TEKNIK KULTIVASI DAN PROSES HILIR m.k Teknologi Bioindustri Departemen Teknologi Industri Pertanian FATETA - IPB

Skema Umum Proses Kultivasi Pengembangan Inokulum Biomassa Pemisahan Sel Cairan Fermentasi Kultur Stok Labu Kocok Bioreaktor Inokulum Supernatan Bebas Sel Sterilisasi Media Formulasi Media Ekstraksi Produk Penanganan Limbah Cair Biorektor Pemurnian Produk Bahan Baku Media Pengemasan produk

Metode kultivasi berdasarkan cara operasi bioreaktor : - curah (batch) - sinambung (continuous) - semi sinambung (fed- batch)   Continuous

Kultur Curah (Batch Neraca Biomassa rX = dX/dt  rx = X  Plot ln X vs t akan memperoleh nilai  Ln X Keterangan :  : laju pertumbuhan spesifik (waktu-1) F : laju alir (vol/waktu) X : konsentrasi biomassa (g/l)  t

Pembentukan Produk : rp == qp X Keterangan : P = konsentrasi produk (g/l) qp = laju spesifik pembentukan produk (g/g sel.jam) k = tetapan laju destruksi produk

Konsumsi Substrat : rs = qs X Keterangan : S = konsentrasi substrat (g/l) ; m = tetapan pemeliharaan sel qs = laju spesifik penggunaan substrat (g/g sel.jam)

Yield Coefficient (Tetapan Rendemen) pembentukan produk berasosiasi dengan pertumbuhan sel Yp/x = dP/dX = rp/rx Yp/s = dP/dS = rp/rs Yx/s = dX/dS = rx/rs rp = Yp/x rx qpX = Yp/x µX  qp = Yp/x µ rp = Yp/s rs qpX = Yp/s qs X  qp = Yp/s qs rx = Yx/s rs µX = Yx/s qs X  µ = Yx/s qs

Laju Spesifik Pembentukan Produk & Penggunaan Substrat (g/g sel.jam)  qp & qs : tidak tergantung konsentrasi sel menggambarkan efektivitas sel dlm mensintesis produk atau penggunaan substrat berguna untuk membandingkan efektivitas hasil suatu fermentasi Laju Volumetrik (Q ; g/l.jam) :  Qp & Qs tergantung dari konsentrasi sel menggambarkan laju sintesis produk atau kebutuhan substrat untuk tiap satuan volume bioreaktor Pola Pembentukan Produk : Pembentukan produk berasosiasi dengan pertumbuhan (growth associated) = Metabolit Primer Pembentukan produk tidak berasosiasi dengan pertumbuhan (non growth associated) = Metabolit Sekunder Campuran keduanya

Aplikasi Kultur Curah : Digunakan untuk memproduksi biomassa, metabolit primer dan metabolit sekunder Untuk produksi biomassa  digunakan kondisi kultivasi yang mendukung pertumbuhan biomassa, sehingga mencapai maksimal Untuk prodiksi metabolit primer  kondisi kultivasi harus dapat memperpanjang fase eksponensial yang dibarengi dengan sintesisi produk Untuk produksi metabolit sekunder  kondisi kultivasi harus dapat memperpendek fase eksponensial dan memperpanjang fase stasioner

Kultur Sinambung Kultur Sinambung Neraca Biomassa : X S F0 (l/jam) S0 (g/l) X0 (g/l) = 0 (umoan steril) F (l/jam) S (g/l) X (g/l) Kultur sinambung dijalankan setelah tercapai Xmaks V Neraca Biomassa : (jam-1) Keterangan : D = laju dilusi = F/V (jml vol kultur yang melalui bioreaktor)

Neraca substrat * Bila tidak ada pembentukan P, Contohnya PST *Konsumsi unt pertumb & pembentukan produk)

Neraca Produk P1 = (Yp/x. µX)/D

Untuk hubungan antara X, S dan D, diperlukan model yang mengekspresikan laju pertumbuhan sebagai fungsi dari substrat pembatas  Model Monod Keterangan : Ks = tetapan “half-rate saturation” = konsentrasi S pada μ = ½ μmaks

Dcrit = laju dilusi kritis (menggambarkan laju dilusi maksimum dari operasi keadaan tunak)  biasanya Dcrit = μmaks D (Ks + S) = Dcrit S D Ks + D S = Dcrit S D Ks = Dcrit S - D S = S (Dcrit – D)

Kurva Pengaruh D pada Kultur Sinambung X,S,P P X S D Kurva Pengaruh D pada Kultur Sinambung

Aplikasi Kultur Sinambung : Merupakan ‘alat’ untuk penelitian fisiologi dan biokimia mikroba, dikarenakan kondisinya mantap, laju pertumbuhan dapat diatur oleh laju alir dan laju pertumbuhan dibatasi oleh konsentrasi substrat pembatas  dapat digunakan untuk penelitian pengaruh substrat pembatas thd kinerja mikroba, untuk perbaikan sistem curah/semi sinambung Untuk isolasi dan seleksi mikroba penghasil enzim menggunakan media diperkaya Untuk produksi biomassa, contoh ICI (Imperial Chemical Industries, kapasitas bioreaktor 3000 m3, substrat metanol) Untuk produksi bir menggunakan bioreaktor menara (tower bioreactor)

