Evaluation and identification of fibro-lytic potential tannin-tolerant microbes from wild herbivores to improve goat browse utilization | Fibro-lytic potential tannin-tolerant microbes in wild herbivores

A Thesis submitted to the Faculty of Science, Agriculture and Engineering in fulfilment of the requirements for the Degree of Doctor of Philosophy in Animal Science in the Department of Agriculture at the University of Zululand, South Africa [2023].

Livestock production in developing countries is constrained by insufficient feed quality to sustain high productivity particularly during dry seasons. During this period, ruminants switch their diet from mixed feeding to principally browsing drought-resistant trees and shrubs containing different types and concentrations of tannins. Consuming high tannin forages reduce nutrient digestibility but small amounts can be beneficial, especially in the presence of rumen bacteria that can tolerate tannins. The objectives of the study were to monitor microbial ecosystems from wild herbivores; manipulate, evaluate and identify tannin tolerant microbes; and create microbial ecosystems with higher fibrolytic potential to use browse species in goats. To achieve these objectives, a series of experiments were conducted. Experiment 1 investigated the effect of condensed tannins on the fibrolytic activity of an microbial ecosystem from goats (G), giraffes (GR), kudus (K), impalas (IM) and a consortia of animals [A1 (G+IM), A2 (G+KD), A3 (G+GR), A4 (GR+IM), A5 (GR+KD), A6 (IM+KD), A7 (G+GR+IM), A8 (G+GR+KD), A9 (G+IM+KD), A10 (GR+KD+IM), A11 (G+GR+KD+IM)]. Fresh faecal samples were collected and mixed with homogenization buffer for crude enzyme extraction. Crude enzyme extracts were precipitated with 60% ammonium sulphate followed by dialysis, and assayed for endocellulase, exocellulase and xylanase activities by incubating with arboxymethyl cellulose, microcrystalline cellulolse and xylan, respectively. An in vitro fermentation study was done by incubating strained faecal fluid with medium containing 1g of Vacchellia sieberiana at different concentrations of condensed tannins [control (feed without adjustment 5.34%), 8 and 10%]. Apparent degradability (APD), True degradability (TD), Neutral detergent fiber degradability (NDFd), Acid detergent fiber degradability (ADFd) and Microbial yield (MY) were measured. Supplementation of V. sieberiana with condensed tannins had significant effect on enzymes specific activities (endocellulase, exocellulase, xylanase) and fibre degradability. Supplemented group had lower enzyme activities, neutral detergent fiber (NDF), acid detergent fiber (ADF), apparent degradability (APD) and true degradability (TD). However, microbial consortia improved (P<0.05) goats’ fibrolytic enzyme activities and dry matter degradability. Therefore, at the end of the in vitro study, microbial consortia [A7 (G+GR+IM), A1 (IM+G), A9 (G+K+I) and A11 (G+GR+KD+IM)] were the most promising and effective microbial ecosystems to enhance goat fibrolytic activities and was thus selected for further studies. Experiments 2 and 3 isolated and identified tannin tolerant bacteria from faecal matter of goats (G), giraffes (GR), kudus (K), impalas (IM) and the best performing consortia [A= A7 (G+GR+IM), B= A1 (IM+G), C= A9 (G+K+IM) and D= A11 (G+GR+K+IM)] on goat fibrolytic activity. Thirteen tannin tolerant bacteria were isolated from the enrichment cultures of faecal microflora through serial dilutions and spread plate method in a medium containing condensed tannins as a sole carbon source and energy. The isolates were identified based on morphological, biochemical characteristics and 16S rDNA gene sequences. A total of 13 Gram-negative and Gram-positive tannin tolerant bacteria designated as G, GR2, A1, B2, D2, C1, GR1, D2, A2, B1, K, I and C2 were isolated and identified as members of the Gram-negative Escherichia sp. (6), Gram positive Enterococcus sp. (5) and Bacillus species (1). A new Gram positive (B1) strain that does have any similarity with reference strain in the NCBI nucleotide database was also identified. In