dietary carbohydrate profile. Spirulina may be a more suitable algae source when compared with Chlorella due to the potential to reduce CH4.

Partial replacement of soybean meal with microalgae biomass on in vitro ruminal fermentation may reduce ruminal protein degradation.

The objective of this study was to evaluate the effects of partially replacing soybean meal (SBM) with algal sources on in vitro ruminal fermentation. Using 6 fermenters in a 3 × 3 replicated Latin square with 3 periods of 10 d each, we tested 3 treatments: a control diet (CRT) with SBM at 17.8% of the diet dry matter (DM); and 50% SBM biomass replacement with either Chlorella pyrenoidosa (CHL); or Spirulina platensis (SPI). The basal diet was formulated to meet the requirements of a 680-kg Holstein dairy cow producing 45 kg/d of milk with 3.5% fat and 3% protein. All diets had a similar nutritional composition (16.0% CP; 34.9% NDF; 31.0% starch, DM basis) and fermenters were provided with 106 g DM/d split into 2 portions. After 7 d of adaptation, samples were collected for 3 d of each period for analyses of ruminal fermentation at 0, 1, 2, 4, 6, and 8 h after morning feeding for evaluation of the ruminal fermentation kinetics. For the evaluation of the daily production of total metabolites and for the evaluation of nutrient degradability, samples from the effluent containers were collected daily. Statistical analysis was performed with the MIXED procedure of SAS with treatment, time, and their interactions considered as fixed effects; day, square, and fermenter were considered as random effects. Orthogonal contrasts (CRT vs. algae; and CHL vs. SPI) were used to depict the treatment effect, and significance was declared when P ≤ 0.05. Fermenters that received algae-based diets had a greater propionate molar concentration and molar proportion when compared with the fermenters fed CRT diets. In addition, those algae-fed fermenters had lower branched short-chain fatty acids (BSCFA) and isoacids (IA), which are biomarkers of ruminal protein degradation, along with lower ammonia (NH3-N) concentration and greater nonammonia nitrogen (NAN). When contrasting with fermenters fed SPI-diets, fermenters fed based CHL-diets had a lower molar concentration of BSCFA and IA, along with lower NH3-N concentration and flow, and greater NAN, bacterial nitrogen flow, and efficiency of nitrogen utilization. Those results indicate that CHL protein may be more resistant to ruminal degradation, which would increase efficiency of nitrogen utilization. In summary, partially replacing SBM with algae biomass, especially with CHL, is a promising strategy to improve the efficiency of nitrogen utilization, due to the fact that fermenters fed CHL-based diets resulted in a reduction in BSCFA and IA, which are markers of protein degradation, and it would improve the efficiency of nitrogen utilization. However, further validation using in vivo models are required.

Detection and quantification of functional genes of cellulose- degrading, fermentative, and sulfate-reducing bacteria and methanogenic archaea.

Cellulose degradation, fermentation, sulfate reduction, and methanogenesis are microbial processes that coexist in a variety of natural and engineered anaerobic environments. Compared to the study of 16S rRNA genes, the study of the genes encoding the enzymes responsible for these phylogenetically diverse functions is advantageous because it provides direct functional information. However, no methods are available for the broad quantification of these genes from uncultured microbes characteristic of complex environments. In this study, consensus degenerate hybrid oligonucleotide primers were designed and validated to amplify both sequenced and unsequenced glycoside hydrolase genes of cellulose-degrading bacteria, hydA genes of fermentative bacteria, dsrA genes of sulfate-reducing bacteria, and mcrA genes of methanogenic archaea. Specificity was verified in silico and by cloning and sequencing of PCR products obtained from an environmental sample characterized by the target functions. The primer pairs were further adapted to quantitative PCR (Q-PCR), and the method was demonstrated on samples obtained from two sulfate-reducing bioreactors treating mine drainage, one lignocellulose based and the other ethanol fed. As expected, the Q-PCR analysis revealed that the lignocellulose-based bioreactor contained higher numbers of cellulose degraders, fermenters, and methanogens, while the ethanol-fed bioreactor was enriched in sulfate reducers. The suite of primers developed represents a significant advance over prior work, which, for the most part, has targeted only pure cultures or has suffered from low specificity. Furthermore, ensuring the suitability of the primers for Q-PCR provided broad quantitative access to genes that drive critical anaerobic catalytic processes.