Kultur Semi Sinambung Kultur ini merupakan kultur curah yang diberi umpan secara sinambung atau sekuensial, tanpa pengeluaran isi bioreaktor, sehingga volume bervariasi selama kultivasi  konsentrasi nutrien bervariasi Keuntungan : Dapat menekan efek represif sumber karbon dan mempertahankan kapasitas aerasi dalam bioreaktor Dapat mencegah efek toksik komponen media Keadaan “quasy-steady state” : Kultur curah dengan pertumbuhan yang dibatasi oleh konsentrasi suatu substrat dan semua nutrien lain berlebih X = Xo + Yx/s ( So– S) Pada saat S ~ 0, akan tercapai Xmaks dan Xo <<X, sehingga Xmaks = Yx/s ( So– S)

Pada saat X = Xmaks , diberi umpan media dengan D < μmaks, maka S segera dikonsumsi begitu memasuki kultur  akumulasi (dS/dt) = 0 Neraca Substrat : Meskipun total biomassa meningkat terhadap waktu, namun konsentrasi sel tetap mengingat volume juga meningkat  dX/dt = 0  μ ~ D (quasy-steady state)

Vo = volume awal & F = laju alir ; t = waktu Semakin lama, nilai D << karena volume semakin meningkat Vo = volume awal & F = laju alir ; t = waktu Pada kultur ini, sisa substrat kecil sebagai akibat konsentrasi sel meningkat

ln VX μ t Penentuan μ pada Kultur Semi-sinambung : Diintegrasikan : ln VX - ln VoXo = μ t ln VX μ t Perbandingan : Kultur sinambung : μ = D Kultur semi Sinambung : D << dan μ << dengan laju yang sama

Fed-batch mode of fermentation has the following features: Advantages: Production of high cell densities due to extension of working time (particularly important in the production of growth-associated products) Controlled conditions in the provision of substrates during the fermentation, particularly regarding the concentration of specific substrates as for ex. the carbon source Control over the production of by-products or catabolite repression effects due to limited provision of substrates solely required for product formation The mode of operation can overcome and control deviations in the organism's growth pattern1 as found in batch fermentation Allows the replacement of water loss by evaporation Alternative mode of operation for fermentations leading with toxic substrates (cells can only metabolize a certain quantity at a time) or low solubility compounds Increase of antibiotic-marked plasmid stability by providing the correspondent antibiotic during the time span of the fermentation No additional special piece of equipment is required as compared with the batch fermentation mode of operation      

       Disadvantages: It requires previous analysis of the microorganism, its requirements and the understanding of its physiology with the productivity It requires a substantial amount of operator skill for the set-up, definition and development of the process In a cyclic fed-batch culture, care should be taken in the design of the process to ensure that toxins do not accumulate to inhibitory levels and that nutrients other than those incorporated into the feed medium become limiting, Also, if many cycles are run, the accumulation of non-producing or low-producing variants may result The quantities of the components to control must be above the detection limits of the available measuring equipment

The use of fed-batch culture by the fermentation industry takes advantage of the fact that the concentration of the limiting substrate may be maintained at a very low level, thus : - avoiding repressive effects of high substrate concentration controlling the organism’s growth rate and consequently controlling the oxygen demand of the fermentation. Saccharomyces cerevisiae is industrially produced using the fed-batch technique so as to maintain the glucose at very low concentrations, maximizing the biomass yield and minimizing the production of ethanol, the chief by-product Hepatitis B surface antigen (HbsAg) used as a vaccine against type B hepatitis has been purified from human plasma and expressed in recombinant yeast, being now produced commercially. Again, the production of the recombinant protein is achieved using fed-batch culture techniques very similar to that developed for Saccharomyces cerevisiae. A cyclic method is used due to reports of superior productivity

Penicillin production is an example for the use of fed-batch in the production of a secondary metabolite. The fermentation is divided in two phases: the rapid-growth phase during which the culture grows at the maximum specific growth rate, and the slow-growth phase in which penicillin is produced. During the rapid-growth phase, an excess of glucose causes an accumulation of acid and a biomass oxygen demand greater than the aeration capacity of the fermentor, whereas glucose starvation may result in the organic nitrogen in the medium being used as a carbon source, resulting in a high pH and inadequate biomass formation. During the production phase, the feed rates utilized should limit the growth rate and oxygen consumption such that a high rate of penicillin synthesis is achieved and sufficient dissolved oxygen is available in the medium Some other examples are the production of thiostrepton from Streptomyces laurentii and the production of cellulase by Trichoderma reesei. The production of thiostrepton uses pH feedback control and the production of cellulase utilizes carbon dioxide production as a control factor