Experiment 4, tannin-tolerant bacterial isolates were screened to produce endocellulase, exocellulase and xylanase enzymes on carboxymethyl cellulose (CMC), microcrystalline cellulose (MCC) and xylan (XY) agar plates, respectively, following the gram’s iodine staining procedure. Enzymes were extracted from each potential isolate and the enzyme activity assay was performed based on 3,5 dinitrosalicylic acid (DNS) method. Temperature, incubation time and pH were then optimised for maximum activity of exocellulase, endocellulase and xylanases. Out of 13 bacterial isolates, two strains (15%) were able to utilize CMC, MCC and XY, five strains (39%) were able to use CMC and XY and six strains (46 %) were not able to use any of these substrates. The optimum temperature for enzyme (cellulase and xylanase) activities was 40°C. However, optimal incubation period and pH varied among bacterial species. In conclusion, fibrolytic microbes were identified from wild ruminant systems and tested on a goat system. Improved fibrolytic activity on goats was confirmed and the study identified 13 tannin tolerant bacteria. The tannin tolerant fibrolytic microbes identified in this study may have evolved in response to the dietary and environmental pressure, especially of the wild systems, as the animals normally consume feed with high tannin concentrations in their natural habitat. Therefore, it is possible that the selective pressures imposed by these diets over time have led to the evolution of specialised microbial communities that are better suited to produce enzymes breakingdown fibrous plant material and extracting nutrients in the presence of tannins. Identifying tannin-tolerant fibrolytic microbes can be used to develop supplements that could help improve the digestive health and nutrient absorption of goats. Future research is needed for fuller understanding of the benefits of modifying domestic animals’ microbiome with tannin-tolerant bacteria from the wild. OKUHUNYUSHWE NGOLIMI LWESIZULU Isifinyezo Ukukhiqizwa kwemfuyo emazweni asathuthuka kuvinjelwa izinga lokudla okunganele ukuze kugcinwe umkhiqizo ophezulu ikakhulukazi ngesikhathi sesomiso. Ngalesi sikhathi, izilwane ezelusayo zishintsha ukudla kwazo zisuke ekudleni okuxubile ziye ekuphequluleni izihlahla ezimelana nesomiso kanye nezihlahlana eziqukethe izinhlobo ezahlukene kanye nokugcwala kwama-tannins. Ukudla ukudla okune-tannin eliphezulu kunciphisa ukugayeka kokudla okunomsoco kodwa amanani aphansi angasiza, ikakhulukazi uma kukhona ama-microorganisms esiswini angamelana namatannin. Izinjongo zocwaningo bekuwukuqapha imvelo ye-microbial evela ezilwaneni ezidla uhlaza zasendle; phatha, uhlole futhi uhlonze amabhaktheriya abekezelela i-tannin; futhi adale ama-microbial ecosystem anamandla aphezulu efibrolytic okusebenzisa izinhlobo zokuphequlula ezimbuzini. Ukufeza lezi zinhloso, uchungechunge lwezilingo lwenziwa. Ukuhlolwa koku-1 kuphenye umthelela wama-tannin ajiyile emsebenzini wefibrolytic we-microbial ecosystem evela ezimbuzini (G), izindlulamithi (GR), i-kudus (K), i-impalas (IM) kanye nenhlanganisela yezilwane [A1 (G+IM), A2 (G+KD), A3 (G+GR), A4 (GR+IM), A5 (GR+KD), A6 (IM+KD), A7 (G+GR+IM), A8 (G+GR+KD ), A9 (G+IM+KD), A10 (GR+KD+IM), A11 (G+GR+KD+IM)]. Amasampula endle amasha aqoqwa futhi axutshwa ne-homogenization buffer ukuze kukhishwe i-enzyme engahluziwe. Ukukhishwa kwe-enzyme engahluziwe kwehliswe nge-60% yeammonium sulphate elandelwa yi-dialysis, futhi yahlolwa imisebenzi yeendocellulase, i-exocellulase kanye ne-xylanase ngokufukamela nge-carboxymethyl cellulose, i-microcrystalline cellulolse ne-xylan, ngokulandelana. Ucwaningo lokuvutshelwa kwe-in vitro lwenziwa ngokufukamela uketshezi lwendle olucindezelwe phakathi nendawo equkethe i-1g ye-Vacchellia sieberiana ekugxilweni okuhlukene kwama-tannins ajiyile [ukulawula (ukuphakela ngaphandle kokulungisa 5.34%), 8 kanye no-10%]. Ukuwohloka okubonakalayo (i-APD), ukuwohloka kweqiniso (TD), ukuwohloka kwefayibha yokuhlanza engathathi hlangothi (NDFd), ukuwohloka kwefayibha yokuhlanza i-Acid (ADFd) kanye nesivuno seMicrobial (MY) kukalwa. Ukwengezwa