Identification of stress-related proteins in Escherichia coli using the pollutant cis-dichloroethylene.

AIMS: To complement our proteome study, whole-transcriptome analyses were utilized here to identify proteins related to degrading cis-1,2-dichloroethylene (cis-DCE). METHODS AND RESULTS: Metabolically engineered Escherichia coli strains were utilized expressing an evolved toluene ortho-monooxygenase along with either (i) glutathione S-transferase and altered gamma-glutamylcysteine synthetase or (ii) a rationally engineered epoxide hydrolase. cis-DCE degradation induced 30 known stress genes and 32 uncharacterized genes. Because of the reactive cis-DCE epoxides formed, we hypothesized that some of these uncharacterized genes may be related to a variety of stresses. Using isogenic mutants, IbpB, YchH, YdeI, YeaR, YgiW, YoaG and YodD were related to hydrogen peroxide, cadmium and acid stress. Additional whole-transcriptome studies with hydrogen peroxide stress using the most hydrogen peroxide-sensitive mutants, ygiW and ychH, identified that FliS, GalS, HcaR, MglA, SufE, SufS, Tap, TnaB, YhcN and YjaA are also involved in the stress response of E. coli to hydrogen peroxide, cadmium and acid, as well as are involved in biofilm formation. CONCLUSION: Seventeen proteins are involved in the stress network for this organism, and YhcN and YchH were shown to be important for the degradation of cis-DCE. SIGNIFICANCE AND IMPACT OF THE STUDY: Six previously uncharacterized proteins (YchH, YdeI, YgiW, YhcN, YjaA and YodD) were shown to be stress proteins.

Comparison of microbial community composition and activity in sulfate-reducing batch systems remediating mine drainage.

Five microbial inocula were evaluated in batch tests for the ability to remediate mine drainage (MD). Dairy manure (DM), anaerobic digester sludge, substrate from the Luttrell (LUTR) and Peerless Jenny King (PJK) sulfate-reducing permeable reactive zones (SR-PRZs) and material from an MD-treatment column that had been inoculated with material from a previous MD-treatment column were compared in terms of sulfate and metal removal and pH neutralization. The microbial communities were characterized at 0, 2, 4, 9, and 14 weeks using denaturing gradient gel electrophoresis and quantitative polymerase chain reaction to quantify all bacteria and the sulfate-reducing bacteria of the genus Desulfovibrio. The cultures inoculated with the LUTR, PJK, and DM materials demonstrated significantly higher rates of sulfate and metal removal, and contained all the microorganisms associated with the desired functions of SR-PRZs (i.e., polysaccharide degradation, fermentation, and sulfate reduction) as well as a relatively high proportion of Desulfovibrio spp. These results demonstrate that inoculum influences performance and also provide insights into key aspects of inoculum composition that impact performance. This is the first systematic biomolecular examination of the relationship between microbial community composition and MD remediation capabilities.

The effect of inoculum on the performance of sulfate-reducing columns treating heavy metal contaminated water.

Sulfate-reducing permeable reactive zones (SR-PRZs) are a passive means of immobilizing metals and neutralizing the pH of mine drainage through microbially mediated reactions. In this bench-scale study, the influence of inoculum on the performance of columns simulating SR-PRZs was investigated using chemical and biomolecular analyses. Columns inoculated from two sources (bovine dairy manure (DM) and a previous sulfate-reducing column (SRC)) and uninoculated columns (U) were fed a simulated mine drainage and compared on the basis of pH neutralization and removal of cadmium, zinc, iron, and sulfate. Cadmium, zinc, and sulfate removal was significantly higher in SRC columns than in the DM and U columns, while there was no significant difference between the DM and U columns. Denaturing gradient gel electrophoresis (DGGE) analysis revealed differences in the microbial community composition among columns with different inocula, and indicated that the microbial community in the SRC columns was the first to reach a pseudo-steady state. In the SRC columns, a higher proportion of the DGGE band DNA sequences were related to microorganisms that carry out cellulose degradation, the rate-limiting step in SR-PRZ energy flow, than was the case in the other columns. The proportion of sulfate-reducing bacteria of the genus Desulfobacterium was monitored using real-time quantitative PCR and was observed to be consistently higher in the SRC columns. The results of this study suggest that the inoculum plays an important role in SR-PRZ performance. This is the first report providing a detailed analysis of the effect of different microbial inocula on the remediation of acid mine drainage.