kwe-V. sieberiana ngama-tannins ajikisiwe kube nomthelela omkhulu emisebenzini ethile yama-enzyme (i-endocellulase, i-exocellulase, i-xylanase) kanye nokuwohloka kwe-fiber. Iqembu elengeziwe lalinemisebenzi ephansi yama-enzyme, i-neutral detergent fiber (NDF), i-acid detergent fiber (ADF), i-apparent degradability (APD) kanye nokonakala kweqiniso (TD). Kodwa-ke, i-microbial consortia yenza ngcono (P<0.05) imisebenzi ye-enzyme ye-fibrolytic yezimbuzi kanye nokuwohloka kwento eyomile. Ngakho-ke, ekupheleni kocwaningo lwe-in vitro, i-microbial consortia [A7 (G+GR+IM), A1 (IM+G), A9 (G+K+I) kanye ne-A11 (G+GR+KD+IM)] kwakuyizinto eziphilayo ezithembisayo nezisebenza kahle kakhulu ze-microbial ecosystem ukuze kuthuthukiswe imisebenzi ye-goat fibrolytic futhi ngaleyo ndlela yakhethelwa izifundo eziqhubekayo. Ukuhlola 2 kanye no-3 amagciwane angakwazi ukumelana ne-tannin ahlukanisiwe avela ezimbuzini (G), izindlulamithi (GR), i-kudus (K), i-impalas (IM) kanye ne-consortia eyenza kahle kakhulu [A= A7 (G+GR+IM), B= A1 (IM+G), C= A9 (G+K+IM) kanye no-D= A11 (G+GR+K+IM)] emisebenzini ye-fibrolytic yembuzi. Amagciwane ayishumi nantathu abekezelela i-tannin ahlukaniswa kumasiko okunothisa we-microflora yendle ngokusebenzisa ukuhlanjululwa okulandelanayo kanye nendlela yokusabalalisa ipuleti endaweni equkethe ama-tannins ajiyile njengomthombo wekhabhoni kanye namandla. Ama-isolate akhonjwe ngokusekelwe ku-morphological, izici ze-biochemical kanye nokulandelana kofuzo kwe-16S rDNA. Isamba samagciwane angu-13 abekezelela i-tannin e-Gram-negative kanye ne-Gram-positive aqokwe njenge-G, GR2, A1, B2, D2, C1, GR1, D2, A2, B1, K, I kanye ne-C2 ahlukaniswa futhi akhonjwa njengamalungu e-Gram. -negative Escherichia sp. (6), i-Gram positive Enterococcus sp. (5) kanye nezinhlobo ze-Bacillus (1). Kuphinde kwahlonzwa uhlobo olusha lwe-Gram positive (B1) olunokufana nereferensi kusizindalwazi se-nucleotide ye-NCBI. Esivivinyweni sesi-4, ama-bacterial isolate abekezelela i-tannin ahlolwa ukuze kukhiqizwe ama-endocellulase, i-exocellulase ne-xylanase enzyme ku-carboxymethyl cellulose (CMC), i-microcrystalline cellulose (MCC) kanye namapuleti e-agar e-xylan (XY), ngokulandelana, kulandelwa inqubo yokungcolisa i-iodine yegremu. Ama-Enzyme akhishwe ku-isolate ngayinye engaba khona futhi ukuhlolwa komsebenzi weenzyme kwenziwa ngokusekelwe ku-3,5 ye-dinitro-salicylic acid (DNS) indlela. Izinga lokushisa, isikhathi sokufukamela kanye ne-pH kwabe sekulungiselelwa umsebenzi omkhulu we-exocellulase, endocellulase kanye ne-xylanases. Kwezihlukanisiwe zamabhaktheriya eziyi-13, izinhlobo ezimbili (15%) zikwazile ukusebenzisa i-CMC, i-MCC ne-XY, izinhlobo ezinhlanu (39%) zikwazile ukusebenzisa i-CMC ne-XY kanye nezinhlobo eziyisithupha (46%) azikwazanga ukusebenzisa noma iyiphi yalezi. amasubstrates. Izinga lokushisa elilungile lemisebenzi ye-enzyme (i-cellulase nexylanase) lalingu-40°C. Kodwa-ke, isikhathi esifanele sokufukamela kanye ne-pH yahlukahluka phakathi kwezinhlobo zamabhaktheriya. Isiphetho, ama-microbes e-fibrolytic ahlonzwa ezinhlelweni ze-ruminant zasendle futhi ahlolwe ohlelweni lwezimbuzi. Umsebenzi owenziwe ngcono wefibrolytic ezimbuzini waqinisekiswa futhi ucwaningo lwathola amagciwane ayi-13 abekezelela i-tannin. Amagciwane e-fibrolytic abekezelela i-tannin ahlonzwe kulolu cwaningo kungenzeka ukuthi avela ngenxa yengcindezi yokudla kanye nemvelo, ikakhulukazi yezinhlelo zasendle, njengoba izilwane zivame ukudla ukudla kunetannin ephezulu endaweni yazo yemvelo. Ngakho-ke, kungenzeka ukuthi izingcindezi ezikhethiwe ezibekwe yilokhu kudla ngokuhamba kwesikhathi ziye zaholela ekuguqukeni kwemiphakathi ekhethekile yamagciwane afaneleka kangcono ukukhiqiza ama-enzyme aphula impahla yezitshalo ze-fibrous kanye nokukhipha imisoco phambi kwama-tannins. Ukuhlonza amagciwane e-fibrolytic abekezelela itannin kungasetshenziswa ukwenza izithasiselo ezingasiza ukuthuthukisa impilo yokugaya ukudla kanye nokumunca izakhamzimba zezimbuzi. Ucwaningo esikhathi esizayo luyadingeka ukuze kuqondwe ngokugcwele izinzuzo zokushintsha imicrobiome yezilwane ezifuywayo ngamagciwane abekezelela ama-tannin avela endle.